Norbert Par

Minet

Dr Lars Petter

Dr Justin Rapporteur

PhD Kollman

Associate Professor, U Washington

Dr Andrew Parker

PhD, Step Pharma Ceo

Dr Alain Fischer

Dr Patrick Revy

Dr Sylvain Latour

Analysis of the regulation of CTPS1 in lymphocyte proliferation and of the mechanism of action of pharmacological inhibitors of CTPS1

Titre : Analyse de la régulation de CTPS1 dans la prolifération lymphocytaire et des mécanismes d'action d'inhibiteurs pharmacologiques de CTPS1

Résumé :

Les cytidine triphosphate synthases (CTPS) sont une famille d'enzymes très conservées réalisant la synthèse de novo de CTP en employant l'UTP, l'ATP et la glutamine comme substrats. Les CTPS forment des dimères inactifs, et des tétramères actifs en présence de leurs substrats (ATP et UTP) ainsi que de leur produit, le CTP. Ces tétramères peuvent ensuite s'assembler sous forme de grandes structures intracellulaires nommées filaments, dont leur rôle dans leur régulation est toujours débattu.

Deux CTPS existent chez l'Homme : CTPS1 et CTPS2. Des travaux antérieurs publiés par notre laboratoire ont pu démontrer le rôle crucial de CTPS1 dans le système immunitaire, en particulier au cours de l'expansion lymphocytaire après stimulation des récepteurs des cellules T en réponse à une infection par des agents pathogènes. De plus, de nombreuses études lient une activité CTPS augmentée au développement et à la progression de cancers. Les données actuellement disponibles suggèrent que CTPS1 est principalement responsable de la synthèse de quantités importantes de CTP afin de répondre aux besoins métaboliques augmentés de cellules activées et proliférantes, dont les cellules tumorales et celles du système immunitaire. CTPS2 pourrait assurer les besoins de CTP à l'état basal étant donné que les patients présentant une déficience en CTPS1 ne semblent pas présenter d'anomalies majeures en dehors du compartiment hématopoïétique. Ainsi, cibler spécifiquement l'activité de CTPS1 semble être une approche thérapeutique valide pour traiter des pathologies comprenant des cancers et certains troubles du système immunitaire.

Nous avons généré et caractérisé des modèles cellulaires de déficiences en CTPS1 et CTPS2 en employant les lignées HEK 293T et Jurkat ainsi que la technologie CRISPR-Cas9. La lignée Jurkat n'exprime pas CTPS2, et l'inactivation de CTPS1 résulte en la perte de toute activité CTPS (CTP synthase) ainsi qu'en l'incapacité de ces cellules à proliférer, conduisant à leur apoptose. CTPS1 et CTPS2 semblent partiellement redondants dans la lignée HEK. Dans les HEK déficientes en CTPS2 (CTPS2-KO), aucun changement en termes d'activité CTPS et de prolifération n'a été détecté.

L'inactivation de CTPS1 conduit à une activité CTPS fortement réduite associée à un défaut de prolifération modéré. Les HEK déficientes pour CTPS1 et CTPS2 ont une activité CTPS nulle. Elles ne sont pas capables de proliférer mais survivent à une privation prolongée en CTP. Chez toutes les lignées étudiées, des approches de complémentation nous ont permis de confirmer que CTPS1 et CTPS2 peuvent restaurer la viabilité et la prolifération de cellules déficientes en CTPS1 et CTPS2. Enfin, nos données suggèrent que CTPS1 et CTPS2 interagissent à l'échelle du tétramère.

Le modèle Jurkat déficient en CTPS a été utilisé afin d'étudier deux mutants de CTPS1 identifiés dans des patients immunodéficients. CTPS1Δ18 (initialement décrit par Martin et al. 2014) résulte de la mutation d'un site accepteur d'épissage, conduisant au saut d'un exon et à l'expression d'un domaine Cterminal alternatif. Nos données confirment que CTPS1Δ18 est un mutant hypomorphe mais actif. Le second mutant, CTPS1Δ19, pour lequel nous n'avions ni données fonctionnelles, ni échantillon de patient, est amputé des 19 derniers résidus de la protéine sauvage. Bien que nos données préliminaires suggèrent que ce mutant est actif et plus stable que CTPS1Δ18, des études plus approfondies seront nécessaires afin de complètement caractériser l'impact de cette mutation.

De nombreuses études ont déjà été publiées au cours desquelles des mutations ciblées ont été introduites dans des CTP synthases afin d'étudier des aspects spécifiques de leurs fonctions et régulations. Ces travaux ont été réalisés chez des espèces procaryotes, eucaryotes unicellulaires et pluricellulaires. Cependant, il est maintenant clair que des différentes significatives dans la régulation des CTPS existent selon les espèces, en particulier à l'échelle du filament. Nos modèles cellulaires nous ont permis de caractériser plus avant l'impact de certaines de ces mutations sur la capacité de CTPS1 à soutenir la prolifération cellulaire, former des filaments et interagir avec CTPS2.

Des formes de CTPS1 et CTPS2 couplées à des étiquettes protéiques ont été largement utilisées dans la littérature dans l'objectif d'étudier la fonction de ces deux protéines dans des modèles cellulaires.

Ces approches nous ont permis d'obtenir d'immenses quantités d'informations concernant le comportement de ces enzymes. Toutefois, elles sont aussi susceptibles d'impacter les propriétés de ces enzymes. Nos résultats mettent en évidence le soin particulier devant être porté lors de l'interprétation de données générées en employant des formes couplées des CTPS.

Ce projet a été établi en partenariat avec une société, Step Pharma, qui développe des inhibiteurs spécifiques de CTPS1. Les CTPS ne sont pas de nouvelles cibles thérapeutiques, mais l'usage des composés déjà existants a été associé à des effets secondaires importants. La plupart d'entre eux sont potentiellement dus à des effets non-spécifiques étant donné qu'ils peuvent impacter l'activité d'enzymes porteuses de domaines, activités et substrats similaires à ceux des CTPS. Cependant, ils peuvent aussi être expliqués par une absence de sélectivité entre CTPS1 et CTPS2, les deux enzymes étant très proches en termes de structure et de fonctions. Nous avons observé que certains composés inhibiteurs développés par Step Pharma présentant une sélectivité importante pour CTPS1 chez l'humain ne conservaient pas cette sélectivité sur les enzymes murines. Par le biais d'analyse de séquences, de travaux de mutagenèse et par la génération de modèles cellulaires, nous avons validé le rôle-clefs de l'isoleucine 250 dans la sélectivité pour CTPS1 des composés de Step Pharma.

Mots clefs : CTP ; CTP synthase (CTPS) ; filaments ; immunodéficiences ; inhibiteurs thérapeutiques. Title : Analysis of the regulation of CTPS1 in lymphocyte proliferation and of the mechanism of action of pharmacological inhibitors of CTPS1

Abstract:

The cytidine triphosphate synthases (CTPS) are a highly conserved family of enzymes responsible for the de novo synthesis of CTP using UTP, ATP and glutamine as their substrate. The CTP synthases form inactive dimers, and active tetramers in presence of their substrates ATP and UTP as well as of their product CTP. These tetramers can further assemble as massive intracellular structures called filaments whose role in their regulation is still debated. There are two CTP synthases in human: CTPS1 and CTPS2. Previous works published in this lab demonstrated the crucial role of CTPS1 in the immune system, especially during lymphocyte expansion after TCR (T-cell receptor) stimulation in response to pathogen infections. Moreover, numerous anterior studies link increased CTP synthase activity to cancer development and progression. The available data suggest that CTPS1 appears be mostly involved in CTP synthesis spikes to sustain the increased metabolic activity of activated and proliferating cells, including immune and tumour cells. The role of CTPS2 would be to support the basal CTP needs as CTPS1-deficient patients do not present major defects outside of the hematopoietic compartment. Thus, specifically targeting the activity of CTPS1 seems to be a relevant therapeutic approach for the treatment of pathologies that include cancers and some immune diseases.

We have generated and characterized CTPS1-and CTPS2-deficient cellular models using the HEK 293T and Jurkat cell lines and the CRISPR-Cas9 technology. Jurkat cells do not express CTPS2, and the inactivation of CTPS1 abrogates their CTPS (CTP synthase) activity and ability to proliferate, leading to apoptosis. CTPS1 and CTPS2 were found to be partially redundant in HEK cells. In CTPS2 knock-out (KO) HEK, no detectable changes in CTPS activity and proliferation were observed. CTPS1-KO HEK were found to have a strongly decreased CTPS activity associated with a moderate decrease in proliferation. CTPS1 and CTPS2-KO HEK were unable to proliferate, yet able to survive a prolonged CTP deprivation despite a null CTPS activity. Exogenous complementation approaches confirmed that both CTPS1 and CTPS2 were able to restore CTPS-deficient cell viability and proliferation. Finally, our data suggest that CTPS1 and CTPS2 might interact at the tetramer level.

The CTPS-null Jurkat model was used to study two CTPS1 mutants expressed in patients with immunodeficiency. CTPS1Δ18 (first described in Martin et al., 2014) results from the mutation of a splice site, leading to exon skipping and to the expression of an alternative C-terminal domain. Our data confirms that CTPS1Δ18 is an hypomorphic, yet active mutant. The second mutant, CTPS1Δ19, for which we had neither functional data nor patient samples, lacks the last 19 residues of the wild-type protein. Although our preliminary data suggests that his mutant is active and more stable than CTPS1Δ18, further studies are required to fully characterize the impact of this mutation.

Many studies have already been published using mutants targeting specific domains of CTPS1 to study specific aspects of its function and regulation. These works have been realized in species ranging from bacteria to unicellular and pluricellular eukaryotes. However, it is now clear that there are significant differences in the regulation of the CTP synthases among species, in particular at the filament level. Our cellular models have allowed us to further characterize the impact of mutations previously studied in the literature on the capacity of CTPS1 to sustain cell proliferation, form filaments and interact with CTPS2.

Forms of CTPS1 and CTPS2 associated with protein tags have been largely used in the literature to study the function of both proteins in cellular models. Such approaches have provided us with tremendous amounts of information regarding the behaviour of the enzymes. However, they are also susceptible have an impact on the properties of the enzymes. Our results highlight the care that must be taken when using and interpretating data generated using tagged forms of CTP synthases. This project has been established in collaboration with a company, Step Pharma, who develops CTPS1-specific inhibitors. CTP synthases are not new therapeutic targets, but existing compounds are also associated with severe adverse effects. Most of them are potentially due to off-target effects as they also impact the activity of other cellular enzymes sporting similar domains, activities, and substrates than the CTP synthases. However, they may also be partially explained by an absence of selectivity between CTPS1 and CTPS2, both enzymes being structurally and functionally closely related. Interestingly, Step Pharma small molecule inhibitors that were shown to be highly selective for human CTPS1 in cellular and enzyme models unexpectedly lost their selectivity on murine enzymes. Through sequence analysis, mutagenesis and generation of cellular models, we have validated the key role of isoleucine 250 on the selectivity for CTPS1 of Step Pharma compounds.

Keywords :

CTP; CTP synthase; filaments: immunodeficiencies; small molecule inhibitors.

Résumé détaillé :

Les cytidine triphosphate synthases (CTPS) sont une famille d'enzymes très conservée réalisant la synthèse de novo de CTP (cytidine triphosphate) en employant l'UTP (uridine triphosphate), l'ATP (adénosine triphosphate) et la glutamine comme substrats ainsi que le GTP (guanosine triphosphte) en tant qu'effecteur allostérique. En retour, le CTP exerce un rétrocontrôle négatif sur l'activité de ces enzymes. Ces enzymes sont donc au coeur de la balance des nucléotides.

En dépit de son rôle important, notamment dans la synthèse des acides nucléiques, des phospholipides et dans la sialylation des protéines, le CTP est le nucléotide le moins abondant dans les cellules. Sa synthèse dépend de deux voies, une voie mineure dite « de sauvetage » exploitant les produits de dégradation des acides nucléiques, et une voie majeure dite « de novo » dépendant de l'activité des CTP synthases.

Les CTP synthases forment des dimères inactifs. En présence de leurs substrats (ATP et UTP) ainsi que de leur produit, le CTP, ces dimères se dimérisent, formant des tétramères qui représentent la forme active de l'enzyme. Plus récemment, il a été démontré que ces tétramères peuvent ensuite s'assembler sous forme de grandes structures intracellulaires nommées filaments, dont leur rôle dans leur régulation est toujours débattu. Deux CTP synthases existent chez l'Homme : CTPS1 et CTPS2. Des travaux antérieurs publiés par notre laboratoire ont pu démontrer le rôle crucial de CTPS1 dans le système immunitaire, en particulier au cours de l'expansion lymphocytaire après stimulation des récepteurs des cellules T en réponse à une infection par des agents pathogènes. De plus, de nombreuses études lient une activité CTP synthase augmentée au développement et à la progression de cancers. Les données actuellement disponibles suggèrent que CTPS1 est principalement responsable de la synthèse de quantités importantes de CTP afin de répondre aux besoins métaboliques augmentés de cellules activées et proliférantes, dont les cellules tumorales et celles du système immunitaire. CTPS2 pourrait assurer les besoins métaboliques en CTP à l'état basal étant donné que les patients présentant une déficience en CTPS1 ne semblent pas présenter d'anomalies majeures en dehors du compartiment hématopoïétique.

Ainsi, cibler spécifiquement l'activité de CTPS1 semble être une approche thérapeutique valide pour traiter des pathologies comprenant des cancers et certains troubles du système immunitaire.

Nous avons généré et caractérisé des modèles cellulaires de déficiences en CTPS1 et CTPS2 en employant les lignées HEK 293T (issue d'un rein embryonnaire humain) et Jurkat (leucémie T) ainsi que la technologie CRISPR-Cas9. La lignée Jurkat n'exprime pas CTPS2, et l'inactivation de CTPS1 résulte donc en la perte de toute activité CTP synthase. Ces cellules restent bloquées en phase G1 du cycle cellulaire, traduisant l'incapacité de ces cellules à proliférer et conduisant rapidement à leur apoptose. CTPS1 et CTPS2 semblent partiellement redondantes dans la lignée HEK. En effet, dans les HEK déficientes en CTPS2 (CTPS2-KO), aucun changement en termes d'activité CTPS ni de prolifération n'a été détecté. L'inactivation de CTPS1 dans les HEK conduit à une forte réduction de l'activité CTPS, associée à un défaut de prolifération modéré. Ceci suggère que, dans ces cellules, CTPS2 joue un rôle mineur en présence de CTPS1, mais que son activité est essentielle en son absence. Les HEK déficientes pour CTPS1 et CTPS2 ont une activité CTPS nulle. Elles ne sont pas capables de proliférer, mais contrairement aux Jurkat CTPS1-KO survivent à une privation prolongée en CTP. Les défauts de prolifération et de survie observés dans ces deux lignées semblent être la conséquence directe de la déprivation en CTP de ces cellules. En effet, ils peuvent être respectivement reproduits ou contrés par le traitement de ces cellules par des inhibiteurs spécifiques des CTP synthases (tels que la 3-deazauridine (3DU), un analogue de l'UTP) ou par une supplémentation en cytidine, un des substrats de la voie de sauvetage du CTP. Chez toutes les lignées étudiées, des approches de complémentation nous ont aussi permis de confirmer que CTPS1 et CTPS2 peuvent restaurer la viabilité et la prolifération de cellules déficientes en CTPS1 et CTPS2. Nous nous sommes aussi intéressés aux filaments de CTPS1 et de CTPS2. Pour ce faire, nous avons employé des marquages intracellulaires des formes endogènes de CTPS1 et CTPS2 ainsi que des formes porteuses d'une étiquette GFP (protéine fluorescente verte) (nommées ci-après GFP-CTPS1 et GFP-CTPS2) formant spontanément des filaments. Les données de la littérature suggèrent que les CTPS1 et CTPS2 humaines peuvent former des filaments en présence de leur substrats (ATP, UTP), tandis que seul CTPS2 peut former des filaments en présence du CTP (produit). Conformément à ce qui était attendu, le traitement de nos cellules avec de la 3-DU, un analogue du substrat UTP, induit la formation de filaments métaboliques de CTPS1 et CTPS2. Dans ces mêmes conditions, tandis que CTPS1 s'assemble sous forme de filaments dans des HEK exprimant ou non CTPS2, il ne nous a pas été possible de détecter des filaments de CTPS2 en l'absence de CTPS1. L'expression d'une forme de CTPS1 porteuse d'une étiquette GFP dans des HEK n'exprimant pas CTPS1 permet de restaurer et d'induire la formation de filaments endogènes de CTPS2 en présence ou non de 3-DU. Il semble donc que, dans ces cellules, la présence de CTPS1 soit nécessaire à la formation de filaments métaboliques de CTPS2. Enfin, nous avons pu démontrer par spectrométrie de masse et coimmunoprécipitation que CTPS1 et CTPS2 interagissaient directement. Cette interaction est conservée en employant des formes de CTPS1 et CTPS2 porteuses d'une mutation bloquant totalement la formation de filaments. La co-expression de ce mutant de CTPS1 déficient pour la formation de filaments avec la protéine de fusion GFP-CTPS2 réduit fortement la formation de filaments. De plus, l'expression stable de ces mutants dans des cellules déficientes en activité CTP synthase restaure leur capacité à proliférer.

Ces données suggèrent donc que l'interaction entre CTPS1 et CTPS2 dans la cellule se produit à l'échelle du tétramère, et confirment que ces mutants sont actifs et capables de fournir suffisamment de CTP pour soutenir la prolifération cellulaire en dépit de leur incapacité à former des filaments. En prenant en considération des études récentes ayant mis en évidence des différences au niveau des mécanismes de régulation de CTPS1 et CTPS2, il est donc envisageable que cette interaction permette de moduler la régulation ainsi que l'activité de ces deux enzymes de manière beaucoup plus fine.

Le modèle Jurkat déficient en CTPS a été utilisé afin d'étudier deux mutants de CTPS1 identifiés dans des patients immunodéficients. CTPS1Δ18 (initialement décrit par Martin et al. 2014) résulte de la mutation d'un site accepteur d'épissage, conduisant au saut d'un exon et à l'expression d'un domaine Cterminal alternatif. Nos données confirment que CTPS1Δ18 est un mutant hypomorphe mais actif. Tout comme GFP-CTPS2, et contrairement à GFP-CTPS1, GFP-CTPS1Δ18 est capable de former des filaments en présence de cytidine/CTP. Ceci suggère que cette mutation pourrait aussi modifier sa régulation, en accord avec des données de la littérature impliquant le domaine C-terminal des CTP synthases dans leur capacité ou incapacité à former des filaments. Le second mutant, CTPS1Δ19, pour lequel nous n'avions ni données fonctionnelles, ni échantillon de patient, est amputé des 19 derniers résidus de la protéine sauvage. L'étude de ce mutant a donc nécessité sa génération à partir des données de séquençage du patient, ainsi que la génération de modèles cellulaires. L'expression de ce mutant dans des Jurkat déficientes en CTPS1 restaure la prolifération de ces dernières. Bien que nos données préliminaires suggèrent que ce mutant est actif et plus stable que CTPS1Δ18, des études plus approfondies seront nécessaires afin de complètement caractériser l'impact de cette mutation, notamment dans des cellules primaires exprimant CTPS2.

De nombreuses études ont déjà été publiées au cours desquelles des mutations ciblées ont été introduites dans des CTP synthases afin d'étudier des aspects spécifiques de leurs fonctions et régulations. Ces travaux ont été réalisés chez des espèces diverses, procaryotes, eucaryotes unicellulaires et pluricellulaires. Cependant, il est maintenant clair que des différentes significatives dans la régulation des CTP synthases existent selon les espèces, en particulier à l'échelle du filament. Nos modèles cellulaires nous ont permis de caractériser plus avant l'impact de certaines de ces mutations sur la capacité de CTPS1 à soutenir la prolifération cellulaire, former des filaments et interagir avec CTPS2.

Ces données, bien que préliminaires, permettent d'infirmer ou de confirmer l'impact de certaines de ces mutations, apportant par ce biais un nouvel éclairage sur certaines données issues de la littérature.

Des formes de CTPS1 et CTPS2 couplées à des étiquettes protéiques ont été largement utilisées dans la littérature dans l'objectif d'étudier la fonction de ces deux protéines. Ces approches nous ont permis d'obtenir d'immenses quantités d'informations concernant le comportement de ces enzymes dans des modèles cellulaires. Toutefois, elles sont aussi susceptibles d'impacter les propriétés de ces enzymes. Par exemple, la tendance naturelle de la GFP à former des dimères favorise la formation spontanée de filaments et cette étiquette est susceptible de déstabiliser ces protéines de fusion. Nos résultats mettent en évidence le soin particulier devant être porté lors de l'interprétation de données générées en employant des formes couplées des CTPS.

Ce projet a été établi en partenariat avec une société, Step Pharma, qui développe des inhibiteurs spécifiques de CTPS1. Les CTP synthases, de manière générale, ne sont pas de nouvelles cibles, et des composés impactant de manière plus ou moins spécifique l'activité synthase ont déjà été identifiés, développés, et employés à des fins thérapeutiques. Cependant, l'usage des tels composés a été associé à des effets secondaires importants. La plupart d'entre eux sont potentiellement dus à des effets nonspécifiques étant donné qu'ils peuvent impacter l'activité d'enzymes porteuses de domaines, activités et substrats similaires à ceux des CTP synthases. Cependant, ils peuvent aussi être expliqués par une absence de sélectivité entre CTPS1 et CTPS2, les deux enzymes étant très proches en termes de structure et de fonctions. Des données récemment publiées démontrent que CTPS2 est plus sensible au rétrocontrôle négatif de son activité exercé par le CTP, tandis que nos données démontrent que CTPS2 est aussi plus sensible à une inhibition par un analogue d'un de ses substrats, la 3-DU. Il est donc possible que CTPS2 soit la cible principale des composés anti-CTP synthase déjà existants, la plupart d'entre eux étant des analogues de son produit, le CTP, ou d'un de ses substrats, l'UTP. Nos modèles cellulaires déficients en CTPS1 ou CTPS2 nous ont permis de valider la sélectivité pour CTPS1 des composés développés par Step Pharma. De plus, nous avons aussi observé que certains de ces composés impactaient la formation de filaments de CTPS1, et de manière probablement indirecte ceux de CTPS2.

De manière inattendue, il a aussi été observé lors de tests enzymatiques que certains composés inhibiteurs développés par Step Pharma et présentant une sélectivité importante pour CTPS1 chez l'humain ne conservaient pas cette sélectivité sur les enzymes murines. Par le biais d'analyses de séquences et de modélisation, un résidu candidat susceptible d'être impliqué dans la sélectivité de ces composés a été identifié. L'emploi d'une approche de mutagenèse ciblée suivie par la génération de modèles cellulaires nous a permis de valider le rôle-clefs de l'isoleucine 250 dans la sélectivité pour CTPS1 des composés de Step Pharma.

This thesis is dedicated to Chloé, Florence and Éléanore.

Acknowledgments

Some say that this is the most important part of the thesis -and usually the one that ends up being written at the very last time, after month and month of uninterrupted work and sleep deprivation. This one is no exception, and I hope I won't forget half of the lab (again) like I used to do as I prepared the dreaded Imagine Tuesday Seminars. I guess that writing them in French would feel out of place given that the document is written in something that looks more or less like English.

First of all, I have to start with Sylvain. I am infinitely grateful for all these years. He has given me the chance to work on a great project, to be accompanied by a lovely team (Les "bisounours du troisième", the "care bears"), which ended up as a PhD project. Thank you for the guidance, the insights, the pep talks, and again, for helping me accomplish what I thought was an unreachable dream.

I also thank the other members of my jury who have agreed to review my work, the Dr Jordheim and Dr Kollman for the time you will dedicate to reading my thesis and writing your report, as well as to Alain Fisher, Patrick Revy and Andrew Parker. I hope that this manuscript will not be too underwhelming given the wait. My deepest apologies, and thank you again for your patience.

I also have to acknowledge the ANRT (and Step Pharma!) who funded most of my PhD, as well as Imagine, who gave me the additional time I needed to finish my thesis as well as a second home (although it is possible that my apartment is actually my secondary accommodation at this point…).

Next, the team! I thought this part would be easier to write. There's so much to say, and at the same time it's so hard to find the words to pinpoint what everyone gave to me. I'm sure that I'm going to forget people, as I usually do, and sometimes some of the most important ones, to. My deepest apologies to you… Of course, I have to start with Manu, not only for the CTPS1 project, but also for all the help he provided and his ability to see the little details that can completely make a difference in the interpretation of your results. Claire, head of molecular and caffeinated biology and so much more! Capucine, I listed you this time, sorry about the Tuesday seminar… Christelle, the lab's mother: another kid is leaving the house soon… but he's not going to forget you ;)! Debora, Benjamin, and Camille. We've shared an office for years, and more than that (yes, sometimes probably too much… thank you for taking care of me that one time, and the other times as well). It has been heartbreaking to see you leave the lab but getting to meet you is one of the best outcomes of this PhD. There's so much more I'd like to add, but it would make people jealous if I keep on droning on about you for too long. Sorry for the glove box, and for keeping this this short; but I hope you know how I feel.

Of course, I also shared the same student office with Sarah, whose good mood and legendary laugh still resonates in the walls of Imagine, and Remy, who warned me that ligations just never worked. Indeed. Zineb, our favourite lab kleptomaniac! I know things aren't always easy, and like everyone you have your defects… yet you still manage to turn the bad into good and to provide us with love and care. Keep up the good energy! Gaétane, what can I say? After a few weeks with you, it felt like you were always part of the team. You're funny, you're kind, you're a wonderful person all around, in short: you were meant to be a Team Latour member. Both of you left, but it feels like you're still here (well, Zineb is still a little bit here ;) ).

Cecile, A New Hope (which might mean that my PhD is Revenge of the Sith, not sure it's a good thing):

CTPS1 is in more than capable hands! Please do not forget my invitation when you finally get your Nobel prize…Romane and her adventures… I'm also going to miss you (and them!). Mathieu (Fufu), who apparently wanted to experience sleep deprivation before the final rush of his PhD. Virginie… it's been a few years now that you (and your wonderful pastries) have left the lab, and I hope everything is going well with your new position (and daughter!). Steicy, you're still a Team Latour member, no matter what! You're probably finishing your PhD as well as I'm writing these lines, good luck… I feel your pain. David, our music lover. I'm still convinced that the pianist you were listening to, that one day, was having a stroke.

Akihiro, who manages to be even less talkative than I am (although language is a barrier, and I have to say that I love hearing you and Takeshi speak Japanese in the lab!). Thank you for your kindness and scientific advice! Takeshi, good luck with your project! And since you're complaining about the lack of spices in food in France, you probably should try "trois fois plus de piment" ;).

Anne-Claire, our CTPS activity specialist, who took care of my precious samples, and always with a smile despite the sometimes complicated work conditions. Due to my recurrent memory issues (exacerbated by the intense last few years… and by the age), I'm not even going to try to list all the students that joined the team at one point or another (and I'm probably going to forget some of the greatest ones by doing so), but still, I'd like to thank Aymar, who also participated to the CTPS1 project. He was brilliant, yet he chose to work on something else than CTPS1 (how could you?), giving me the chance, maybe not to shine, but at least to work on this project and write this thesis. Thank you, and I hope everything went well for you. I'm also going to mention this year's batch of students, Roz, Radi and Annaëlle. Students like you give us hope that not everything is lost! Good luck to the new generation, Anne-Laure, Alexandre and Mathieu! The team at Step Pharma: Geoffroy, who was one of the first persons that I met when I arrived at Imagine… I never imagined that this would lead me here. Thank you so much. Helene, I think we actually joined Step at around the same time… thank you for everything, and for bringing a little bit of French in

Step Pharma ;)! Andrew and Philip, we sadly didn't get much time to get to know each other thanks to the COVID restrictions. I also wish you the best for the end of the project. Tim Bourne, thank you for your time at Step Pharma, the advices, your knowledge. And in particular, thank you for that one email you sent me around Christmas a few years ago, an unexpected act of kindness that I desperately needed at the time.

I also thank the people at Sygnature, and in particular Louise, Rebecca, David and Scott. Congratulations for your work! There is so many people at Imagine that I'd like to thank… I'm frustrated as faces, not names, are going through my head. I have to thank Emilie, head of human resources, a great person that actually makes you feel you're more human than resource. I'd like to thank Malika as well, for being so adorable and caring, and Christine, who patiently dealt with our administrative issues. And also people of the third floor: the Crow, FRL, DGSI and more recently the Miccio team, good luck! I'm going to miss meeting you in the Imagine corridors, Vincent and its delicate humor, Marie Claude who always needs to analyse a few tubes when the Fortessa planning is full (we still love you!), Marta, the kind of person that just oozes with good energy, Aude, "Salut les voisins!", Laetitia, IncuCyte hero, and so on… Not to forget Patrick and Gaël, the two members of my CSI, for their advices. Patrick, although I didn't manage to do everything we wanted to do, I'm really grateful for the (too) few fascinating discussions we had. And so many others… Jihane, Chadi, Thomas, Béatrice, Jean, Martine, Boussad, and the rest of the teams at Sisene and Matriscience, you were not part of my thesis, but you are part of my story.

Although this thesis is mainly dedicated to Chloé, Florence and Éléanore, there are also a few people that I'd like to mention who didn't make it to the end. Laurence, I failed to keep contact, which is one of my specialties, until it was too late… Thank you for these years. You were one of the kindest persons I've ever known. Mamie Sam and Suzanne, two grandmothers, one of blood and one of heart. Jean Plouët, the chance you gave me also led me here, at the right time, the right place, with the right people. Thank you.

I probably could go on and on, but I have a defence to prepare… I also feel the need to address very special thanks to what used to be my family. They'll never read this, but I don't care. Thank you for the lack of love, trust, and support. It has been such a pleasure to have to fight depression for more than 2 years when I should have been fully focused on my thesis. I'm done being blamed for things I've never done. I was wrong to care about you; at least I know that I owe you nothing.

Luckily, on the opposite end of the spectrum, there is my real family. My wife Florence, my daughter Éléanore, and my "in-law" family (I'm not very fond of this expression -you're so much more than that), in particular Geneviève and Thierry. I've been through the worst moments of my life during the past few years, but all of them have been steadily by my side. It's also thanks to them that I survived. I really wouldn't have made it without them. Thank you for everything. Florence, the never-ending thesis writing is finally coming to an end! Thank you also for reviewing my abstract, and for your patience during these long months (…years…), and for everything else. I love you. Éléanore, maybe you'll read this someday, unless I'm too ashamed to let you. I haven't been the greatest of fathers, especially during these last months (bad parenting probably runs in the family; maybe there's a cohort to be constituted here). In spite of this, you've been an endless source of love (and nonsensical stories). I love you too.

I also thank Marie-Ange, my godmother, Joëlle and Cédric. You know why.

Chloé. You've given me so much. Part of me died with you. The rest will keep on loving you, always.

List of abbreviations

Introduction Nucleotides

Nucleotides are one of the essential molecular bricks used by all living organisms and viruses and result from the association between a nucleoside and up to three phosphate groups. They are divided in two families, depending on the structure of their nucleobase that, once fused with a sugar (ribose or 2deoxyribose), turns them into a nucleoside. Structurally, pyrimidine is the simplest of the two families, consisting in a heterocyclic nitrogenous ring, while purines associate the pyrimidine base with an imidazole ring. Five of those nucleotides are considered the "main" ones: two purines (adenosine and guanosine) and three pyrimidines (cytidine, thymidine and uridine). In RNA, thymidine is substituted for by uridine.

Nucleotides are not only the building blocks of nucleic acids, supporting the genetic information, but are also used for other purposes including the generation and transfer of energy within the cells.

A steady state of nucleotide availability is therefore required for normal cell function, and the quantification of these nucleotides (mainly adenosine, guanosine, uridine, cytidine and thymidine) in normal and pathological tissues and cells has been the subject of numerous studies. Notably, large differences in nucleotide levels between tissues, cell populations and transformed cells have been highlighted in an extensive study published in 1994 by TW Traut, that aggregates data from tens of published research papers accounting for about 600 values in human and mammal cells 1 .

While adenosine/adenosine triphosphate (ATP), unsurprisingly, was found to be abundant in normal and tumor tissues, due to its implication in numerous cellular processes (DNA/RNA synthesis, energy transfer, signal transduction…), cytidine triphosphate (CTP) is conversely present at the lowest levels and therefore often described as the limiting nucleotide in normal cells. Despite its low availability, this nucleotide is no less crucial as it is used for RNA / DNA (as dCTP) synthesis, for the sialylation of proteins 2 , but also as an energy source for phospholipids synthesis 3 , and has even been recently described as a substrate of the enzyme Viperin, used for the synthesis of a cellular antiviral molecule, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP) [START_REF] Gizzi | A Naturally Occurring Antiviral Ribonucleotide Encoded by the Human Genome[END_REF] . Increased or high levels of CTP in tumor extracts were reported by Traut 1 , in accordance with the metabolic requirements of highly proliferating cells.

CTP synthases

Most nucleotides can be synthetized through two pathways: the main ones are the de novo synthesis pathways ("from scratch") that are completed by the salvage pathways that recycle nucleotides from degradation products of nucleic acids) [START_REF] Evans | Mammalian Pyrimidine Biosynthesis: Fresh Insights into an Ancient Pathway[END_REF] . This is the case with the CTP for which most of the cellular needs are fulfilled by the de novo synthesis driven by the CTP synthases (also called synthetases), members of the class I glutamine-dependent aminotransferases (or triad) [START_REF] Goto | Crystal Structures of CTP Synthetase Reveal ATP, UTP, and Glutamine Binding Sites[END_REF] . Although its major role is the generation of CTP using UTP, it has also been described to be able to generate dCTP using dUTP [START_REF] Pappas | Characterization of a Novel DUTP-Dependent Activity of CTP Synthetase from Saccharomyces Cerevisiae[END_REF] .

It comes as no surprise, then, that de novo CTP synthesis and CTP synthases are not only present in mammals, but also universally conserved across species and members of this family of enzymes were identified and described in bacteria [START_REF] Lieberman | ENZYMATIC AMINATION OF URIDINE TRIPHOSPHATE TO CYTIDINE TRIPHOSPHATE[END_REF][START_REF] Trach | Complete Sequence and Transcriptional Analysis of the Spo0F Region of the Bacillus Subtilis Chromosome[END_REF] , archaea 10 , yeast [START_REF] Ozier-Kalogeropoulos | Sequencing and Characterization of the Saccharomyces Cerevisiae URA7 Gene Encoding CTP Synthetase[END_REF][START_REF] Ozier-Kalogeropoulos | Use of Synthetic Lethal Mutants to Clone and Characterize a Novel CTP Synthetase Gene in Saccharomyces Cerevisiae[END_REF] , plants (Arabidopsis thaliana [START_REF] Daumann | Characterization of Filament-Forming CTP Synthases from Arabidopsis Thaliana[END_REF] ; Oryza sativa var. japonica 14 ); Nematodes (Angiostrongylus cantonensis [START_REF] So | Pathways of Pyrimidine Nucleotide Biosynthesis in Gravid Angiostrongylus Cantonensis[END_REF] ) and fishes (Danio rerio [START_REF] Dzaki | CTP Synthase Knockdown during Early Development Distorts the Nascent Vertebral Column and Causes Fluid Retention in Multiple Tissues in Zebrafish[END_REF] ). Moreover, the presence of at least two different CTP synthases has been reported in multiple species, including the humans.

Figure 2: Partial phylogenetic tree of CTPS from various species mentioned in this document

Table 1: Names commonly used for CTPS family members in different species

In humans, the gene coding for CTPS1 is located on chromosome 1 [START_REF] Takahashi | Chromosome Mapping of the Human Cytidine-5'-Triphosphate Synthetase (CTPS) Gene to Band 1p34.1-P34.3 by Fluorescence in Situ Hybridization[END_REF] and its coding sequences are spread out on 19 exons 18 , while the highly homologous CTPS2 (74% of identity), identified later [START_REF] Van Kuilenburg | Identification of a CDNA Encoding an Isoform of Human CTP Synthetase[END_REF] , is located on chromosome X.

A bit of history on the CTP synthases

Identification of the enzyme and the main cofactors and substrates

In 1955, an enzyme purified from E. coli samples was shown to be able to aminate uridine triphosphate to generate cytidine triphosphate, a reaction that also required ATP and ammonium [START_REF] Lieberman | ENZYMATIC AMINATION OF URIDINE TRIPHOSPHATE TO CYTIDINE TRIPHOSPHATE[END_REF] .

A similar activity was then observed in extracts from normal and tumor rat tissues in 1958 [START_REF] Kammen | Amination of Uridine Nucleotides to Cytidine Nucleotides by Soluble Mammalian Enzymes; Role of Glutamine and Guanosine Nucleotides[END_REF] . However, the sequence of the pyrG gene (E. coli), encoding the enzyme responsible for this reaction, the CTP synthase, was not elucidated until 1986 [START_REF] Weng | Nucleotide Sequence of Escherichia Coli PyrG Encoding CTP Synthetase[END_REF] . Further studies in mammalian cells showed that a glutamine analog, the 6-Diazo-5-oxo-L-norleucine (DON), strongly decreased the incorporation of ureidosuccinic acid (carbamoyl aspartate) and orotate into cytosine while uracil was less affected, suggesting that glutamine was involved in the synthesis of a metabolite containing a cytosine moiety using another containing a uracil moiety [START_REF] Eidinoff | Pyrimidine Studies: I. Effect of DON (6-Diazo-5-Oxo-l-Norleucine) on Incorporation of Precursors into Nucleic Acid Pyrimidines[END_REF] . Glutamine was identified as the major ammonium contributor in amino acid synthesis of mammalian cells [START_REF] Salzman | The Utilization of Glutamine, Glutamic Acid, and Ammonia for the Biosynthesis of Nucleic Acid Bases in Mammalian Cell Cultures[END_REF] . These observations led researchers to further investigate the requirement for glutamine in the formation of cytidine nucleotides in rat tumor cell extracts [START_REF] Kammen | The Formation of Cytidine Nucleotides and RNA Cytosine from Orotic Acid by the Novikoff Tumor in Vitro[END_REF][START_REF] Hurlbert | Formation of Cytidine Nucleotides from Uridine Nucleotides by Soluble Mammalian Enzymes: Requirements for Glutamine and Guanosine Nucleotides[END_REF] and E.Coli [START_REF] Chakraborty | Role of Glutamine in the Biosynthesis of Cytidine Nucleotides in Escherichia Coli[END_REF] , which suggested that this enzyme had a glutamine amidotransferase (GAT) function.

The multimeric structure of the CTP synthase

The enzyme responsible for this cytidine triphosphate synthesis using UTP as a substrate was named in 1967 the UTP-ammonia ligase, or CTP synthetase, in a study focused on the kinetics of the reactions catalyzed by this enzyme in E.coli [START_REF] Long | Cytidine Triphosphate Synthetase of Escherichia Coli B. I. Purification and Kinetics[END_REF] . In this study, it was observed that the end product CTP acted as an activator at low concentrations, but as a competitive inhibitor at high concentrations. These results suggested that CTP synthetase consisted in two identical subunits -a deduction that was later challenged. Each subunit was purported to contain all of the necessary sites for catalysis, with three sites for ATP, UTP and GTP and one for the nitrogen donor, the presence of all of them being required for the reaction to occur. They subsequently established a model in which the CTPS dimer switched between an active and an inactive state based on the availability of substrates and products, with some of the nucleotides being able to substitute for others.

Two years later, Levitzki and Koshland showed that CTPS exhibited a negative cooperativity for the effector GTP and the substrate glutamine, meaning that the binding of GTP could negatively impact the binding of another glutamine/GTP molecule [START_REF] Levitzki | COOPERATIVITY IN REGULATORY ENZYMES[END_REF] . Shortly after, the purified E.coli CTP synthetase was found to be a dimer of about 105 kDa, with a monomer size of about 50kDa. The incubation of the enzyme with ATP, UTP, Mg2+ and glutamine led to the apparition of a 210kDa aggregate (the tetramer form), although only ATP, UTP and Mg2+ appeared to be required for this phenomenon to occur [START_REF] Levitzki | The Subunit Structure and Subunit Interactions of Cytidine Triphosphate Synthetase[END_REF] . The formation of this tetramer was conserved with lower concentrations of the enzyme and a treatment with the glutamine analog DON. This reaction, which did not appear to be reversible, produced ADP and Pi in equal amounts, and converted UTP into an equivalent amount of CTP, with a phosphorylated UTP intermediate being generated in the process, while there was no sign of GTP being degraded. The binding of a maximum of two DON molecules per tetramer suggested that only half of the sites were able to react with glutamine at a given time [START_REF] Levitzki | Half-of-the-Sites Reactivity and Conformational States of Cytidine Triphosphate Synthetase[END_REF] , a finding that was confirmed much later [START_REF] Willemoës | Competition between Ammonia Derived from Internal Glutamine Hydrolysis and Hydroxylamine Present in the Solution for Incorporation into UTP as Catalysed by Lactococcus Lactis CTP Synthase[END_REF] . The same study also suggested that the nascent NH3 generated during glutamine hydrolysis was delivered to the active site to directly react with UTP -using what would years later be shown to be an "ammonia tunnel" [START_REF] Endrizzi | Crystal Structure of Escherichia Coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets † , ‡[END_REF][START_REF] Mccluskey | Pinching" the Ammonia Tunnel of CTP Synthase Unveils Coordinated Catalytic and Allosteric-Dependent Control of Ammonia Passage[END_REF] . This ligand-induced tetramer formation was observed again in a study showing the sequential and cooperative ATP and UTP binding [START_REF] Levitzki | Ligand-Induced Dimer-to-Tetramer Transformation in Cytosine Triphosphate Synthetase[END_REF] .

This study was also focused on the role of GTP and suggested that in the absence of ATP and UTP, the dimer of CTP synthase could still exert its glutaminase activity, albeit at a lower rate than in its tetramer form. It appeared that the role of GTP was limited to the promotion of the glutaminase activity of the CTP synthase, making it an allosteric effector [START_REF] Levitzki | Ligand-Induced Dimer-to-Tetramer Transformation in Cytosine Triphosphate Synthetase[END_REF] . The nucleotide requirement for tetramer formation were found to be conserved in the mammal enzyme [START_REF] Mcpartland | Cytidine 5'-Triphosphate Synthetase of Calf Liver. Size, Polymerization, and Reaction Stoichiometry[END_REF] .

Regulation of the CTP synthase activity

Although the identity of the substrates and cofactors were well established at this point, little was known about the involvement of the end product CTP in the de novo CTP synthesis reaction, although a role of CTP in the feedback control of pyrimidine de novo synthesis 36 and salvage 37 had already been reported.

Kinetics data from the calf liver CTP synthase suggested that CTP inhibited the activity of CTP synthase at high concentrations but could have an activating effect at low doses. Low concentrations of CTP improved CTP synthase dimerization in the presence of subsaturating amounts of UTP, while high CTP concentrations did not impact the tetramerization of the enzyme, suggesting that the inhibitory effect was not due to the disruption of CTPS tetramer formation [START_REF] Mcpartland | Cooperative Effects of CTP on Calf Liver CTP Synthetase[END_REF] . CTP was also shown to regulate the UTP/CTP pool balance in a murine T lymphoblast cell line, S49 [START_REF] Aronow | Biochemical Phenotype of 5-Fluorouracil-Resistant Murine T-Lymphoblasts with Genetically Altered CTP Synthetase Activity[END_REF] .

Structural studies

The elucidation of the nucleotide sequence of the pyrG gene in E.coli [START_REF] Weng | Nucleotide Sequence of Escherichia Coli PyrG Encoding CTP Synthetase[END_REF] and of the gene coding for the human CTP synthase 18 led to a new understanding of the CTP synthase structure. Using these preliminary data, Weng hypothesized that the CTP synthase accomplished its enzymatic activity through the association of its now clearly identified C-terminal glutamine amidotransferase (GAT) domain with a domain responsible for the aminator function of the enzyme [START_REF] Weng | Nucleotide Sequence of Escherichia Coli PyrG Encoding CTP Synthetase[END_REF] . Further studies showed that the conserved region between residues 346 and 355 (366-375 in human) had an important structural role, with single residue changes not only impacting the activity of the GAT domain and the stability of the protein, but also the structure of what was then called the aminator domain. This in turn suggested that they interacted to transfer the NH3 generated during glutamine hydrolysis to this uncharacterized domain [START_REF] Weng | Structural Role for a Conserved Region in the CTP Synthetase Glutamine Amide Transfer Domain[END_REF] .

The human CTPS sequence was then found to be highly conserved compared to the bacterial sequence [START_REF] Yamauchi | Molecular Cloning of the Human CTP Synthetase Gene by Functional Complementation with Purified Human Metaphase Chromosomes[END_REF] .

The crystal structure of E.Coli [START_REF] Endrizzi | Crystal Structure of Escherichia Coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets † , ‡[END_REF] and Thermus thermophilus [START_REF] Goto | Crystal Structures of CTP Synthetase Reveal ATP, UTP, and Glutamine Binding Sites[END_REF][START_REF] Goto | Purification and Preliminary X-Ray Characterization of CTP Synthetase from Thermus Thermophilus HB8[END_REF] CTP synthase tetramers were elucidated in the early 2000s. These structures revealed that the synthase/amidoligase (AL) active site was constituted of 3 monomers and identified the localization of the "tunnel" used by the nascent ammonia generated during the glutaminase reaction to reach the 4-phospho-UTP intermediate. Although there are currently no complete crystal structures of the human enzymes, the high degree of sequence conservation of the enzyme among species, its essential functions along with the currently available cryo-electron microscopy data suggest that the structure itself is also highly conserved.

The "Filament Era"

In the last ten years, the ability of CTPS to form intracellular filaments, which has been observed in numerous species, has been a hot topic of research. The existence of this spectacular structure resulting from the organized assembly of CTPS proteins was first reported in 2010 by in three independent studies [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF][START_REF] Liu | Intracellular Compartmentation of CTP Synthase in Drosophila[END_REF][START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] . However, the assembly of GFP-tagged Ura7 (S. cerevisiae CTPS1) foci in yeast reaching the stationary phase of growth, that disassembled upon medium replenishment, could also be considered as the first observation of higher-order CTPS protein assembly [START_REF] Narayanaswamy | Widespread Reorganization of Metabolic Enzymes into Reversible Assemblies upon Nutrient Starvation[END_REF] .

While working on the identification of P-bodies (processing bodies), cytoplasmic structures composed of translationally repressed mRNAs and proteins related to mRNA decay [START_REF] Luo | P-Bodies: Composition, Properties, and Functions[END_REF] , JL Liu discovered that one of the anti-P-bodies antibodies he was using cross-reacted with a yet-unknown thick filamentous structure in male gonads, located on the sperm tails. This structure was also present on follicle cells, nurse cells and oocytes. Due to their elongated and curved shapes, he named these structures "Cytoophidium", meaning "Cell snakes" in Greek. These structures had shapes and localizations comparable to those formed by a protein taken from a bank of GFP-tagged drosophila proteins [START_REF] Buszczak | The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies[END_REF] , and were also recognized by the same cross-reacting antibody: CTP synthase. These filaments of very variable length and thickness were found to be bundles of thinner filaments [START_REF] Liu | Intracellular Compartmentation of CTP Synthase in Drosophila[END_REF][START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] . Around that time, CTPS filaments were also described in Caulobacter crescentus (a prokaryote organism) [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] , Saccharomyces cerevisiae (unicellular eukaryote) and rat hippocampal neurons (pluricellular eukaryote) [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF] . Bacterial CTPS filaments were shown to participate to the curvature of C. crescentus cells and were lost after a treatment with the glutamine antagonist DON [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] , unlike Drosophila and Human CTPS filaments which were found to be induced by the same treatment [START_REF] Chen | Glutamine Analogs Promote Cytoophidium Assembly in Human and Drosophila Cells[END_REF] . Mutation of the UTP binding site in bacteria (G147A, which corresponds to G148A in human CTPS1/CTPS2) had no impact on filament formation, while the disruption of the glutaminase domain activity by the C388G (C399 in human CTPS1/CTPS2) mutation strongly reduced filament formation [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] . In the yeast, mutating the CTP-binding site (E161K) led to a strongly decreased filament formation associated with an increase in foci formation. Supplementation with both ATP and CTP led to an increase in foci formation. Treating the cells with AMP-PNP (adenosine 5'-[β, γ-imido]triphosphate), a nonhydrolyzable analogue of ATP that had previously been shown to inhibit CTPS tetramerization 50 also strongly decreased filament formation, suggesting that tetramers were required for filament formation [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF] . These early, sometimes discrepant findings illustrated the ubiquity of CTPS filament formation, but also the complexity of their regulation.

CTPS1 as a therapeutic target?

Targeting the CTPS activity is not a new therapeutic approach. For instance, elevated CTPS activities are frequent in tumor cells. However, the lack of knowledge regarding the role of CTPS1 and CTPS2 in human cells complicates the use of such approaches, as CTP synthesis in normal cells is already a bottleneck despite its involvement in key processes.

We can therefore close this brief historic overview by mentioning the recent description of a cohort of patients affected by a mutation in the CTPS gene, that despite strongly reduced CTPS1 protein and activity levels, leading to impaired immune responses, exhibited no other major functional defect in nonhematopoietic compartments [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] . This puts CTPS1 back into the spotlight as a key player in immune responses and suggests that CTPS1-selective compounds could be a viable and safer therapeutic alternative to dampen abnormal immune responses or control CTPS1-dependent tumor cell growth. The CTP synthases possess two domains connected by a small α-helical linker region, a synthetase (ammonia-ligase, AL) and a glutamine amidotransferase (GAT) [START_REF] Weng | Nucleotide Sequence of Escherichia Coli PyrG Encoding CTP Synthetase[END_REF] , followed by a small C-terminal regulation domain containing multiple phosphorylation sites and where the main sequence differences between CTPS1 and CTPS2 reside [START_REF] Kassel | Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation[END_REF] .

Structure of the CTPS family of enzymes

Figure 5: Comparison of the C-terminal domains of CTPS1 and CTPS2 between different mammal species

The C-terminal domain is poorly conserved between CTPS1 (top) and CTPS2 (bottom). However, the Cterminal part of CTPS1 is conserved between species. The same applies for CTPS2.

During the production of CTP, UTP is phosphorylated by ATP in the synthetase domain [START_REF]Mechanistic Investigations of Escherichia Coli Cytidine-5'-Triphosphate Synthetase. Detection of an Intermediate by Positional Isotope Exchange Experiments[END_REF] . Meanwhile, glutamine is hydrolysed by the glutamine amidotransferase domain through the formation of a γ-glutamyl S-ester-enzyme intermediate [START_REF] Weng | Nucleotide Sequence of Escherichia Coli PyrG Encoding CTP Synthetase[END_REF][START_REF] Levitzki | Cytidine Triphosphate Synthetase. Covalent Intermediates and Mechanisms of Action[END_REF] using the thiol function of cysteine 399, yielding glutamate and ammonia (NH3), with GTP acting as an allosteric effector during this step [START_REF] Levitzki | Ligand-Induced Dimer-to-Tetramer Transformation in Cytosine Triphosphate Synthetase[END_REF][START_REF] Bearne | Inhibition of Escherichia Coli CTP Synthase by Glutamate γ-Semialdehyde and the Role of the Allosteric Effector GTP in Glutamine Hydrolysis[END_REF] by altering the structure of a lid ("lid L11" 57 ), although it was also suggested that 4-phospho-UTP could also play this role [START_REF] Willemoës | Steady-State Kinetics of the Glutaminase Reaction of CTP Synthase from Lactococcus Lactis: The Role of the Allosteric Activator GTP in Coupling between Glutamine Hydrolysis and CTP Synthesis[END_REF][START_REF] Willemoës | Substrate Inhibition of Lactococcus Lactis Cytidine 5′-Triphosphate Synthase by Ammonium Chloride Is Enhanced by Salt-Dependent Tetramer Dissociation[END_REF] .

The resulting ammonia is funnelled using a so-called "ammonia tunnel" [START_REF] Mccluskey | Pinching" the Ammonia Tunnel of CTP Synthase Unveils Coordinated Catalytic and Allosteric-Dependent Control of Ammonia Passage[END_REF][START_REF] Iyengar | Aspartate-107 and Leucine-109 Facilitate Efficient Coupling of Glutamine Hydrolysis to CTP Synthesis by Escherichia Coli CTP Synthase[END_REF][START_REF] Lunn | Alternative Substrates for Wild-Type and L109A E. Coli CTP Synthases[END_REF] towards the synthetase site where it reacts with the phosphorylated UTP, displacing the newly added phosphate group to turn it into CTP, releasing Pi 62 .

While the bacteria enzyme was found to be able to use exogenous NH3 [START_REF] Lieberman | ENZYMATIC AMINATION OF URIDINE TRIPHOSPHATE TO CYTIDINE TRIPHOSPHATE[END_REF] , it appears that the mammalian enzyme mostly relies on L-glutamine hydrolysis [START_REF] Salzman | The Utilization of Glutamine, Glutamic Acid, and Ammonia for the Biosynthesis of Nucleic Acid Bases in Mammalian Cell Cultures[END_REF] and that some potential amino donors (L-arginine, potassium phosphate or NH4HCO3) could synergistically improve this step [START_REF] Mclaren | Apparent Synergism between Amino Donors for CTP Synthesis in Chinese Hamster Fibroblasts[END_REF] . CTP excess induces a rotation of the tetramers, shifting the structure towards a product-bound state (as reported in the human [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] and D. melanogaster enzymes [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] ) and creating a constriction preventing the NH3 from entering the ammonia tunnel.

Dimeric and tetrameric structures

As the structure and most of the sequence of CTP synthases are conserved across evolution, so are some of the regulatory mechanisms. The CTP synthases are allosterically regulated by GTP, their substrates (ATP, UTP) and their product (CTP). In absence of ligands, the CTP synthases can exist as monomers [START_REF] Anderson | CTP Synthetase from Escherichia Coli: An Improved Purification Procedure and Characterization of Hysteretic and Enzyme Concentration Effects on Kinetic Properties[END_REF] , although CTP synthase is usually observed as dimers in absence of ATP, UTP and GTP.

The dissociation of dimers as monomers is observed in presence of low concentrations of enzyme or absence of ligands and is associated with a lag phase in CTP production after enzyme levels increase or substrate replenishment, suggesting that the dimerization reaction is slow and that the monomers are inactive [START_REF] Mclaren | Apparent Synergism between Amino Donors for CTP Synthesis in Chinese Hamster Fibroblasts[END_REF][START_REF] Anderson | CTP Synthetase from Escherichia Coli: An Improved Purification Procedure and Characterization of Hysteretic and Enzyme Concentration Effects on Kinetic Properties[END_REF] .

In presence of the substrates (ATP/UTP/Mg2+), the CTPS dimers dimerize into tetramers that are commonly described as being the active form of these enzymes [START_REF] Levitzki | The Subunit Structure and Subunit Interactions of Cytidine Triphosphate Synthetase[END_REF][START_REF] Thomas | Purification of Cytidine-Triphosphate Synthetase from Rat Liver, and Demonstration of Monomer, Dimer and Tetramer[END_REF] , and disassemble in absence of substrates in most species, one notable reported exception being the Lactococcus lactis CTP synthase that might be stabilized by ionic interactions [START_REF] Willemoës | Substrate Inhibition of Lactococcus Lactis Cytidine 5′-Triphosphate Synthase by Ammonium Chloride Is Enhanced by Salt-Dependent Tetramer Dissociation[END_REF] . In E.coli, the dimers and tetramers are mostly stabilized by polar and hydrophobic contacts at the synthase domains interface [START_REF] Endrizzi | Crystal Structure of Escherichia Coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets † , ‡[END_REF] .

Neither glutamine nor GTP appear be required for tetramer formation (Saccharomyces cerevisiae [START_REF] Pappas | Nucleotide-Dependent Tetramerization of CTP Synthetase from Saccharomyces Cerevisiae[END_REF] ).

Tetramer existence in absence of substrates has also been observed in the cases of L. lactis CTPS [START_REF] Willemoës | Substrate Inhibition of Lactococcus Lactis Cytidine 5′-Triphosphate Synthase by Ammonium Chloride Is Enhanced by Salt-Dependent Tetramer Dissociation[END_REF][START_REF] Wadskov-Hansen | Cloning and Verification of the Lactococcus Lactis PyrG Gene and Characterization of the Gene Product, CTP Synthase*[END_REF] , E. coli CTPS [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF][START_REF] Mccluskey | Biophysical Analysis of Bacterial CTP Synthase Filaments Formed in the Presence of the Chemotherapeutic Metabolite Gemcitabine-5'-Triphosphate[END_REF] and human CTPS2 using a mutant (V352C) that is unable to disassemble once aggregated as filaments [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] . While the glutaminase domain has been shown to be active in dimeric form, at least in bacteria [START_REF] Levitzki | Ligand-Induced Dimer-to-Tetramer Transformation in Cytosine Triphosphate Synthetase[END_REF][START_REF] Willemoës | Substrate Inhibition of Lactococcus Lactis Cytidine 5′-Triphosphate Synthase by Ammonium Chloride Is Enhanced by Salt-Dependent Tetramer Dissociation[END_REF] , the synthetase domain seems to require tetramer formation [START_REF] Endrizzi | Crystal Structure of Escherichia Coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets † , ‡[END_REF][START_REF] Endrizzi | Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex † , ‡[END_REF] .

Recent data generated using the D. melanogaster enzyme suggest that the binding of GTP depends on the prior binding of the substrates (ATP and/or UTP) that induces a rotation between the glutaminase (GAT) and synthetase (AL) domain [START_REF] Zhou | Structural Basis for Ligand Binding Modes of CTP Synthase[END_REF] . The global shift induced by the substrates binding to the CTPS tetramer "creates" the GTP binding site and aligns two cavities ("Ammonia tunnel", [START_REF] Endrizzi | Crystal Structure of Escherichia Coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets † , ‡[END_REF][START_REF] Mccluskey | Pinching" the Ammonia Tunnel of CTP Synthase Unveils Coordinated Catalytic and Allosteric-Dependent Control of Ammonia Passage[END_REF][START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] ) that channel the NH3 released from the glutamine hydrolysis towards the phospho-UTP intermediate. Then, the binding of glutamine stabilizes the F373 residue that can then interact and potentially stabilize the binding of GTP.

At this point, GTP is positioned at the entry of a cleft, blocking this passage through which exogenous NH3 can enter the ammonia tunnel while simultaneously ensuring that only the NH3 generated during the glutamine hydrolysis reaches the 4-phospho-UTP [START_REF] Zhou | Structural Basis for Ligand Binding Modes of CTP Synthase[END_REF] .

Filaments, cytoophidia, or rod and rings structures

CTPS tetramers can, in a reversive manner, assemble into higher order structures: large intracellular filaments that are also called rods and rings [START_REF] Carcamo | Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells[END_REF] or cytoophidia [START_REF] Liu | Intracellular Compartmentation of CTP Synthase in Drosophila[END_REF] . The existence of such filaments has been reported in all reigns of life (Bacteria (E. coli, C. crescentus [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] ), Archea [START_REF] Zhou | CTP Synthase Forms Cytoophidia in Archaea[END_REF] , single-celled (S. pombe [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] ; S. cerevisiae [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF] ) and pluricellular eukaryotic organisms (Drosophila 44 ; Zebrafish 75 ; Rattus norvegicus [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF] ; H. sapiens [START_REF] Chen | Glutamine Analogs Promote Cytoophidium Assembly in Human and Drosophila Cells[END_REF] ) including plants (A. thaliana [START_REF] Daumann | Characterization of Filament-Forming CTP Synthases from Arabidopsis Thaliana[END_REF] ), arguing for a major role in the regulation of these enzymes.

Interestingly, both the binding of the substrate (UTP) or the product (CTP) impact filament formation. However, while the global mechanism appears to be conserved, the way nucleotide binding affects filament formation appears to be species-dependent. These filaments are part of a family of "membraneless organelles" whose members are often unrelated [START_REF] Park | Structures, Functions, and Mechanisms of Filament Forming Enzymes: A Renaissance of Enzyme Filamentation[END_REF] .

Reversible filament formation of an enzyme involved in nucleotide metabolism was first reported for Inosine Monophosphate Deshydrogenase (IMPDH) using inhibitors of the enzyme (mycophenolic acid (MPA), mizoribine, and VX-944). The dissociation of these filaments can be induced using guanosine or GTP supplementation [START_REF] Ji | Regulation of the Interaction of Inosine Monophosphate Dehydrogenase with Mycophenolic Acid by GTP*[END_REF] and filament formation upregulates enzymatic activity [START_REF] Keppeke | IMP/GTP Balance Modulates Cytoophidium Assembly and IMPDH Activity[END_REF] . Early data suggested that CTPS1 and IMPDH2 were part of the same filaments as both were induced using glutamine and guanosine analogs, including DON [START_REF] Carcamo | Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells[END_REF] . However, no direct interaction between CTPS and IMPDH has been reported thus far, and an array of data shows that the CTP synthases are able to form filaments in absence of IMPDH [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF][START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF][START_REF] Keppeke | Assembly of IMPDH2-Based, CTPS-Based, and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction[END_REF][START_REF] Schiavon | Compositional Complexity of Rods and Rings[END_REF] and that CTPS filaments can spontaneously separate from IMPDH filaments [START_REF] Chang | Interfilament Interaction between IMPDH and CTPS Cytoophidia[END_REF] . The fact that DON and Acivicin induce IMPDH filament formation could also be the consequence of the inhibition of glutamine-utilizing enzymes other than the CTP synthases [START_REF] Schiavon | Compositional Complexity of Rods and Rings[END_REF] , although the parallel assembly, the induction of CTPS filament formation by IMPDH inhibitors such as ribavirin [START_REF] Keppeke | Assembly of IMPDH2-Based, CTPS-Based, and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction[END_REF] and the modification of the morphology of CTPS1 filaments colocalized with IMPDH2 filaments following selective IMPDH2 filament disruption [START_REF] Chang | Interfilament Interaction between IMPDH and CTPS Cytoophidia[END_REF] still suggests that there might be a link between them through yet-to-be-identified partners.

Kinetic studies [START_REF] Chang | Interfilament Interaction between IMPDH and CTPS Cytoophidia[END_REF][START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF] have shown that small CTPS tetramers assemble as small foci, then elongate. These thin filaments can then associate with other CTPS filaments side-by-side or fuse end-to-end, yielding large, micrometer-scale structures than can end up curved or even ring-shaped. It has also been suggested that dimers can assemble as CTPS filaments, although there is currently no structural data of this hypothetical structure [START_REF] Noree | Common Regulatory Control of CTP Synthase Enzyme Activity and Filament Formation[END_REF] .

Mechanisms of regulation

Transcriptional regulation

Cytidine nucleotides were shown to repress the expression of CTPS in B. subtilis, a mechanism later shown to be dependent on CTP levels [START_REF] Meng | Regulation of Transcription of the Bacillus Subtilis PyrG Gene, Encoding Cytidine Triphosphate Synthetase[END_REF][START_REF] Meng | Cis-Acting Sequences of Bacillus Subtilis PyrG MRNA Essential for Regulation by Antitermination[END_REF][START_REF] Meng | Attenuation Control of PyrG Expression in Bacillus Subtilis Is Mediated by CTP-Sensitive Reiterative Transcription[END_REF][START_REF] Jensen-Macallister | Regulation of PyrG Expression in Bacillus Subtilis: CTP-Regulated Antitermination and Reiterative Transcription with PyrG Templates in Vitro[END_REF] . Conversely, decreased CTP pools were shown to increase the expression of L. lactis CTP synthase [START_REF] Jorgensen | CTP Limitation Increases Expression of CTP Synthase in Lactococcus Lactis[END_REF] . While such a regulation method is not applicable to other species as they involve a prokaryote-specific mechanism, it has been reported that glutamine deprivation in HeLa cells induced CTPS1 mRNA expression, although the increase was moderate (2-fold) and was not associated with increased protein levels [START_REF] Calise | Glutamine Deprivation Initiates Reversible Assembly of Mammalian Rods and Rings[END_REF] .

In mammals, CTPS has been shown to be a target of TWIST, a transcription factor involved in embryonic morphogenesis [START_REF] Feng | Gene Expression Profiling in TWIST-Depleted Gastric Cancer Cells[END_REF] , as well as of the proto-oncogene MYC [START_REF] Liu | Global Regulation of Nucleotide Biosynthetic Genes by C-Myc[END_REF][START_REF] Aughey | The Interplay between Myc and CTP Synthase in Drosophila[END_REF] .

The expression of CTPS1 is upregulated in T and B -lymphocytes following their activation with some immune stimuli including stimulation by antigen receptors of T and B lymphocytes (TCR and BCR), CD40

and LPS. Downstream pathways involved are currently being investigated in our lab [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] .

Regulation by phosphorylation

Numerous sites of phosphorylation are present in both CTPS1 and CTPS2, most of them being located at the C-terminal tail of the proteins: (Olsen et al., 2006) Some kinases involved in the phosphorylation of yeast CTP synthase were first identified in the laboratory of Georges Carman. Yang and colleagues [START_REF] Yang | Phosphorylation of CTP Synthetase from Saccharomyces Cerevisiae by Protein Kinase C ( * )[END_REF][START_REF] Yang | Regulation of Yeast CTP Synthetase Activity by Protein Kinase C ( * )[END_REF] first showed that Protein Kinase C (Pkc1p, here shortened as PKC) had the ability to phosphorylate CTPS on serine and threonine residues (S36, S330, S354, and S454 [START_REF] Park | Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces Cerevisiae[END_REF] ), although whether PKC directly phosphorylated CTPS or acted through a partner was unclear.

Sites CTPS1 K100 Y106 S562 (T566)(Y567)(S568) S571 S573 S574 S575 S578 T581 CTPS2 (Y3) (T7) (S21) (S22) (T25) Y83 (S562)(S563)(S564) (Y567) S568 S571 S574
Further studies showed that he resulting phosphatidylserine and diacylglycerol-dependent phosphorylation by the PKC 97 increased CTPS activity. The enhanced activity in these conditions seemed mediated by an increased affinity for ATP and was associated with a decreased cooperativity between the substrates ATP and UTP and a decreased sensitivity to CTP feedback inhibition. However, part of these data is not transposable to the human enzymes as some sites are not conserved (S330, S354, S454).

Protein Kinase A (PKA) was also shown to be involved in the phosphorylation of yeast CTP synthase on a different serine residue which, similarly to what was observed with PKC, results in an increased affinity for ATP while decreasing the cooperativity between ATP and UTP and the sensitivity to CTP-induced inhibition [START_REF] Yang | Phosphorylation and Regulation of CTP Synthetase from Saccharomyces Cerevisiae by Protein Kinase A[END_REF] . The target residue, not conserved in the human enzymes, was later identified as S424 by sitedirected mutagenesis [START_REF] Park | Identification of Ser424 as the Protein Kinase A Phosphorylation Site in CTP Synthetase from Saccharomyces Cerevisiae[END_REF] and shown to be phosphorylated by PKC as well 100 .

Complete dephosphorylation of the enzyme resulted in a sharp decrease (80%) in enzymatic activity [START_REF] Yang | Phosphorylation of CTP Synthetase from Saccharomyces Cerevisiae by Protein Kinase C ( * )[END_REF][START_REF] Yang | Phosphorylation and Regulation of CTP Synthetase from Saccharomyces Cerevisiae by Protein Kinase A[END_REF] that was associated with impaired tetramer formation leading to the accumulation of inactive dimers [START_REF] Pappas | Nucleotide-Dependent Tetramerization of CTP Synthetase from Saccharomyces Cerevisiae[END_REF] .

Re-phosphorylation of the enzyme using PKA, PKC or both partially restored the full protein phosphorylation and tetramer formation (resp. 30% for PKA, 40% for PKC and 60% for the combination) that was associated with a partial, but globally correlated, increase in CTPS activity [START_REF] Pappas | Nucleotide-Dependent Tetramerization of CTP Synthetase from Saccharomyces Cerevisiae[END_REF][START_REF] Yang | Phosphorylation of CTP Synthetase from Saccharomyces Cerevisiae by Protein Kinase C ( * )[END_REF][START_REF] Yang | Phosphorylation and Regulation of CTP Synthetase from Saccharomyces Cerevisiae by Protein Kinase A[END_REF] , suggesting that other kinases might be involved in the phosphorylation of CTP synthases.

Human CTPS1 and CTPS2 expressed in this yeast model are also phosphorylated by yeast and bovine PKA, resulting in an increased enzymatic activity 101 . Loss of the human CTPS1 phosphorylation site T455 by site-directed mutagenesis was associated with increased intracellular concentrations of CTP and an increase in the choline-dependent phosphatidylcholine synthesis 102 , while the loss of the S462 site induces a loss of activity 103 , showing that the phosphorylation of CTPS1 can both positively and negatively regulate its activity.

However, these results obtained in yeast were challenged when it was shown that the modulation of PKA and PKC activity in a human cell line (HEK 293) had no impact on CTPS1 phosphorylation under the conditions tested, while serum deprivation induced its phosphorylation on S571, S574 and S575 in a Glycogen Synthase Kinase 3 (GSK3)-dependent manner, inhibiting its activity 104 . Moreover, the phosphorylation of S575 (by a yet unidentified kinase) seems required to prime for the phosphorylation of the S571. S575 phosphorylation of CTPS1 leads to its association with Pin1 (Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1), a protein that catalyzes the cis to trans isomerization of phosphorylated serine or threonine followed by proline residues, changing the conformation of targeted proteins 105 , although no detectable changes in activity or expression of CTPS1 associated with Pin1 could be observed 106 . GSK3β is overexpressed in muscle biopsies of myotonic dystrophy type 1 patients (DM1) 107,108 , dysregulating its target genes, including CTPS1 109 . S571 and S574 are conserved in CTPS2. While low serum also induced CTPS2 phosphorylation on both sites along with the S568, only the latter was shown to have an impact on CTPS2 activity. Despite leading to an apparent increase in the affinity to UTP, the casein kinase 1-dependent phosphorylation of the S568 decreased the activity of the enzyme through a reduction of the activity of the glutaminase domain [START_REF] Kassel | Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation[END_REF] . Higgins et al., 2007) In S. cerevisiae, staurosporine, a non-specific inhibitor of protein kinases, was found to strongly increase Ura7 filament formation [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF] . However, the contribution of phosphorylation is this model is unclear as targeted mutations on previously identified phosphorylation sites had a moderate impact on filaments [START_REF] Noree | Common Regulatory Control of CTP Synthase Enzyme Activity and Filament Formation[END_REF] .

Disruption of mTOR and its downstream target S6K1 (ribosomal protein S6 kinase beta-1)) decreases CTPS filament formation 110 . A similar effect was observed in S. cerevisiae when TOR, the S. cerevisiae homolog of TOR, is inhibited 111 . Indeed, the selective disruption of mTORC1 and mTORC2 highlighted several differentially phosphorylated sites between both conditions in enzymes involved in pyrimidine synthesis, including notably the C-terminal of CTPS1 112 . S6K1 was also found directly associated with CTPS1 by immunoprecipitation, suggesting that it might phosphorylate CTPS1 110 .

Finally, disruption of dAck (drosophila Activated Cdc42-associated kinase) in D. melanogaster decreases CTPS filament formation, although dAck did not phosphorylate CTPS1, suggesting that it may act on CTPS filament formation through the phosphorylation of an unidentified partner 113 .

Regulation by ubiquitination

Ubiquitination of CTPS1 has been reported in S. cerevisiae 114 , D. melanogaster 115,116 and H. sapiens 117 .

Human CTPS1 was also identified as a substrate of the HRD1 RING E3 Ubiquitin Ligase 118 .

Protein ubiquitination mediated by Cbl, another E3 ubiquitin-protein ligase, has been shown to positively regulate filament formation in D. melanogaster follicular cells, increasing CTP synthase activity. However, the available data suggest that the impact of Cbl on CTPS filament formation and activity was not through a direct ubiquitination of CTPS 115 . Interfering with the ubiquitination pathways in drosophila S2 cells and human HEp2 cells decreased filament formation, while glutamine-deprivation filament formation was associated with a decrease of CTPS ubiquitination in drosophila S2 cells, suggesting that, at least in drosophila, ubiquitination of potential protein partners was required for filament formation, while ubiquitination of CTPS itself was negatively associated with filament formation 116 . Similarly, DON-induced filament formation was shown to decrease CTPS ubiquitination, which might protect the protein from proteasome-mediated degradation 119 .

Feedback regulation

Finally, as mentioned earlier, the product CTP regulates the activity of CTP synthase. At low concentrations, CTP increases the binding of UTP to the enzyme, increasing the activity of the enzyme [START_REF] Mcpartland | Cooperative Effects of CTP on Calf Liver CTP Synthetase[END_REF][START_REF] Thomas | Purification of Cytidine-Triphosphate Synthetase from Rat Liver, and Demonstration of Monomer, Dimer and Tetramer[END_REF] . At high concentrations, however, CTP inhibits the enzyme [START_REF] Long | Cytidine Triphosphate Synthetase of Escherichia Coli B. I. Purification and Kinetics[END_REF]120 . The IC50 of CTP for human CTPS1 and CTPS2 was shown to be close to the lower end of the physiological concentrations of CTP, suggesting that CTP levels tightly regulate the activity of both enzymes [START_REF] Kassel | Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation[END_REF] . However, according to recent data the IC50 of CTP for human CTPS2 is five-times lower than for CTPS1 121 .

Structural studies showed that, while the CTP binding site partially overlaps the UTP binding site through their common triphosphate moiety, the cytosine moiety of the CTP is located in a different pocket, explaining how it is able to compete with UTP while being sensitive to mutations affecting the pocket in which the cytosine moiety can reside [START_REF] Endrizzi | Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex † , ‡[END_REF] .

Intriguingly, ADP [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] and CTP [START_REF] Zhou | Structural Basis for Ligand Binding Modes of CTP Synthase[END_REF]121 seem to be able to also occupy the ATP binding site in some CTP synthases including human CTPS2, suggesting the existence of another layer of regulation.

Filament formation

The recent discovery of the ability of CTP synthases to form micron-scale filaments in response to various stimuli [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF][START_REF] Liu | Intracellular Compartmentation of CTP Synthase in Drosophila[END_REF][START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] has opened a new field on research on the regulation of this family of enzymes. Usually present in the cytoplasm, there structures have also been observed in the nuclei of cells from various species (S. pombe 122 , D. melanogaster 123 and H. sapiens [START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF] ).

Early data generated with the C. crescentus and E. coli homologs of CTP synthase (encoded by the pyrG gene) showed that CTP synthase filament formation was induced both by high concentrations of the enzyme and by CTP binding, decreasing the activity of the enzyme [START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF] and that a UTP-binding deficient mutant (G147A) was still able to form filaments [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] . Additionally, the CTPS tetramers assembled in filaments were shown to be "locked" in an inactive conformation [START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF] . This led to the hypothesis that the filaments were a storage form of the enzyme, as they allowed the cell to inactivate the enzyme in excess when the CTP pools were sufficient, without degrading it. As filament formation is reversible, this would allow the cell to quickly reactivate the CTP synthase protein pool, should the CTP pool decrease or the need for CTP increase. CTPS filaments having already been observed for both bacterial and eukaryotic enzymes, it was assumed that this mechanism was also conserved across species.

These early conclusions were challenged by data obtained from other models, in particular D. melanogaster. Catalytically active filaments were observed in drosophila during oogenesis during which high amounts of CTP are required to sustain intensive DNA and RNA synthesis. Disruption of filament formation by decreasing the tyrosine kinase dAck activity (drosophila Activated Cdc42-associated kinase) induced membrane defects due to perturbed phospholipid synthesis and decreased fly fertility 113 . D.

melanogaster CTPS filaments can be detected using an antibody targeting phosphorylated S36 of CTPS [START_REF] Liu | Intracellular Compartmentation of CTP Synthase in Drosophila[END_REF] , a conserved residue (in human CTPS1, not CTPS2) whose phosphorylation had been associated with an increased enzymatic activity in S. cerevisiae [START_REF] Park | Phosphorylation of CTP Synthetase on Ser36, Ser330, Ser354, and Ser454 Regulates the Levels of CTP and Phosphatidylcholine Synthesis in Saccharomyces Cerevisiae[END_REF] , suggesting that an active form of the enzyme was incorporated in the filaments, although it is unknown if the disruption of this phosphorylation site has any impact on filament formation. Disruption of CTPS filament formation using a CTPS mutant dominantly blocking filament formation (H355A) negatively affects drosophila egg production 124 .

Other discrepancies between species (like bacteria and mammals) and CTP synthases from the same species (like human CTPS1 and CTPS2) have also been observed using mutagenesis approaches. For instance, multiple separate studies in the bacteria C. crescentus and E. coli, the budding yeast S. cerevisiae, the fly D. melanogaster and the mammal H. sapiens compared the impact of targeted mutations, including one disrupting the UTP binding site (G147A/G148A) and one disrupting the CTP binding site (E155K/E160K/E161K). G148A has no impact on filament formation in C. crescentus [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] , while it increases filament formation in S. cerevisiae [START_REF] Noree | Common Regulatory Control of CTP Synthase Enzyme Activity and Filament Formation[END_REF] , and impairs filament formation in H. sapiens CTPS1 125 .

Another example is the E161K mutation that abolishes filament formation in E. coli, S. cerevisiae and D. melanogaster (although foci were observed in the last two cases) [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF][START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF]126 whereas it has no impact on human CTPS1 filament formation 113 .

These differences could result from minute changes in the sequence of the enzymes, as well as from the presence of absence of post-translational modifications or partners that may reflect differential regulations between species and/or be associated with particular culture conditions. Along these lines, the role of filaments may have evolved from bacteria to mammals.

CTPS filament formation is impacted by stresses

GFP-tagged Ura7-expressing S. cerevisiae reaching the stationary phase of growth assembled as foci [START_REF] Narayanaswamy | Widespread Reorganization of Metabolic Enzymes into Reversible Assemblies upon Nutrient Starvation[END_REF] or filaments [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF] that could be reverted by glucose-containing medium replenishment, suggesting that nutrient deprivation induced filament formation. Nutrient stress has also been shown to induce filament formation in a cell cycle-independent way in D. melanogaster. These filaments disassemble when the cells exit quiescence, suggesting that they represent an inactive form of the enzyme 126 .

Further studies in human and murine cell lines showed that deprivation of glutamine, and simultaneous glutamine and serum deprivation induced filament formation [START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF] . In this condition, the glutamine and serum deprivation-induced influx of exogenous histidine was shown to be required for filament formation 125 .

Interestingly, although previous data suggested that the human CTPS1 filaments, constituted of tetramers in the active conformation, were a more active form of the enzyme [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] , the stress-induced filaments (Earle's balanced salt solution, no serum) observed here were less active. The CTPS1 protein levels were also higher in presence of histidine than in its absence, suggesting that filament formation protects the protein from degradation, improving the ability of cells to reinstate normal cell growth following nutrient replenishment. CTPS1 filament formation in response to glutamine (and serum) deprivation appeared to be mediated by the stress response pathway involving GCN2 (general control nonderepressible 2) / ATF4 (Activating transcription factor 4) / MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase).

This pathway allows the metabolism to adapt to nutritional stress, including nucleotide depletion, simultaneously decreasing the amount of reactive oxygen species (that disrupt filament formation) through MTHFD2 and favouring histidine influx through ATF4, in fine leading to the methylation-dependent (at least in this condition) formation of CTPS1 filaments 125 .

In hepatocellular carcinoma, levels of Hsp90 (Heat-shock protein 90) and CTPS filament are increased and correlate with tumor grade 127 . While there is no direct link between them, another study in S. pombe found that knocking down heat-shock proteins decreased filament formation 128 , suggesting that the modulation of filament formation by heat-shock proteins could be an evolutionary conserved response to stress.

Filament formation adds another layer of regulation of the enzymatic activity

Overexpression of CTPS has been shown to increase filament formation in multiple species (C. crescentus [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] ; E. coli 79 ; Drosophila 129 ; mouse [START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF] and human 130 ), while conversely knocking down CTPS protein levels decreases filament formation [START_REF] Chen | Glutamine Analogs Promote Cytoophidium Assembly in Human and Drosophila Cells[END_REF] . CTPS1 filaments have been observed in a variety of cancers, with filament formation specifically associated with the tumor tissue in hepatocellular carcinoma and increased with the tumor grade. Since the authors of the study had no expression level data, it is unclear whether this was due to increased CTPS1 protein levels in the transformed cells or to other factors such as nutritional stress, a common occurrence in tumors 127 .

Although CTPS filament formation is a very conserved mechanism, the impact of filamentation itself on the enzymatic activity is still widely debated. CTP synthases overexpression only results in minor increases in CTPS enzymatic activity and CTP pools 126 and has been associated with increased filament formation in multiple species (Drosophila 126,129 ; Bacteria 79 ), suggesting that filament formation might negatively regulate their enzymatic activities to preserve the nucleotide pools equilibrium. Indeed, CTPS overexpression in D. melanogaster during the development impairs brain development, leading to smaller brains and optic lobe defects 131 . GFP-tagged CTPS1 in mouse embryonic brains is present in diffuse form during the early G1 phase, while the cell prepares itself for division, which includes synthetizing a sufficient pool of nucleotides, while it is assembled as filaments during the other phases of the cell cycle, suggesting that decreased filament formation is required to promote CTP synthesis. However, the overexpression of GFP-CTPS1 also impaired further mitotic activity of the neuronal progenitor cells, inhibiting neuronal proliferation and migration 132 . Indeed, a filamentation-deficient E. coli mutant (E277R) of CTPS was shown to have a much higher CTPS activity in presence of high levels of CTP compared to the wild-type enzyme, suggesting that filament formation improves the product-induced feedback inhibition mechanism [START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF] .

However, recent data showing that some eukaryotic CTP synthases can form at least two kinds of filaments (formed of substrate-or product-bound tetramers) (human CTPS2 [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] ; D. melanogaster CTPS [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] add another layer of complexity (see below). Substrate-bound human CTPS1 filaments were shown to be more active than the unbound enzyme [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] . A similar result was obtained using the D. melanogaster enzyme in the same conditions. Conversely, human CTPS2 filament formation in the active, substrate-bound conformation did not modify the kinetic parameters of the enzyme. However, filament formation by the inactive, product-bound human CTPS2 increased the cooperativity of CTPS2 regulation by CTP, allowing a fast and efficient inactivation of the enzyme to adapt to metabolic changes [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] . Unsurprisingly, productbound D. melanogaster CTPS filaments are also inactive [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] .

Altogether, these results suggest that filament formation is a way to quickly adapt to metabolic changes.

Filaments could be a storage form of the CTP synthases [START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF]125 , maintaining a pool of enzyme in an inactive (mainly in prokaryotes [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF][START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF]125 or active (mainly in eukaryotes [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] ) conformation depending on the enzyme and on the cellular context, and protecting it from degradation as an added benefit 119,125 . In some cases, like for the human CTPS1 and CTPS of D. melanogaster, filaments might be a complex meant to optimize CTP production [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] . Finally, it could be a remarkably fast way to respond almost instantaneously to minute changes in nucleotide concentrations, well-suited for an enzyme like CTPS2 that would be meant to ensure balanced nucleotide levels in basal conditions [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] , since nucleotides can still freely diffuse into the CTPS filaments [START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF] .

Figure 9: CTPS filament formation is modulated by conformational changes induced by substrate and product binding

The sole filament-forming drosophila CTP synthase has a similar behaviour to human CTPS2. (based on Lynch et al., 2017).

Differences between species and CTPS family members

The CTP synthases have been studied in a multitude of models. Despite their relatively high sequence conservation, similar structures and conserved regulation modes by their substrates and products, some key differences exist and have been highlighted, not only by comparative studies between species, but also by comparing the behaviour of CTP synthases from the same species.

During the early exploration of filament formation in C. crescentus, it was discovered that a treatment with the glutamine analog DON disrupted filament formation [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] . This could have been an early indication of the tetramer requirement for filament formation as DON had been reported to disrupt bacterial CTPS tetramers 133 . Conversely, in drosophila and human, CTPS filament formation is induced by DON and azaserine, another glutamine analog [START_REF] Chen | Glutamine Analogs Promote Cytoophidium Assembly in Human and Drosophila Cells[END_REF]126 .

However, new data comparing bacterial and human CTPS1 showed that the structure of the CTPS1 filaments was different between the two species. Binding of the substrate (UTP) or the product (CTP) induces conformational changes in both enzymes, but conversely to the bacteria, it is the substrate-bound (UTP) form of the human enzyme that is integrated into filaments, while it is the product-bound (CTP) form in the bacteria. The comparison of the native enzyme (assembled as filaments in presence of ATP, UTP and GTP) with a filamentation-deficient mutant (H355A, a residue at the tetramer-tetramer interface) suggests that filamentation increases the enzymatic activity of CTPS1 [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] . Histidine 355 is located in a conserved insert that appeared early in eukaryotic evolution and has also been shown to disrupt filament formation of the drosophila [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF]124 and mouse 119 enzymes.

However, despite their relatively conserved sequences, including the aforementioned eukaryotic insert (containing H355), the CTP synthases of S. cerevisiae (budding yeast) and S. pombe (Fission yeast) were also shown to respond differently to nutritional stress. While the S. pombe enzyme mostly forms filaments during the early to mid-exponential phase of growth, only to decrease during the latter half and disassemble at the stationary phase 128 , the S. cerevisiae enzyme is mostly assembled as filaments during the stationary phase or after glucose deprivation, suggesting that in this model, filament formation is associated with a quiescent state [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF][START_REF] Noree | Common Regulatory Control of CTP Synthase Enzyme Activity and Filament Formation[END_REF] .

Two studies published in 2019 highlighted an even more fascinating aspect of filament formation regulation: the filament formation of human CTPS2 and of the sole D. melanogaster CTP synthase could be induced both by the substrates (S-state tetramers) and the products (P-state tetramers) [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] .

While the S-state filaments were similar to what was described for human CTPS1 [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] , the P-state filaments were also constituted of tetramers, although the structure of these filaments was slightly altered due to the product-binding induced changes. The Histidine 355 residue at the tetramer-tetramer interface was shown to be as crucial to CTPS2 filament formation as it is for CTPS1. Both laboratories attributed this specific ability of CTPS2/D. melanogaster CTPS to form P-state filaments to the C-terminal domain of both proteins that is poorly ordered and more flexible, not creating any steric hindrance that could block tetramer association. However, while the deletion of the C-terminus had no impact on human CTPS2 filament formation in both states [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] , it led to a decreased filament length in D. melanogaster, suggesting that may have a stabilizing role in this species [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] . Conversely, structural analysis and deletion of the Cterminus of human CTPS1 suggested that it was sterically incompatible with the P-state tetramer filament formation but was required to stabilize S-state filaments [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] .

Interestingly, through the generation of a mutant leading to the formation of disulfide cross-links at the filament interface of human CTPS2 (V352C, interacting with C362), causing irreversible filament formation in the absence of a reducing agent, Lynch and Kollman demonstrated that substrate-bound and productbound filaments could dynamically switch without prior dissociation, depending on nucleotide availability.

In absence of ATP and UTP, V352C CTPS2 was also shown to assemble into filaments whose shape corresponded to that of the substrate-bound filaments, suggesting that CTPS2 was able to form tetramers in absence of substrates (and products) [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] . Assembly of a CTP synthase in tetramer form in absence of substrates had previously been observed in E.coli 133 . This crucial difference in the determinants for filament formation was shown to impact the regulation of CTPS2 activity by CTP. In presence of its substrates, CTPS2 was found to be as active as the filamentation-deficient H355A mutant, unlike CTPS1, while the inhibitory effect of CTP was shown to be stronger on the filamentation-deficient CTPS2, suggesting that substrate-induced CTPS2 filament formation decreased the product-induced feedback inhibition threshold. Interestingly, reaching the CTP inhibition threshold, which is 5 times lower for CTPS2 than for CTPS1 121 , leads to a rapid inactivation of the enzyme, suggesting that the conformational changes induced by CTP binding propagate along the filament and that the activity of CTPS2 is tightly regulated by CTP levels [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] . The role of the N-terminal part of CTP synthases in filament formation is poorly explored. A study in drosophila identified conserved residues at the N-terminus of D. melanogaster CTPS and H. sapiens CTPS1 (K2, Y3, A20) (also conserved in CTPS2), that were shown to be crucial for filament formation as their disruption by mutagenesis (K2E, Y3E, A20R) abolished filament formation. Interestingly, while the A20R mutant (both in D. melanogaster CTPS and H. sapiens CTPS1) assembled as foci, filament formation could still be induced by a treatment with the glutamine analog DON 134 . As recent data suggests that the determinants of filament formation between human CTPS1 and CTPS2 are different, the behaviour of the latter being closer to that of the D. melanogaster enzyme [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] , it would be interesting to further explore the specific role of this region in the human CTP synthases.

Association of the CTP synthases with proteins and protein complexes

Association of enzymes involved in purine biosynthesis has been reported in the literature 135 . Such functional associations between enzymes associated with pyrimidine biosynthetic pathways would also make sense. While studying the impact of serum starvation-induced GSK3 phosphorylation of CTPS1, Higgins identified, along with Pin1 and tubulin, an association of CTPS1 with cytoplasmic dynein and CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) 106 . Dyneins are a family of proteins relying on ATP hydrolysis to generate force and movement inside and outside the cell 136 , that were also shown to be able to use CTP as an energy source 137,138 , while CAD is an enzyme involved in the first three steps of the pyrimidine biosynthesis pathway 139 . Nucleoside diphosphate kinase (NDPK), an enzyme that converts nucleosides diphosphates (other than ATP) into nucleosides triphosphates, was also reported to associate with tubulin 140 , suggesting a functional colocalization of pyrimidine biosynthetic enzymes around the tubulin network 106 . The D. melanogaster (dAck) and human Ack1 (activated cdc42-associated kinase 1) have been found associated with some CTPS filaments. Interestingly, a loss-of-function dAck mutant (dAck86) failed to associate with CTPS filaments and was associated with a reduced phospholipid composition and ovarian RNA levels, suggesting that the CTPS activity was reduced. Additionally, the authors observed a disturbed filament organization, including a modification of the filament structure (smaller and thinner filaments) and the inappropriate persistence of filaments in late-stage egg chambers. This suggests that dAck modulates filament formation and activity, although not direct phosphorylation of CTPS by dAck was observed in this model 113 . S6K1 associates with CTPS1, suggesting that it might phosphorylate CTPS1 as mentioned earlier 110 .

Even though no direct association has been reported, the colocalization of CTP synthase filaments and other filament-forming metabolic enzymes has been reported in multiple species, including inosine-5'monophosphate-dehydrogenase 2 (IMPDH2) [START_REF] Carcamo | Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells[END_REF][START_REF] Keppeke | Assembly of IMPDH2-Based, CTPS-Based, and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction[END_REF] , asparagine synthetase (ASNS) 141,142 and △1pyrroline-5-carboxylate synthase (P5CS), an enzyme of the proline synthesis pathway 143 . These assemblies could reflect functional gathering of enzymes sharing comparable functions (for instance, IMPDH is the first committed step in guanosine nucleotides synthesis 144 ) or an optimization of their functions and regulations through shared substrates and products.

GTP

CTP and CTP Synthases in physiology Crucial role of the CTP synthase activity during development

The importance during early development of the CTP synthase activity was initially demonstrated in newborn and adult rat brains 145,146 . CTPS activity in the young rat brain (1-day-old) was found to be 4 times higher than in the corresponding adult tissue. Interestingly, CTPS activity was found to be 6-fold higher in the hemispheres compared to the cerebellum in adult rat brain, suggesting that RNA synthesis was decreased in the cerebellum. This was indeed in accordance with data previously published showing that the activity of the Mg2+-dependent RNA polymerase of cerebellar nuclei was lower than in nuclei isolated from other brain regions 147 . It was then hypothesized that the variations observed during the rat development could also be related to the involvement of cytidine nucleotides in lipid metabolism, although it was admittedly unclear in their model based on the available data.

The existence of multiple CTP synthase family members in most organisms (two in most currently described eukaryotic models, and even five [START_REF] Daumann | Characterization of Filament-Forming CTP Synthases from Arabidopsis Thaliana[END_REF] or six 148 in plants with distinct properties) highlights the importance of CTP synthesis and suggests that this could be a safeguard to preserve minimal CTPS activity levels, although each enzyme is probably involved in specific processes.

For instance, Saccharomyces cerevisiae expresses two CTP synthases (URA7 and URA8, named thereafter CTPS1 and CTPS2 for simplification purposes), and cells deficient for one of the enzymes appear to be viable (with CTP concentrations of respectively 78% and 36% of the control in cells deficient for CTPS1 versus CTPS2), while the double KO is lethal, suggesting that their role is at least partially redundant [START_REF] Ozier-Kalogeropoulos | Use of Synthetic Lethal Mutants to Clone and Characterize a Novel CTP Synthetase Gene in Saccharomyces Cerevisiae[END_REF] . However, not only were the levels of expression of CTPS2, as quantified by mRNA levels, half of that of CTPS1, but the enzyme appeared to be more sensitive to CTP inhibition compared to CTPS2, suggesting that the CTPS2 activity levels were more tightly regulated 149 . Similarly, CHO (Chinese Hamster Ovary) cells deficient for CTPS1 were cytidine-dependent, suggesting that CTPS2 cannot compensate for the absence of CTPS1 in this cell line, although the expression of CTPS2 in this cellular model was not verified at the time 150 .

CTPS2 might be the main provider of CTP during development, as patients expressing weak levels of an hypomorphic CTPS1 mutant present with no major defects, developmental or other, outside of the hematopoietic compartment. This suggests that CTPS2, along with the low contribution of the weakly expressed mutated CTPS1 and of the salvage pathway, can supply sufficient amounts of CTP to sustain normal development [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] .

Recent in vitro and clinical data suggest that CTPS1 may have a key role during decidualization, the adaptation of the endometrium to accommodate a pregnancy. A decreased expression of CTPS1 was observed in decidua tissues of severe pre-eclampsia (pregnancy complication associated with a high blood pressure and proteinuria) patients. In vitro, induction of decidualization in human embryonic stem cells increases the expression of CTPS1 associated with a relocation of the protein over time from the cytoplasm to the nucleus. Interestingly, CTPS1 does not seem directly involved in cell proliferation in this model, but instead sequentially interacts with ATPS during the first steps of decidualization and then with STRESS-70 during the later stages to regulate ATP level 151 .

Phospholipid synthesis

Kennedy and Weiss, while working on the biosynthesis of some phospholipids, identified in 1956 an asyet-unknown cofactor required for the synthesis of lecithin from phosphatidylcholine: the cytidine triphosphate. Through the formation of CDP-choline, CTP was also shown to be involved in the synthesis of sphingomyelin 152 and some triglycerides 153 . It was later showed that CTP levels controlled the balance between CTP-dependent phospholipid synthesis and neutral lipid synthesis (phosphatidic acid, diacylglycerol and triacylglycerol) 154 and the expression in Saccharomyces cerevisiae of a CTP synthase deficient for CTP feedback regulation (E161K) was shown to increase the synthesis of phosphatidylcholine, phosphatidylethanolamine and phosphatidate while simultaneously decreasing that of phosphatidylserine 155 , confirming these data. Ostrander et al., 1998) Unsurprisingly, it was also found in yeast that the positive impact of the phosphorylation by PKA and PKC on the activity of URA7 also impacted phospholipid synthesis 100,156

Protein glycosylation

Discovered in 1936 by Gunnar Blix 158 , the sialic acids are a family of sugars that can be appended to the terminal end of glycoproteins through a CMP-coupled intermediate 159,160 . The earliest report of the existence of a sialic acid-containing nucleotide was in the late fifties 161,162 . The synthesis of this nucleotide was shown to require CTP [START_REF] Roseman | Enzymatic Synthesis of Cytidine 5′-Monophospho-Sialic Acids[END_REF]163 , as inhibiting CTPS activity also reduced sialic acid regeneration in HL-60 cells 164 . Sialylation of proteins has been associated with cell fate decision, neurodevelopment and to the maintenance of stem cell pluripotency, but most importantly, Sialylated proteins have also been recognized as tumor markers 165 and are suspected to be involved in the epithelial-mesenchymal transition (EMT) 166 . Moreover, they are negatively charged, providing charge repulsion, which physiologically prevents unwanted adhesion, but in the case of metastasis might favour dissemination 167 . Human dolichol kinase also requires CTP to phosphorylate dolichol as dolichyl monophosphate, a carrier lipid involved in N-glycosylation of proteins 169 .

Highly proliferating tissues and cell populations

Increased CTP synthase activity levels are also observed in highly proliferative tissues. A high CTP synthase activity, akin to what is observed in hepatomas from the same species, was reported in regenerating rat livers 170 .

Conversely, fibroblasts were found to have a low level of CTPS activity, relying mostly on the salvage pathway to synthetize CTP, a finding that was attributed to the low proliferative potential of this cell type 171 .

Nucleus

CMP-Neu5Ac

Golgi

Cytosol

Figure 16: Human CTPS1 and CTPS2 expression data extracted from BioGPS

As the signal intensity cannot be compared between different probesets, this only highlights that CTPS2 levels of expression across tissues (and included cell lines) are relatively homogeneous. CTPS1 is strongly overexpressed in some blood cells populations and lymphoma/leukemia cell lines.

Lymphocytes and other blood cell populations

As key effectors in the immune response, lymphocytes need to be able to quickly proliferate in response to stimuli such as CD3, CD28 and IL-2 stimulation. The stimulation of T lymphocytes with anti-CD3 and IL-2 leads to a significant increase in the nucleotide pools compared to resting cells, especially for CTP (21 ± 5 in resting cells versus 346 ± 106 pmol/106 cells in anti-CD3 stimulated T lymphocytes.) 172 , associated with strongly increased CTPS activity levels [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] . Assessment of the CTP synthase activity in multiple blood cell populations showed that the highest activity levels were found in thrombocytes (6.48 nmol CTP/mg/h), monocytes (2.23) and lymphocytes (1.69) 173 . It appears that resting cells rely on the salvage pathway to support their metabolic requirements, while the de novo activity is required for nucleotide pool expansion 174 . CTPS1 was here identified as the main source of CTP synthase activity in multiple immune cell populations including T cells and a key contributor to the immune response [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] .

Antiviral immunity

While studying the mechanism of action of interferon-induced inhibition of the replication of human cytomegalovirus (HCMV), Chin and Cresswell identified a new protein whose expression was induced both by interferon stimulation and the HCMV envelope protein glycoprotein B: the "virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible", or Viperin 177 . This protein was initially shown to inhibit virus replication by interacting with various viral proteins 178 . It was later discovered that its antiviral activity also relied on its ability to synthetize an antiviral ribonucleotide using CTP as a substrate, the 3'deoxy-3',4'-didehydro-CTP (ddhCTP), that specifically acts as a chain terminator for the RNA-dependent RNA polymerases of some viral species [START_REF] Gizzi | A Naturally Occurring Antiviral Ribonucleotide Encoded by the Human Genome[END_REF] .

Protein deamidation

Protein deamidation has been suggested to play a role in some biological processes, including the innate immune response, and is used as a strategy by some pathogens to escape the immune response 179 .

Recent data suggest that some viruses, including SARS-CoV2, activate CTPS1 which in turn deaminates IRF3 (Interferon Regulatory transcription Factor 3). Deaminated IRF3 is unable to bind to the promoters of its targets, possibly muting interferon induction and impairing the innate immune response, highlighting another potential activity of the enzyme through its glutaminase domain 180 .

CTP synthases and cell cytoskeleton

In C. crescentus (also known as C. vibroides), crescentin, a bacterial cytoskeletal protein 181 interacts with CTPS. The interaction between CTPS and crescentin was shown to regulate cell curvature [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF] , although the role of crescentin-induced cell curvature is poorly understood 182,183 . However, this is so far the only example of an effect of CTPS filaments on cell cytoskeleton.

While purified CTP synthase enzymes can form filaments [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF][START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF][START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF] , the bundling of thin filaments formed by chains of tetramers into large filaments might require the presence of cytoskeleton proteins or some other proteins. Indeed, the presence of small gaps of GFP staining in GFP-tagged CTPS filaments suggests that this might be the case [START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF] . In eukaryotes, filaments assemble in a dynamic way, through the assembly of smaller aggregates that elongate and end up "fusing" side-by-side or end-to-end [START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF] . Moreover, the study of the polarized distribution of CTPS filaments in D. melanogaster shows that neither filament formation nor filament localization are random processes. The sole D. melanogaster CTPS is located basolaterally in follicle cells (in 99% of the cells) and the knockdown of some of the proteins involved in cell polarity (Stardust, Crumb and aPKC (atypical protein kinase C)) disturbed the CTPS filament location 184 . CTP synthase filaments have been observed in the cytoplasm and in the nucleus of some cells, including human cells [START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF]122,185 . Interestingly, the morphology of the filaments differs between the cytoplasm and the ? ? ?

nucleus, the cytoplasmic filaments appearing more curved [START_REF] Gou | CTP Synthase Forms Cytoophidia in the Cytoplasm and Nucleus[END_REF] . Although this could be explained by the size difference, it is also possible that the presence or absence of some partners in the two cellular compartments could impact the shape of these filaments. Similarly, CTPS filaments in apoptotic drosophila egg chambers have an increased curvature, suggesting that some components of the filaments affecting their shape might be disrupted or degraded 186 . The CTP synthases have also been found associated with cytoskeleton proteins in multiple species, although these associations appear to be very species-dependent. Tubulin was found associated with CTPS1 in a co-IP experiment 106 and CTPS filaments in D. melanogaster were later observed co-localized with the tubulin network, but not with the centrioles [START_REF] Liu | Intracellular Compartmentation of CTP Synthase in Drosophila[END_REF] . However, other studies found no enrichments of tubulin and actin in IMPDH2/CTPS1 filaments and no association with centrosomes [START_REF] Carcamo | Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells[END_REF]187 . Similarly, the two CTP synthases of S. cerevisiae Ura7 and Ura8 did not colocalize with the tubulin network, although they colocalized with each other [START_REF] Noree | Identification of Novel Filament-Forming Proteins in Saccharomyces Cerevisiae and Drosophila Melanogaster[END_REF] .

The Japonica rice (Oryza sativa var. japonica) CTPS1 was also found to interact with tubulin, suggesting an involvement with microtubules during early development stages [START_REF] Yoon | CTP Synthase Is Essential for Early Endosperm Development by Regulating Nuclei Spacing[END_REF] .

Following glutamine deprivation, SNAP29 (synaptosome-associated protein 29) is displaced towards the cytokeratin network and interacts with CTPS in a human cell line (HEp-2, a HeLa cell line derivative). The stress-induced filaments are associated with the cytokeratin network and disruption of the latter or knockdown of SNAP29 disassembles CTPS filament, suggesting that SNAP29 is required for CTPS filament formation and maintenance 187 .

CTP and CTP Synthases in pathology Diabetes

Decreased levels of CTP and CTP synthase activity (respectively 63% and 53% of the normal pool) were observed in a rat diabetes model (induced by an alloxan treatment, a glucose analog is known to be cytotoxic to beta cells 188 ). An insulin treatment for 2 days restored both levels to the normal range, while a prolonged treatment with insulin led to an increase of the CTP levels of respectively 792% and ~4 fold the CTP and CTP synthase activity levels 189 .

Intriguingly, the CTP synthetase activity levels were found increased after diabetes induction by streptozocin (alkylating agent, toxic to the pancreas beta cells 190 ), yet unchanged in another model, unilateral nephrectomy (accelerating the development of diabetes mellitus 191,192 ).

Vascular repair

CTPS1 expression is upregulated in neointimal smooth muscle cells (SMCs), that overexpand following vascular injury, causing thickening and leading to abnormal, overconstricted blood vessel formation.

Treatment with CPEC has been shown to reduce smooth muscle cells proliferation, improving reendothelialization following injury 193,194 . However, it was later shown that CPEC actually has a dual role in vascular repair since, as a cytosine derivative, it is also able to stimulate the adenosine receptors A1 and A2a. This in turn activates downward signaling pathways and induces smooth muscle cell redifferentiation, modifying their behaviour towards a pro-angiogenic phenotype 195 .

Cancer

The malignant transformation of cells ultimately leading to cancer require the development of capabilities that allow the cell to escape the constrains of physiological cell growth, including the deregulation of metabolic pathways 196 including nucleotide synthesis 197 . As the highly controlled CTP levels are a bottleneck for cell proliferation, most notably due to the role of CTP in nucleic acids, phospholipids (cell membranes) synthesis and protein glycosylation 198 , improving CTP supply lines seems to be a requirement for fast-growing tumor cells. Along these lines, dysregulation and increase in CTP synthase activity have been associated with the process of malignant transformation [199][200][201] while CTPS1 knockdown has been associated with reduced tumor growth in a drosophila model 202 .

A study published in 2019 linked high CTPS2 expression with improved survival outcome in osteosarcoma patients. Additionally, lower expression levels were also observed in metastatic tumor samples. However, as pointed out by the authors, the clinical information was scarce, and the number of samples relative to the metastatic and non-metastatic group was small. Increased resistance to 5-Fluorouracil (5-FU) in CTPS2-low tumors had previously been reported 203 ; a high expression of CTPS2 could therefore have provided a therapeutic benefit to the patients by decreasing endogenous UTP levels, therefore increasing the sensitivity to the analog 5-FU (See "CTP synthase as a biomarker for drug sensitivity or resistance") 204 .

In 1978, among a panel of rat and human hepatomas that all exhibited increased levels of UDP kinase activity 205 , the enzyme producing UTP from UDP, Williams et al. identified a rapidly growing cell line (hepatoma 3924A or MH-3924A). Despite the high UDP kinase activity levels in this cell line, UTP concentration was not increased, while the CTP levels were increased 170,206 . Analysis of the whole panel of tumors showed an increased CTP synthase activity that was correlated with the individual tumor growth rates 170 . Such an increased was reported in human colon 207 , kidney 208 , lymphoproliferative disorders 209 and leukemia 210 , murine Ehrlich ascites tumor cells 211 as well as in many other human and murine tumor tissues 212 . Indeed, multiple factors identified in tumor cells and associated with tumorigenesis regulate CTPS1 expression. TWIST, a transcription factor whose expression in adult tissues has also been correlated with tumorigenesis due to its role in the epithelial-mesenchymal transition and its anti-apoptotic activity 213,214 , induces CTPS expression [START_REF] Feng | Gene Expression Profiling in TWIST-Depleted Gastric Cancer Cells[END_REF] . The protooncogene Myc also induces the expression of genes involved in nucleotide synthesis, including CTPS1 in a human cell line model [START_REF] Liu | Global Regulation of Nucleotide Biosynthetic Genes by C-Myc[END_REF]215 . More recently, CLIC1 (chloride intracellular channel protein 1), a protein first isolated and cloned from a human myelomonocytic cell line (U937) 216 that is frequently associated with a poor prognosis for cancer patients 217,218 has been shown to upregulate the expression of CTPS1 219 . Hypoxia-induced factor-1α (HIF-1α), a factor frequently upregulated in tumors was shown to bind in the region of the CTPS1 promoter In a gemcitabine-resistant cell line, inducing its expression 220 .

The dysregulation of nucleotide synthetic pathways leads to the alteration of nucleotide pools, a phenomenon that is associated with mutagenic events that can lead to malignant transformation of cells 221 . As such, altered CTP synthase activity levels could disrupt the delicate equilibrium of nucleotide pools. Indeed, inactivation of a yeast homolog of CTPS1, Ura7, was shown to cause the activation of DNA damage responses and increase mutations rates that could be rescued by the proofreading function of the DNA polymerase Polε. Most of the transitions observed were G:C to A:T, further confirming that the misincorporation of bases was due to the CTP pool reduction 222 . Conversely, a dysregulation of the CTPS activity leading to increased CTP levels led to increased mutation rates 223 .

A precise physiological or therapeutic control of CTP levels is therefore essential.

Immunodeficiency

Blocking CTP synthesis with cyclopentenyl cytosine (CPEC) decreases T cell proliferation and cytotoxicity, leading to anergy, highlighting the importance of the CTPS activity during the immune response 224 .

As mentioned previously, a CTPS1 deficiency leading to impaired immune responses has been described in several publications [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF]225,226 . All of them result from the same homozygous mutation, and so far, no other CTPS1 mutants have been reported. Consistent with previous studies, the basal level of CTP synthase activity was low in control (and patient) resting lymphocytes. However, unlike the controls, activation of the patient peripheral blood mononuclear cells (PBMCs) and T cell blasts failed to induce an increase in the CTPS activity and CTPS1 expression levels, leading to the impaired activation and expansion of the cells and compromising the immune response [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] . Current treatment options for CTPS1 deficiencies are limited to hematopoietic stem cell transplantations 225,227 . EBV infection of B cells was shown to upregulate the expression of both CTPS1 and CTPS2 at the mRNA and protein levels, while most immune stimuli had a moderate impact on CTPS2 protein levels, suggesting that CTPS2 may play an important role in EBV infection of B cells, in particular in the specific case of CTPS1 deficiencies 228 .

As the physiological requirement of a CTPS1-dependent expansion of the lymphocytes CTP pool to proliferate is probably conserved in autoimmune diseases, specifically inhibiting CTPS1 to dampen the abnormal response could be a promising therapeutic strategy [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] .

Microbial infections

As all living organisms and viruses rely on nucleotides for their metabolic needs and for the transmission of genetic information, the study of the CTP metabolism and of the CTP synthases from various microbial species not only gave precious information regarding the highly conserved structure and function of this family of proteins, but also new therapeutic avenues.

Parasites

There are multiple examples of parasites relying on de novo CTP synthesis to sustain their growth, which could be an adaptation to the limited CTP supply from the host.

For instance, early studies of the purine and pyrimidine metabolic enzymes in Plasmodium falciparum suggested that these cells had a low to absent CTP synthase activity (and no CTP salvage pathway could be detected in these cells either), suggesting that this parasite could strictly depend on the host CTP supply 229 . However, later studies confirmed the existence of an active CTP synthase gene in these cells 230 .

The list goes on: Chlamydia trachomatis, an obligate intracellular bacterium, is auxotrophic for all ribonucleosides triphosphate with the exception of the CTP synthetase activity. As this bacterium is able to incorporate exogenous cytidine from the host 231,232 , the conservation of the CTP synthase activity might be an adaptation of the parasite to the limited intracellular CTP pool. Rickettsia prowazekii, another obligate intracellular bacterium, was also shown to be able to import ATP, GTP and UMP from the host, but not CTP, instead relying on its own de novo CTP synthesis ability 233 . Trypanosoma brucei, that is also devoid of a CTP synthase pathway, can also synthetize CTP de novo, albeit slowly, leading to low CTP pools 234,235 . A similar requirement for de novo CTP synthase activity was also reported for Toxoplasma gondii 236 .

Viral replication

Viruses strictly rely on the host metabolic machinery to replicate, which includes dependency on the nucleotide pool and synthesis machinery as their small genomes usually cannot fit genes involved in the construction of metabolic pathways 237 . Unsurprisingly, many CTP synthase inhibitors or cytidine analog (Carbodine (carbocyclic cytidine analog), CPEC (cytidine analog) [238][239][240] ) were found to have antiviral properties as they limit the viral replication through nucleotide depletion, mainly due to the use of CTP in viral genome synthesis and membrane phospholipids.

As a result, some viruses have been reported to induce CTPS1 or CTPS2 expression in infected cells. Some data suggest that HIV-1 impairs de novo nucleotide synthesis, and pyrimidine in particular, to repress the immune response 241 . This impedes the ability of T lymphocytes to proliferate and leads to metabolic cell death following activation. However, it has more recently been shown that HIV-1 it can also induce CTPS1 expression 242 . Indeed, further decreasing the already low intracellular levels of the nucleotide would also be a double-edged sword, impairing its own replication. EBV has also been shown to drive CTPS1 transcription through its EBV latency III growth program, along with CTPS2 and other members of the pyrimidine de novo synthesis and salvage pathways 228 . Finally, SV-40-transformed cells (Simian Virus 40) have higher CTPS expression and activity levels, suggesting that this virus also drives CTPS expression [START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] .

The viral dependence on the host nucleotide pool, and CTP in particular, probable drove the apparition of defence mechanisms such as Viperin, a member of the radical S-adenosyl-L-methionine (SAM) family, that converts CTP into ddhCTP. The resulting CTP analog can consequently be used by the RNA-dependent RNA-polymerases of some virus families, acting as a chain terminator to impede viral replication [START_REF] Gizzi | A Naturally Occurring Antiviral Ribonucleotide Encoded by the Human Genome[END_REF] .

The limited CTP pool in human cells, along with other cytosine-mediated antiviral processes like CpG methylation, recognition of CpG-containing viral sequences, and ddhCTP-induced replication termination, have been suggested to drive virus evolution 237 . As mentioned above, nucleotide pool imbalance favours mutations, and the limiting nucleotides can end up being replaced by other nucleotides during replication.

Incidentally, the SARS-CoV2 outbreak is a striking illustration of the impact of the limited CTP pool in human cells on virus evolution. A recent study showed that, although the cytosine content was globally lower than that of all other nucleotides in coronaviruses (17.9% vs 21.5-33.3%), the disequilibrium was further highlighted when comparing strains of established epidemic coronaviruses in humans (13-16.7%) to spillover-related viruses (ie viruses originating from another reservoir) (18.4-20.3%), with a codon usage bias disfavouring C-containing codons over alternatives when possible 243 . This can be explained by the selective advantage conferred by the replacement of cytosine by other, more abundant nucleotides, facilitating the replication of viral genomes and decreasing the risk of developing further, more deleterious mutations due to the lack of intracellular C nucleotides.

SARS-CoV2 has also recently been shown to generate polypeptides that bind CTPS1, increasing its activity. An unexpected consequence of this hyperactivation of CTPS1 is, as mentioned earlier, the deamidation through CTPS1 of IRF3, decreasing its activity to impede the immune response 180 .

The CTPS activity, mainly through CTPS1, appears to be a double agent in the context of viral infections, as it is needed for the immune system to fight the infection, while it simultaneously fuels viral replication, and potentially dampens the innate immune response, although this activity might require the specific interaction with viral proteins 180 .

CTP Synthases as therapeutic targets

As nucleotides and nucleotides synthesis are central to all known life forms, targeting nucleotide supply to impede the proliferation or replication of prokaryotic (Chlamydia trachomatis 244,245 ; Giardia intestinalis 246 ; Streptococcus pneumoniae 247 ; Mycobacterium tuberculosis [248][249][250][251] ; Toxoplasma gondii 236 ; Bacillus subtilis 252 ) or eukaryotic parasites (Trypanosoma brucei 234,235,[253][254][255][256][257] ; Trypanosoma evansi 258 or viruses 238,239,259 ) and tumor cells has been a prolific research field, especially as the CTPS activity [START_REF] Traut | Physiological Concentrations of Purines and Pyrimidines[END_REF]170,208,210,260 and CTPS expression levels 261 were found to be often elevated in highly proliferating cells such as tumors.

Interestingly, it has been shown that antimetabolites, including CTP synthase inhibitors, could induce tumour cell differentiation through the impairment of DNA synthesis [262][263][264] .

Targeting CTPS1 could also provide therapeutic benefits in the management of stent thrombosis following percutaneous coronary intervention due to its implication in smooth muscle proliferation during vascular repair 194 and the interest in generating CTPS1-specifig inhibitors has been rekindled in recent years 252,265- 267 .

CTPS-targeting compounds

The requirement for nucleotides (ATP, UTP, and to a lesser extent GTP) suggested that the use of analogs could impede the activity of CTP synthases. Although multiple compounds were serendipitously identified and later found to be active on the CTP synthases, there were already studies as early as in 1982 aiming to get a better insight on the substrate specificity of CTP synthases that could facilitate the development of CTPS-inhibiting analogs 268 .

The most well-known compounds impacting CTP synthesis will be introduced in this next part.

Glutamine analogs : DON (6-Diazo-5-oxo-L-norleucine)

DON is a naturally occurring glutamine analog that was isolated and characterized in 1956 from the culture broth of an unidentified peruvian streptomyces 269,270 . DON irreversibly binds to the glutamine active sites of glutamine-utilizing enzymes 271,272 , in the case of CTP synthase with a maximum 2 units per tetramer 273 , suggesting that the binding of one unit of DON in a CTPS dimer impedes the fixation of another unit.

Although commonly described and used as a CTPS inhibitor, its list of targets includes Carbamoyl phosphate synthase (CAD), involved in the early steps of pyrimidine nucleotides synthesis, and enzymes involved in purine biosynthesis [START_REF] Eidinoff | Pyrimidine Studies: I. Effect of DON (6-Diazo-5-Oxo-l-Norleucine) on Incorporation of Precursors into Nucleic Acid Pyrimidines[END_REF]274 . However, significant adverse effects, including nausea, diarrhea and thrombocytopenia complicate its use as a therapeutic agent 272 .

Acivicin ((alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) Acivicin, discovered in 1972, is another naturally occurring glutamin analog that is produced by Streptomyces sviceus 275 . Similarly to DON, acivicin competitively and irreversibly inhibits glutaminedependent amidotransferases, including CTP synthetase [275][276][277][278][279] .

Azaserine

Azaserine is a serine derivative isolated from a strain of Streptomyces fragilis 280 . It is a glutamine analog that was shown to inhibit purine synthesis 281 , certain glutamine-requiring reactions (including CTPS) 282 and glutamine transport in a rat renal brush-border membrane vesicles model 283 .

Uridine analog: 3-deazauridine (4-hydroxy-1-(β-D-ribo-pentofuranosyl)-2-pyridone)

3-deazauridine (3-DU) is a uridine/UTP analog that needs to be metabolized as 3-deazauridine triphosphate (3-DU-TP) to compete with UTP for binding on the CTP synthases, inhibiting their activity.

This compound was shown to exhibit a potent inhibitory effect on the growth of microbial and tumor cells in vitro, on tumor models in vivo and on viral replication [284][285][286][287][288] . Early studies studying the mechanism of action and metabolization of the compound 284,285,289,290 showed that the toxic effect of the compound was not mediated by incorporation into nucleic acids.

However, although the compound was first synthetized in 1968 291 , CTP synthase was only identified as the target in 1974 292,293 . 3-DU was shown to block some leukemic cells in S phase 294 and to induce cell death in the HL-60 cell line 295 .

It was also suggested that 3-DU could inhibit the salvage of uridine as well as the de novo pyrimidine synthesis by mimicking the UMP-induced Carbamoyl phosphate synthase 2 inhibition 296 . The fact that its effect can be fully countered through the CTP salvage pathway by using cytidine suggests that its effect is CTPS-specific.

3-DU has also been shown to have a potent antiviral activity against HIV-1 by increasing mutations, leading to the apparition of mutation types that were absent in absence of drugs, which could be a consequence of nucleotide pool imbalance 259 .

Cytidine analogs: CPEC (Cyclopentenyl cytosine)

CPEC is a cytidine analog and antagonist 297 , derived from Neplanocin A, an antibiotic compound isolated from a culture of Ampullariella regularis A11079 298 . This compound, initially generated to reduce the toxicity of the original molecule, was found to be a strong antiviral and antitumor compound 299 , impairing DNA synthesis and inducing differentiation or senescence in breast cancer through the induction of p53 263,300 .

CPEC is metabolized as CPEC-TP and appears to be a non-competitive CTPS inhibitor 301 . Incorporation of CPEC into nucleic acids, and especially RNA, has been reported 297,302 .

However, despite promising results in vitro and in vivo, notably on non-lymphocytic and lymphocytic leukemia 301,303 , the drug was found to have highly deleterious secondary toxic effects including hypotension leading to the death of some patients 304,305 .

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC)

Gemcitabine is a deoxycytidine analog whose metabolite (gemcitabine triphosphate) can be incorporated into DNA 306 and RNA 307 , acting as a chain terminator. It was also shown to bind CTP synthase with a higher affinity than CTP 308 , inhibiting its activity and depleting CTP and dCTP pools, while leading to an accumulation of UTP 309 .

Carbodine (4-amino-1-[(1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl]pyrimidin-2one)

Carbodine is a carbocyclic analog of cytidine used as an antiviral agent. As it does not seem to be incorporated into viruses nucleic acids 260 , and its effects are reverted by cytidine supplementation, its target appears to be the CTP synthases 238 .

Antiviral or anti-parasitic effect of CTPS targeting compounds

The extracellular parasite Trypanosoma brucei has been reported to have very low intracellular CTP levels 234 due to a slow CTP synthase activity 235 . In vivo studies using a murine model of Trypanosoma brucei infection showed that adequate doses of DON and Acivicin could significantly reduce the amount of parasites in the blood while being low enough to avoid compromising the hosts nucleotide pool 253 , although further studies in comparison with a derivative compound, 3-Bromoacivicin, showed that this treatment was insufficient to avoid a re-emergence of the parasite 255 .

Targeting CTP synthase to potentiate other drugs

Numerous studies show that using CTPS inhibitors like the 3-DU in combination with compounds targeting other nucleotide synthesis pathway could improve pre-existing and future therapeutic avenues 310 .

With toxic nucleotide analogs / DNA/RNA replication inhibitors

Azacytidine and 5-aza-2-deoxycytidine are cytidine analogs that have been used as anti-tumor agents, notably at high doses where their incorporation into DNA appears to be toxic, leading to cell death. It was shown that inhibiting CTPS activity through the use of 3-deazauridine could improve the toxicity of such compounds by depleting the CTP and dCTP pools, increasing the odds of incorporation of these cytidine analogs at low concentrations into nucleic acids 311,312 .

3-deazauridine has also been shown to potentiate the activity of Zalcitabine and Lamivudine, two cytidine analogues acting as chain terminator of DNA synthesis, on HIV-1 replication 313,314 with the same mechanism of action.

Similarly, a CPEC or 3-DU pretreatment was shown to sensibilize the cells to Cytarabine (deoxycytosine analog) 302,302,[315][316][317] , Decitabine (cytidine analog) 318 and Gemcitabine (deoxycytidine triphosphate analog) 319 , again by improving the incorporation rates of these toxic analogs.

With nucleic acid synthesis inhibitors

High concentrations of thymidine were shown to inhibit the rate of DNA synthesis during the G1/S transition and to decrease dCTP pools 320 , kill tumor cells in vitro 321 and cause tumor graft regression in athymic mice 322 . Synergistic effects of 3-DU and thymidine were reported, but the mechanism of action was not identified, although the authors suggested that the target could be ribonucleotide reductase, the enzyme converting (ribo)nucleotides into deoxyribonucleotides 323 .

Dibromodulcitol is an alkylating agent, causing single strand breaks 324 . The combination of dibromodulcitol and acivicin was shown to be synergistic, an effect attributed to the depletion of the CTP pool and inhibition of DNA synthesis 325 . Similarly, actinomycin, a DNA intercalant that inhibits RNA polymerase-mediated elongation 326 , was also shown to synergize with Acivicin 277 .

CTP synthase as a biomarker for drug sensitivity or resistance?

Low CTPS2 expression in colorectal cancer has been associated with resistance to 5-fluorouracil (5-FU) 203 . 5-FU, an uracil analog, inhibits thymidylate synthase whose metabolites can be incorporated into DNA and RNA 327 . Decreased levels of CTPS2 lead to a decreased consumption of UTP, that in turn competes with 5-FU during RNA synthesis, decreasing its ability to induce toxic effects. Intriguingly, earlier published data showed that a hamster cell line with increased levels of CTP synthase activity due to the loss of CTP-induced negative feedback was less sensitive to 5-FU. The author suggested that this might be due to the accumulation of CTP, leading to the inhibition of earlier steps in the pyrimidine salvage and de novo pathways, in fine decreasing the anabolism of 5-FU 328 .

Similarly, an association between elevated levels of CTP synthase activity and resistance to treatments with cytidine analogs including cytarabine (cytosine arabinoside) and decitabine (5-aza-2'-deoxycytidine) has been reported 209,294,329 as "normal" CTP competes with the analogs.

Conversely, increased CTPS2 expression, that is associated with increased proliferation, was found to correlate with sensitivity to platinum-based (cisplatin and carboplatin) antitumor compounds since they cause DNA damage (which depends on cell proliferation) 330 .

Known side effects to compounds targeting CTPS activity?

Although usually effective to treat autoimmune diseases, tumor development, viral and parasite infections, treatments using nucleotide antagonists cause diverse side effects, notably due to induction of nucleotide pool imbalance. For instance, part of the mutagenic effect of BrDU (Bromodeoxyuridine) was shown to be mediated by a transient depletion of the dCTP pool 331 . The disruption of this equilibrium is known to facilitate transitions (replacement of a base with another one from the same backbone (purine/purine or pyrimidine/pyrimidine), for instance cytidine to thymidine) and transversions (replacement of a purine with a pyrimidine and conversely) 332 , mutagenic events that could ultimately lead to carcinogenesis 221 . In most cases, however, dNTP imbalance causes cell death 333 thanks to the degradation of DNA by cellular safeguards such as dNTP imbalance-induced endonucleases 334,335 . The glutamine antagonists azaserine and DON were shown to cause somatic crossing over, point mutations and segmental loss, although this effect was not directly linked to CTPS inhibition 336 . As mentioned earlier, a phase I trial with CPEC led to the death of some patients due to hypotension 304,305 . Side effects such as myelosuppression, skin erythema, stomatitis as well as digestive troubles (vomiting, diarrhea ) were also identified in a phase I-II study of 3-DU 337 . Hence, a selective and finely tuned inhibition of CTP synthase activity is crucial to achieve therapeutic benefits.

Aim of my thesis

Previous works of the lab of Sylvain Latour have demonstrated the crucial role of CTPS1 in the immune system, especially during T lymphocyte expansion after TCR stimulation in response to pathogen infections. These data, along with anterior studies linking increased CTP synthase activity to cancer development and progression, suggested that the inhibition of CTP synthase activity, and CTPS1 in particular, could be a relevant therapeutic approach to treat immune diseases associated with abnormal T-cell expansion such as autoimmunity, GVHD (graft versus host disease) or T-cell lymphomas.

CTP synthases are not new therapeutic targets, but the compounds already developed were also associated with severe adverse effects, most of them potentially due to a poor specificity leading to offtarget effects. These compounds often interfere with the activity of other cellular enzymes with similar domains, activities, or sharing the same substrates. Another problem associated with the compounds is their absence of selectivity between CTPS1 and CTPS2, both enzymes being structurally and functionally very closely related. The identification of CTPS1 as the potentially most interesting therapeutic target based on the human CTPS1 immunodeficiency gave a second chance to CTP synthase-targeting therapies and provided a rationale to develop selective inhibitors of CTPS1.

Step Pharma was created in 2014 to develop therapeutic small molecules that selectively inhibit CTPS1. However, the cells of the patients failed to upregulate the protein levels of CTPS1, leading to proliferative defects and to the inability to generate an appropriate immune response (Martin et al., 2014). After the publication of these data, eleven more patients were identified [225][226][227] , of which five were studied in our lab, allowing for a better characterization of their phenotypes [START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] .

Another, isolated Indian patient was brought to our attention. Born from consanguineous parents, she carried a different mutation of the CTPS1 gene, a 10 base pair deletion in exon 18. The resulting frameshift creates an early stop that leads to the loss of 19 residues, thereafter designated as CTPS1Δ19. Her phenotype (a life-threatening disseminated chickenpox and chronic EBV infection with lymphoproliferation) suggested that her symptoms could also be attributed to a CTPS deficiency.

However, she needed urgent care and got a bone marrow transplant at 17 years old.

To further characterize the CTPS1Δ18 mutant as well as the CTPS1 variant from the Indian patient, we have expressed both mutants in our CTPS-null Jurkat T cell model.

Results

Part of the data presented here (Figure 24, Figure 25) has been reproduced from the article published in 2020 in JCI insight (Martin et al., 2020) which can be found in annex.

Both mutants identified in the patients affect the C-terminal part of the protein

The coding sequence of human CTPS1 is spread out over 19 exons. However, the presence of a stop codon in exon 18 prevents the expression of the residues coded by this region. CTPS1Δ18 results from a mutation in a splice donor site located at the junction of intron 17-18 and exon 17 (c.1692-1G>C, p.T566Dfs26X), resulting in the replacement of exon 18 by exon 19 and its subsequent expression (Figure 22). CTPS1Δ19 (c.1714_1724del, p(Gly572ProfsTer2) results from the deletion of 10 base pairs in exon 18. The resulting deletion induces a frameshift, replacing the last 20 residues of the protein by a single proline, for a total loss of 19 residues (hence the CTPS1Δ19 name). Interestingly, when transiently expressed in HEK, both mutants exhibit a decreased apparent molecular weight after migration on a denaturing gel (Figure 23B). While this could be expected for the shorter CTPS1Δ19 mutant (572aa vs 591aa for the wild-type protein), the difference observed for CTPS1Δ18 (590aa vs 591aa) suggests that the removal of the domain coded by exon 18 has an impact on the size of the protein. This might be explained by several potential and described phosphorylation sites present in the sequenced coded by exon 18 (Figure 22B).

Exogenous expression of the CTPS1Δ18 mutant in CTPS1-KO cells restores their proliferation

CTPS1-KO Jurkat cells (Figure 24A, B) are dependent on cytidine supplementation (source of CTP generated through the salvage pathway) and are unable to proliferate in absence of cytidine (Figure 24C). Thus, they represent a potent tool to verify if the identified mutants are active. CTPS1-KO Jurkat, maintained in presence of cytidine, were transduced with pLenti 7.3 vectors containing the coding sequences of GFP plus or minus wild-type CTPS1 or CTPS1Δ18, generated as described previously [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF] . Interestingly, despite comparable GFP levels, indicating similar transduction efficiencies, CTPS1-KO Jurkat cells complemented with the CTPS1Δ18 mutant (Jurkat Δ18 ) expressed lower levels of the protein compared to cells complemented with the wild-type CTPS1 (Jurkat CTPS1 ) (Figure 24B). In accordance with the reduced protein levels, while both cell populations were able to expand in absence of cytidine supplementation, Jurkat Δ18 had a delayed expansion compared to Jurkat CTPS1 . Interestingly, Jurkat Δ18 and Jurkat CTPS1 expansion accelerated over time. This acceleration was associated with an increase in GFP (Figure 24D,E) and CTPS1 levels (Figure 24E). Cytidine supplementation prevented the increase in GFP and CTPS1 protein levels (Figure 24D), confirming that the increase in GFP and CTPS1 levels were caused by the cytidine deprivation-induced selective pressure. Co-culture of Jurkat Δ18 and Jurkat CTPS1 initially maintained with cytidine with CTPS1-KO Jurkat confirmed the slower expansion rate of Jurkat Δ18 as well as the absence of selective advantage of complemented cells in presence of cytidine supplementation (Figure 24F).

CTPS1Δ18 has a preserved enzymatic activity

To verify if CTPS1Δ18 can restore CTPS1-KO Jurkat cell proliferation and CTPS activity, Jurkat Δ18 and Jurkat CTPS1 were cultured in absence of cytidine until the stabilization of GFP levels. As expected, GFP levels in the Jurkat Δ18 population stabilized at a higher level compared to the Jurkat CTPS1 population.

(Figure 24D). Co-culture of these selected populations with CTPS1-KO Jurkat confirmed that both populations had similar proliferation rates (Figure 25B) and CTPS activity levels (Figure 25C), validating that CTPS1Δ18 has an enzymatic activity at least equivalent to that of WT CTPS1. Taken together, these data suggest that the decreased CTPS activity observed in the patient is due to the low stability of the CTPS1Δ18, not to a decreased activity.

GFP-tagged CTPS1Δ18 assembles as filaments

Recent studies on human CTPS1 and CTPS2, as well as on D. melanogaster CTPS filament formation have highlighted the role of the C-terminal domain of the CTP synthases in filament formation [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] .

We have therefore studied the impact of the Δ18 mutation on the ability of GFP-CTPS1 to form filaments. However, we failed to detect CTPS1 filaments in Jurkat Δ18 . As the mutant protein levels are low in this model, we generated a GFP-tagged form of CTPS1Δ18 and stably overexpressed it in CTPS1 and

CTPS2-KO HEK cells (double KO) (Minet et al. draft).

As previously observed in CTPS1-KO cells stably overexpressing GFP-CTPS1 and GFP-CTPS2, both GFP-CTPS1 and GFP CTPS2 form filaments in absence of any treatment. While GFP-CTPS1 filament formation was induced by the UTP analog 3-DU and reduced by cytidine supplementation, none of these treatments had obvious effects on GFP-CTPS2. Intriguingly, while GFP-CTPS1Δ18 also formed filaments, its behaviour was similar to GFP-CTPS2 in that we observed no obvious modification of filament formation by any of the conditions we tested (Figure 26A). As the C-terminal domain of CTP synthases has been reported to be involved in in filament formation, stabilizing (in D. melanogaster [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] ) or impeding filament formation in the product-bound conformation of the CTPS1 tetramer [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] , it is possible that the abnormal C-terminal end of this mutant does not prevent filament formation in the product-bound state (in our case, in presence of cytidine, converted to CTP by the salvage pathway), like CTPS2. We also occasionally observed a very specific, nest-like filamentation pattern in some cells complemented with GFP-CTPS1Δ18 consisting in the accumulation of very thin filaments as well as the presence in some cells of large numbers of very short filaments (Figure 27). While it is possible that the abnormal C-terminal domain impedes the assembly of smaller filaments into larger structures, it cannot be excluded that it also affects the interaction between CTPS1 and some other proteins involved in the recruitment and assembly of chains of tetramers (fibers) into larger structures.

We previously observed that neither CTPS1 nor CTPS2 form detectable filaments in HEK in normal culture conditions (Minet et al. draft). While both proteins assembled as filaments after a treatment with 3-DU, not detectable or very rare CTPS2 filaments were present in CTPS1-KO HEK cells in this condition.

Expression of GFP-CTPS1 restored CTPS2 filaments, which were found colocalized with GFP-CTPS1 filaments, even in absence of 3-DU induction (Minet et al. draft). Similarly, stable overexpression of GFP-CTPS1Δ18 in CTPS1-KO HEK restored CTPS2 filament formation, and we also observed the presence of rings of GFP-CTPS1Δ18/CTPS2. All CTPS2 filaments and ring structures were colocalized with GFP-CTPS1Δ18. (Figure 26). This suggests that CTPS2 can spontaneously assemble along CTPS1 (or CTPS1Δ18) filaments. However, as CTPS2 associates with CTPS1 at the tetramer level (Minet et al.

draft), it is also possible that CTPS2 is integrated into CTPS1 filaments. The presence of GFP-CTPS1Δ18 filaments in absence of any treatment or after a treatment of the cells with 3-DU or cytidine suggests that, like GFP-CTPS2, GFP-CTPS1Δ18 tetramers can assemble as filaments in the product-bound and substrate-bound forms. This is particularly interesting as a CTPS1 mutant lacking its last 33 C-terminal residues was reported to be unable to form filaments when bound to its substrates, suggesting that this part of the protein was required to stabilize the filament. Conversely, it has been suggested that the Cterminal domain of CTPS1 could be sterically incompatible with product-bound tetramer filament formation, and rare pairs of tetramers have been observed on purified enzymes is this condition [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] . In the case of GFP-CTPS1Δ18, it is possible that the new C-terminal domain removes this barrier, possibly provides a new stabilization interface compatible with both filament states, or that the affinity between the tetramers is boosted by the presence of the GFP tag [START_REF] Chang | Interfilament Interaction between IMPDH and CTPS Cytoophidia[END_REF] . This also raises the possibility that such a mutant might be able to form filaments that can dynamically switch between active and inactive conformations depending on cellular substrate/product levels as reported for CTPS2, increasing the cooperativity of the regulation of the enzyme [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] .

Generation of the CTPS1Δ19 mutant

As the carrier of the CTPS1Δ19 mutation required urgent care, we were not able to retrieve material from the patient. We therefore generated the mutant using a wild-type CTPS1 cDNA by introducing the relevant mutation (c.1714_1724del, p(Gly572ProfsTer2)) by site-directed mutagenesis. The resulting mutated cDNA was cloned in a lentiviral pLVX vector along with the previously described CTPS1Δ18 mutant and wild-type CTPS1 and CTPS2. Transient expression levels of both mutants were similar to that of the control CTPS1 in double KO HEK despite similar transfection efficiencies (Figure 23A,B). This was unexpected for CTPS1Δ18 as expression the pLenti 7.3 vector gave lower protein levels compared to the wild-type protein in Jurkat cells [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF] . It is possible that these differences could be due to the different promoters between both vectors (CMV (cytomegalovirus) for the pLenti 7.3, EF-1α (elongation factor-1α) for the pLVX). The EF-1α vector present in the pLVX drives higher levels of expression of the transgene, potentially stabilizing the mutant protein.

CTPS1Δ19 restores the proliferation of Jurkat cells

The pLVX vectors containing the coding sequences of CTPS1, CTPS2, CTPS1Δ18 and CTPS1Δ19 were then packaged in lentiviral particules and unconcentrated culture supernatants used to transduce CTPS1-KO Jurkat with a low efficiency (Figure 28C). As expected, cytidine deprivation led to a sharp decrease in the amount of living cells as non-transduced cells were rapidly eliminated through the lack of proliferation and induction of apoptosis (Minet et al. draft) (Figure X7A), while cytidine supplementation prevented cell selection (Figure X7B). Subsequent expansion of the cells in absence of cytidine was associated with an increase in the amount of mCherry-positive cells. All cells were mCherry-positive at the end of the selection for all populations, including CTPS1Δ19. The differences in the percentages of mCherry-positive cells at the end of the selection can be explained by quantification issues as mCherry levels vary between the constructs, also reflecting differences in cell selection due to selective pressure (Figure 28D, D17 and D24). Interestingly, as previously observed for similar experiments (Minet et al. draft), the expansion of the cells transduced with the CTPS2 vector was delayed compared to the other constructs. Extended culture in absence of cytidine led to a further selection of the cells (Figure 28D). The cells transduced with the CTPS2 and CTPS1Δ18 vectors were selected toward mCherry-high populations. In the case of the mutant CTPS1Δ18, this can be explained by stability issues requiring higher expression levels to yield optimal CTPS1 levels. This is confirmed by the analysis of its expression by western blot where, despite higher mCherry reporter levels as observed by cytometry, CTPS1Δ18 protein levels are lower (Figure 28E,F). While it is also possible that CTPS2 is less stable than CTPS1, we showed that the increased mCherry levels with this construct is explained by lower enzymatic activity of CTPS2 (compared to CTPS1). Conversely, while mCherry-high cells were present in the CTPS1 and CTPS1Δ19 populations at early selection stages, further expansion led to a progressive decrease in the amount of mCherry-high cells towards more moderate expression levels (Figure 28D). While this phenomenon is slower for the CTPS1Δ19, this might suggest that the mutant is active and that, like CTPS1, its overexpression could actually decrease the growth of the cells or possibly trigger regulation mechanisms that would repress its activity.

Conclusion and discussion

Part of the CTPS1Δ18 data (Figure 24 and Figure 25) comes from the Martin et al. article published in 2020 in JCI insight [START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] that can be found in the annexes.

CTPS1Δ18 is an hypomorphic, yet active CTPS1 variant

This project stems from the discovery and characterization in 2014 of a cohort of immunodeficient patients whose phenotype was attributed to a CTPS1 deficiency. The data presented at the time made it clear that the phenotype of the patients was due to the CTPS1Δ18 homozygous mutation, but there were admittedly some limitations. In particular, it was unclear whether the deficiency was due to a total absence of CTPS1 as the antibodies available failed to detect the mutant protein in the patient samples. The CTP synthase enzymatic activity in patient samples was significantly lower than that in the control samples; however, the enzymatic test does not discriminate between the relative activities of CTPS1 and CTPS2. The identification of new patients, along with the development of new antibodies and the emergence of CRISPR-Cas9 as a widespread tool for the development of knock-out cellular models provided us with new opportunities to better characterize the pathology along with the functional consequences of this mutation.

CTPS1Δ18 was not detectable in patient samples in the first study. However, this mutant was transiently expressed and detected control HEK cells, suggesting that the protein might be completely unstable or present, but below the detection level in patient samples [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF] . The availability of a new, specific anti-CTPS1 antibody later allowed the detection of the mutant protein in the cells of the parents along with the wildtype CTPS1. Intriguingly, the levels of the mutant protein in the heterozygous parent samples were elevated compared to the afflicted children, and similar to those of the wild-type protein [START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] . Along with the successful expression of this mutant in control HEK cells (that express both CTPS1 and CTPS2), this suggest that the presence of wild-type CTPS1 stabilizes the mutant protein.

Indeed, while we have successfully complemented CTPS1-KO Jurkat cells with CTPS1Δ18, we have also observed a selection over time of cells expressing higher levels of reporter compared to cells complemented with wild-type CTPS1. This suggests that there is a positive selection towards cells that have integrated multiple copies of the vector. However, this high expression was not associated with increased protein levels of CTPS1Δ18 as similar amounts of CTPS1 and CTPS1Δ18 were detected in the complemented Jurkat cell populations by the end of the selection (see Figure 25,[START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] ). In this model, the expression of GFP and CTPS1/CTPS1Δ18 are driven by two different promoters (respectively SV40 and CMV) and a partial loss of expression of CTPS1Δ18 might explain such a difference. However, transgene extinction is a progressive phenomenon and decreased CTPS1Δ18 levels were already observed at the beginning of the selection despite elevated CTPS1 levels. Overexpression might therefore be required to counter the instability of this mutant in absence of the endogenous form. A second transduction of Jurkat cells complemented with CTPS1Δ18 with wild-type CTPS1 could further validate the stabilization hypothesis.

As filament formation has been suggested to protect CTPS1 from degradation 119,125 , it would also be interesting to verify if CTPS1 (and CTPS2) can form filaments in activated T-cell blasts of controls as well as of the heterozygous parents, potentially stabilizing CTPS1Δ18 in the structure.

CTPS1Δ19 is a new, active CTPS1 variant

At first sight, the CTPS1Δ19 mutant appeared to be a cousin of CTPS1Δ18 -a CTPS1 variant, affecting the regulatory C-terminal domain of the protein, identified in an immunodeficient patient. The critical state of the patient prevented the analysis of samples before transplantation, and we were left with the identifiers of the mutations. The mutant had to be generated from scratch for it to be studied in our cellular models. Intriguingly, while the enzymatic activity of this mutant remains to be evaluated due to technical issues, it successfully restored the proliferation of CTPS-null Jurkat as well as HEK (data not shown).

While our preliminary cell complementation data suggest that this mutant is stable and active, it is possible that the vector used in this study biases the results as the strong EF1α promoter leads to a strong overexpression of the proteins of interest, potentially stabilizing the transgenes, as observed for CTPS1Δ18 (Figure 23B). While we also observed a counter-selection over time of mCherry-high cells in the CTPS1Δ19 population, similarly to what we observed with the corresponding CTPS1 construct, this selection was delayed, suggesting that CTPS1Δ19 might represent an intermediate between CTPS1 and CTPS1Δ18 due to potential differences in stability and activity (Figure 28D). This could be further explored using co-culture assays with control and CTPS1-KO cells, similarly to what we did using cells complemented with the CTPS1Δ18 mutant (Figure 24 and Figure 25) as well as activity measurements on cell extracts. Finally, we currently lack information regarding the ability of CTPS1Δ19, or of a GFPtagged form of this mutant, to form filaments. Filament formation has been reported to increase the enzymatic activity of CTPS1, and the deletion of the C-terminal domain of CTPS1 has been reported to impede its ability to form filaments. A potential inability of the CTPS1Δ19 mutant to form filaments could decrease its ability to increase its enzymatic activity following the upregulation of its expression during T cell activation.

Limits of our model

While the data obtained using our CTPS-null Jurkat cellular model allowed us to corroborate the data obtained on patient cells for the CTPS1Δ18 mutant and suggested that CTPS1Δ19 may not be as deleterious as CTPS1Δ18, the approach used to study this mutant was also different. As mentioned earlier, the strong overexpression driven by the EF1α promoter is susceptible to yield expression levels that could be sufficient to stabilize the transgene. Using a promoter driving lower expression levels of these transgenes, and CTPS1Δ19 in particular, could give us additional clues as we do not know the physiological levels of this mutant in patient cells. Beyond these points, there are several unknowns for CTPS1Δ19 that we need to consider. We lack data on primary cells as the patient had to be transplanted; the activity of this mutant needs to be assessed; we do not know either if it is able to assemble as filaments, or if its interaction with potential partners, including CTPS2, is impacted. If it is able to form filaments, it cannot be excluded that the absence of part of its C-terminal domain could remove the barrier to assemble product-bound filaments. Although this is very hypothetical, a capacity of this mutant to assemble, or at least integrate product-bound CTPS filaments could also make it more sensible to cooperative regulation through conformational spread 338 , in particular if it assembles with CTPS2. As CTPS2 is five-fold more sensitive to CTP levels than CTPS1 121 , this association could also make it more sensitive to CTP feedback inhibition, in fine decreasing its ability to sustain the metabolic needs of highly proliferating cells such as lymphocytes.

In this situation, the strength our CTPS-null cellular model is also its weakness. While it allows us to evaluate independently the activity of CTP synthase mutants in a cellular context with no interference from the endogenous forms, it might also mask some of the effects these mutants would have in normal cells due to the potential impact of the modification of their C-terminal domain on their interaction with CTPS2. Evaluating the impact of the expression of both mutants on the growth of cells strictly depending on CTPS2 expression (as the CTPS1-KO HEK model), for instance by competition assays, could allow us to strengthen our early findings. This could also allow us to understand the eventual contribution of CTPS1Δ19 to the pathology as well as shed some light on the role of the C-terminal domain in the interaction between both proteins.

Chapter 2 -Characterization of a collection of GFP-tagged CTPS mutants from the literature Introduction

Part of this project consisted in testing new pharmacological inhibitors of CTPS1 and to try to understand their mechanism of action, partly through the identification of the site of action of the compounds. For this purpose, we have generated a series of CTPS1 mutants extracted from the literature impacting different regions of the protein on which we planned to test the compounds.

Multiple CTPS mutants have been generated and studied over the years in species ranging from bacteria (E. coli) to human. These studies provided us with invaluable information regarding the structure-function relationship of CTPS1 and CTPS2, and in particular on the impact of the alteration of specific regions on filament formation. However, it is also now known that, while most currently studied CTPS enzymes share substrates, products and the ability to assemble as micrometer-scale filaments, there are also large differences in the behaviour of the enzymes between species, and even within the same species. This is particularly true for the determinants of filament formation and their potential impact on enzymatic activity.

Although we were able to identify a key residue involved in the selectivity of the small inhibitory molecules developed by the industrial partner through a different approach (see chapter 4), further study of these mutants in our cellular models could expand our knowledge regarding the consequences of such mutations on proliferation and filament formation. This collection of previously described CTPS1 mutants from the literature (Figure 29, Table 3) has been studied as two semi-independent series.

Figure 29: Map of the mutations selected for this study Table 3: List of the mutations selected for this study

Results

N-terminal GFP-tagged CTPS1 and CTPS2 are active

N-terminal GFP-tagged forms of CTPS1 and CTPS2 were generated by cloning the coding sequence of both enzymes into C1 EGFP vectors. After linearization, to improve their integration, C1 EGFP (empty, or containing either GFP-CTPS1 or GFP-CTPS2) vectors were electroporated in control and CTPS1-KO HEK and the evolution of the populations, cultured in absence of cytidine, followed over time. After a sharp initial decrease corresponding to the dilution of cells that did not integrate the vectors, a progressive expansion of the CTPS1-KO cells expressing GFP-CTPS1, and to a much lower extent GFP-CTPS2 was observed, while no expansion of the GFP-positive cells was observed for the GFP (empty) vector or for any of the control populations (Figure 30). Similarly, the transfection of double KO HEK cells with the linearized C1 EGFP vector (with GFP-CTPS1, GFP-CTPS1Δ18 and GFP-CTPS2) restored cell proliferation (Figure 30). While variations in transfection and integration efficiencies do not allow us to extrapolate more information from these experiments, they confirm that GFP-tagged CTPS1, CTPS1Δ18 and CTPS2 are active.

Figure 30: Complementation of CTPS1-KO or double-KO HEK with GFP or N-terminal GFP-tagged CTPS1

or CTPS2 Follow-up of control, CTPS1-KO or double KO HEK transfected with linearized C1 EGFP vectors and cultured in complete DMEM in absence of cytidine. Each point represents a passage. A) Control and CTPS1-KO cells. The sharp decrease up to day 17 is due to the dilution and subsequent loss of the cells that did not integrate the various constructs. B) Double-KO HEK.

Transient expression of the first series of constructs

All of the GFP-CTPS1 mutants generated by site-directed mutagenesis as well as the GFP-tagged CTPS1 and CTPS2 constructs are well overexpressed in double KO HEK. However, we also observed for all the mutants as well as for the controls the presence of a large number of bands on western blot, indicative of a degradation of the fusion proteins, independently of the mutations introduced, suggesting that the tag destabilizes the fusion proteins (Figure 31). All GFP-tagged CTPS1 excepted H355A formed filaments in absence of any treatment. Treatment with 400µM of 3-deazauridine to induce filament formation also failed to induce H355A filament formation (Figure 32). However, the presence of filaments in some conditions is intriguing, especially in the case of the G148A and R294D CTPS1. Both were expected to disrupt filament formation, which in the case of the former is due to the alteration of the UTP binding site, probably hindering its ability to assemble as tetramers as the UTP binding site is altered. Conversely, since the reported filament loss in E. coli and C. glutamicum [START_REF] Mccluskey | Biophysical Analysis of Bacterial CTP Synthase Filaments Formed in the Presence of the Chemotherapeutic Metabolite Gemcitabine-5'-Triphosphate[END_REF]339 was due to the alteration of the CTP

Stable complementation of double KO HEK cells with GFP-tagged CTPS1 mutants (series 1)

To assess the activity of the various mutants, linearized vectors were transfected into double KO HEK cells. As expected, culture of the cells in presence in presence of cytidine prevented any selection, while cytidine deprivation led to the selection of cells expressing active constructs. GFP alone, D70A (Mg 2+ /ATP binding site disruption), G148A (UTP binding site disruption), E155K (Prevention of CTP production) and C399A (disruption of the glutaminase active site) failed to restore the proliferation of double KO cells.

While there is an apparent increase in the proportion of GFP-positive cells for G148A at day 30, this is probably a technical artifact. As this population failed to proliferate in absence of cytidine supplementation, this might reflect stress-induced autofluorescence. This experiment also confirms that the removal of the C-terminal domain (R560X or P563X) does not abolish the activity of the enzyme, and that filament formation (H355A) is not required for the activity of CTPS1 (Figure 33).

GFP-CTPS1 P563X

No cytidine 200µM cytidine

Time (days) % of GFP-positive cells

Transient expression of the second series of constructs

We have previously demonstrated that CTPS1 associates with CTPS2 independently of filament formation, as H355A CTPS1 disrupts filament formation, even with a GFP tag (Minet et al. draft). However, it is currently unknown if this assembly happens at the dimer or at the tetramer level. We decided to generate mutants (V114F, M157I at the monomer-monomer interface, G152E, R164H at the dimer-dimer interface) previously used in a study in D. melanogaster 126 to identify the precise step during which the interaction initially takes place.

We also generated H3552 CTPS2 to validate its inability to assemble as filaments. As with the first series, all of the mutants were well expressed in double KO HEK (Figure 34) and formed filaments (excepted H355A CTPS2), as previously reported. While decreased filament formation for V114F and M157I (monomer-monomer interface mutants) has been described in D. melanogaster, this effect was unclear on the human enzyme. While filament formation was partially expected as it had already been described in the literature for the D. melanogaster enzyme 126 , this is surprising as dimers are not expected to form CTPS filaments in human cells, much less monomers. However, it is also possible that the tendency of the GFP tag to dimerize compensates for a potentially decreased affinity between monomers and dimers.

As expected, A20R did not form filaments but instead presented punctate structures 134 .

Figure 34: Transient expression of the second series of GFP-tagged CTPS1 mutants in double KO HEK

A) Western blot, 48h post-transfection. B) 40x images, 48h post-transfection (72h for A20R), GFP only.

Stable complementation of double KO HEK cells with GFP-tagged CTPS1 mutants (series 2)

We then assessed the activity of these mutants using our double KO HEK model and linearized vectors.

As observed for H355A CTPS1, H355A CTPS2 is active as it is able to restore the proliferation of our CTPS-deficient cellular model, confirming that the activity of CTPS1 and CTPS2 can be decoupled from their ability to form filaments. We also tested A20R CTPS1 which also failed to restore cell proliferation, suggesting that this mutant impacts the activity of CTPS1.

Surprisingly, in particular in the case of the dimerization mutants V114F and M157I, all of the other mutants restored the proliferation of double KO HEK (Figure 35). This is especially intriguing as the levels of GFP remained low in the populations (Figure 35B). This suggested that the protein levels for the various constructs was also low, which was confirmed by western blot (Figure 35C,D). The low levels of GFPpositive cells might also be explained by the presence of a high amount of GFP-low cells who cannot be easily discriminated from negative cells, potentially even indicating a high CTP synthase activity. Once again, it is possible that, although these mutations are expected to destabilize the monomer-monomer and dimer-dimer interface, the presence of the GFP tag conversely increases the affinity between subunits by its own tendency to dimerize, favouring the formation of dimers and active tetramers.

Figure 35: Generation of stable double KO HEK expressing the second series of GFP-tagged CTPS1 mutants

Impact of mutations expected to disrupt dimer, tetramer and filament formation on the association between CTPS1 and CTPS2

Even though the previously published results along with the data presented here suggest that the effect of the mutations, if any, on dimer and tetramer formation is incomplete at best, we decided to assess the impact of the dimer and tetramer mutation mutations on the interaction between CTPS1 and CTPS2. GFP alone, GFP-CTPS1 and the H355A (deficient for filament formation), V114F, M157I (mutation of the monomer-monomer interface), G152E and R164H (dimer-dimer interface mutants) GFP-tagged CTPS1 mutants were expressed in control HEK. Unlike the double KO HEK we previously used, these cells express endogenous CTPS1 and CTPS2 which are susceptible to associate with and modify the behaviour of the mutants. Filament formation was therefore verified 48h post-transfection by microscopy (Figure 36A).

As previously reported, while V114F and M157I CTPS1 formed shorter filaments, only H355A failed to assemble as filaments, even in presence of the endogenously expressed CTPS1 and CTPS2. The GFPtagged proteins were then immunoprecipitated and previously identified associated proteins analysed by western blot (Figure 36). All of the proteins, GFP excepted, were found associated with CTPS2. While the CTPS1 signal was variable between the lanes, potentially accounting for experimental variations in transfection and immunoprecipitation efficiencies, CTPS2 was found associated with all the mutants.

We also tested the interaction with SUGP1, a splicing factor that is thought to play a role in mRNA splicing that we previously found specifically associated with GFP-tagged CTPS1 (Minet et al. draft) SUGP1 was found associated with all the CTPS1 mutants. However, we failed to detect an association between SUGP1 and endogenous CTPS1 by immunoprecipitation. Although the only antibody that specifically immunoprecipitated CTPS1 was found to be non-specific as it also immunoprecipitated CTPS2 in CTPS1-KO HEK (data not shown), this preliminary data suggests that the interaction we observed with the GFP-tagged proteins might not be physiological, or too weak to be detected by this approach.

We cannot exclude that the GFP-tagged CTPS1 mutants interact with endogenous CTPS1 and CTPS2 in a manner that requires neither filaments nor classical subunit-subunit interaction (classical dimer/tetramer formation). Yet, this suggests that none of the monomer and dimer interface mutants disrupt the expected interfaces in the human protein.

Conclusion and discussion

Our models provide us with a new tool to study CTPS1 and CTPS2 mutants with no interference from endogenous forms, and with an absolute dependency on CTP supplementation or complementation with an active CTP synthase. Although our results are preliminary and require further exploration, this approach allowed us to confirm or infirm the part of the hypothetical effects of some mutants already described in the literature.

While most of the constructs expected to be inactive due to the disruption of domains crucial for the CTPS activity (ATP/Mg2+ binding domain, UTP binding domain, glutaminase domain) failed to restore the proliferation of our CTPS-deficient model, mutants initially described for the D. melanogaster enzyme and designed to impact the monomer-monomer and dimer-dimer interfaces were found to be at least partially active. This is of particular interest as perturbed (but not fully disrupted) filament formation by the monomer interface mutants, and preserved filament formation for the dimer interface mutants led the authors who generated these mutants to suggest that inactive dimers may have been the base unit of filaments in D.

melanogaster. Although we cannot exclude that these mutations could have a different impact on the human enzyme, the gross morphology of the V114F filaments is similar to what had been reported for the D. melanogaster enzyme 126 . Additionally, our cellular model proves that at least part of the activity of the GFP-tagged forms of these mutants is preserved, which should not be the case if dimer and tetramer formation was really disrupted if we consider that the CTPS tetramers represent the active form.

While we successfully managed to immunoprecipitate endogenous CTPS2, we have not currently identified an antibody able to efficiently and specifically immunoprecipitate untagged CTPS1. In our GFP immunoprecipitation assay (Figure 36B), even though the anti-CTPS1 antibody detects a band at the expected molecular weight for CTPS1 in the immunoprecipitated proteins blot (Figure 36B, black arrow), we cannot claim with certainty that endogenous CTPS1 associates with the GFP-tagged mutants as the GFP IP also enriches the fraction in GFP-tagged CTPS1 degradation products. However, the absence of a strong signal at the expected molecular weight for CTPS1 in double KO HEK cells expressing GFPtagged CTPS1 mutants (Figure 31) could indicate that this band, or at least part of the signal associated with this band originates from endogenous CTPS1. This should not have been observed, or the signal should at least have been strongly decreased with the V114F or M157I mutants if these really disrupted dimer formation. The use of another, monomeric tag appears to be the simplest approach to validate these early results and validate the association, of lack thereof, of these mutants with wild-type CTPS1.

Similarly, the G148A mutant, designed to disrupt UTP binding, is as expected inactive. While this mutant did not seem to form more filaments as reported for the S. cerevisiae enzyme (Noree et al., 2014), the presence of a few filaments was unexpected as the absence of UTP binding suggests that this mutant should not be able to form tetramers. Indeed, a N-terminal Flag-tagged form of human CTPS1 G148A was found to be unable to form filaments 125 . As previously mentioned, at least part of these discrepancies could be explained by increased polymerization in the presence of the GFP tag. CTP synthase tetramer formation in absence of substrates has been reported in multiple species (L. Lactis [START_REF] Willemoës | Substrate Inhibition of Lactococcus Lactis Cytidine 5′-Triphosphate Synthase by Ammonium Chloride Is Enhanced by Salt-Dependent Tetramer Dissociation[END_REF][START_REF] Wadskov-Hansen | Cloning and Verification of the Lactococcus Lactis PyrG Gene and Characterization of the Gene Product, CTP Synthase*[END_REF] , E. coli [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF][START_REF] Mccluskey | Biophysical Analysis of Bacterial CTP Synthase Filaments Formed in the Presence of the Chemotherapeutic Metabolite Gemcitabine-5'-Triphosphate[END_REF] and human CTPS2 [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] ). While these tetramers are probably labile species in absence of nucleotide binding, it is possible that the GFP tag could act as a "magnet" of sorts by bringing GFP-tagged subunits in closer proximity. This, however, does not explain the interaction between the GFP-tagged dimer/tetramer mutants and endogenous CTPS1 or CTPS2. It is possible that these mutations work better when two proteins (or four) carrying the same mutation try to assemble, and that the affinity between the mutated proteins and the wild-type proteins is less affected.

At this point, though, the identification of new, validated mutants that would disrupt dimer and tetramer formation would probably be the easiest path, especially in the light of the most recent structural data on human CTPS1 and CTPS2 [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] .

Another mutant of particular interest is E161K CTPS1 (mutation of the CTP binding site) that was found to form punctate structures in D. melanogaster 126 . Even though we did not observe such a localization pattern in our cellular model, recent data have shown that the D. melanogaster enzyme is able to assemble as filaments in the presence of substrates and of products, like human CTPS2 [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] . The mixed diffuse/filament pattern, similar to GFP-CTPS1, that we observed does not contradict this finding. Diffuse protein could reflect cells in which substrates levels are lower, while cells containing filaments could be cells in which substrates levels are higher, due to different proliferation states and expression levels. In this case, diffuse staining could just reflect a lack of substrates, not a CTP-induced dissociation. However, it would be interesting to validate the ability, or lack thereof in this case, of CTP/cytidine to disrupt filament formation of this mutant. It would be equally interesting to test the impact of this mutation on CTPS2 since it has been reported to be able to assemble as filaments in its substrate-and product-bound conformations, like the D. melanogaster enzyme.

Chapter 3 -Impact of N-terminal tags on CTPS1 and CTPS2 Introduction

Protein tags are frequently used to study various proteins. Most studies on CTPS filament formation use fluorescent tags, for their ease of use, especially in the case of proteins such as CTPS1 that assemble as organized structures under specific conditions. However, while these tags can be directly observed in living cells, their large sizes along with their intrinsic properties are susceptible to disturb the normal behaviour of the proteins they are fused to, modify their activity and stability, and hinder their interaction with other cellular proteins.

On the other hand, smaller tags such as His-or DYK/FLAG-tags are designed to minimize their impact on the protein of interest, sometimes offering cleavage sites for endonucleases, to remove the tag for further studies or protein purification. While they can also be used to visualize the protein localization, such uses require the detection of the tag with specific molecules such as antibodies and are used at endpoint as staining the cells to detect the intracellular tagged proteins usually requires prior fixation.

Several studies have been focused on CTPS1 variants (CTPS1Δ18 and CTPS1Δ19) and mutants that affect the C-terminal end of the protein. Additionally, the C-terminal end of CTPS1 contains multiple phosphorylation sites (Figure 22) and recent studies have highlighted the crucial role of the C-terminal domain of the CTPS enzymes [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF] . For this reason, we have generated and used N-terminal GFP, DsRed and DYK-tagged forms of CTPS1 and CTPS2 in our models. This chapter summarizes some of the findings we obtained from these studies that reveal some of the advantages, but also limitations of the use of these tagged forms.

Results

Impact of the N-terminal GFP tag on the stability and the activity of the proteins

We have used GFP-tagged forms of CTPS1 and CTPS2 enzymes as tools to visualize the localization of the proteins in transfected cells following various treatments (chapter 1 and chapter 2; Minet et al. draft).

While GFP-tagged CTPS1, CTPS1Δ18 and CTPS2 were shown to be able to restore the proliferation of double KO HEK (devoid of CTPS activity and dependent on supplementation with exogenous CTP (Minet 22), the presence of degradation bands as observed by western blot suggests that these proteins are unstable (Chapter 2). Additionally, the behaviour of these proteins appeared to be affected by the presence of the GFP tag as both GFP-CTPS1 and GFP-CTPS2 spontaneously form filaments in HEK cells (Figure 26A), which is not the case for the endogenous proteins (Figure 26B), although GFP-CTPS1 was still responsive to treatments inducing or decreasing filament formation (Figure 26A). Unmodified GFP tags dimerize 340 and that introducing a mutation that disrupts GFP dimerization in GFP-tagged CTPS1 strongly decreases filament formation [START_REF] Chang | Interfilament Interaction between IMPDH and CTPS Cytoophidia[END_REF] .

et al. draft)) (Figure
We also observed the presence of degradation bands with both GFP-tagged and DsRed-tagged CTPS proteins (Figure 38A), suggesting that both tags, of comparable molecular weight (around 27kDa), destabilize the fusion proteins. We also observed higher levels of GFP in the GFP-CTPS2 populations compared to the GFP-CTPS1 populations (Figure 38B), consistently with the observations that CTPS2 expression levels are higher in complemented populations (than that of CTPS1) regardless of the presence or not of a tag (Minet et al. draft; Figure 28). Conversely, GFP detection by western blot revealed lower levels of GFP-CTPS2 compared to GFP-CTPS1 (Figure 38C). This suggests that GFP-CTPS2 is less stable than GFP-CTPS1. We observed important variations in the activity levels of CTPS1-KO HEK stably complemented with GFP-CTPS1 and GFP-CTPS2, in contrast to control and CTPS1-KO HEK cells in which CTP synthase activity levels were rather stable and relatively homogeneous across measurements (Figure 39). These data may suggest that the GFP tag impacts the activity of the enzymes, and that this effect is at least partially mediated by the instability of the fusion proteins, although we cannot exclude that these measurements were also affected by culture conditions.

Figure 39: GFP-tagged CTPS1 and CTPS2 activity is highly variable in stable cell lines

A) Averaged values for all measurements (samples from the same group at each time point and replicates ± SEM) for each group. B) Averaged values for each group at each measurement (± SEM if replicates were available).

Generation of DYK-tagged CTPS1 and CTPS2

The observed degradation of GFP-tagged CTPS1 and CTPS2 as well as the high variability in activity levels makes difficult the comparison of their relative levels of enzymatic activity in cell extracts. To try to have a better evaluation of their activity associated with their relative expression levels, we added a small tag at the N-terminus, the DYK tag (Commercial name FLAG-tag ® , Sigma-Aldrich) consisting of 8 residues (that can be specifically recognized by commercially available monoclonal antibodies).

In the N-terminal GFP-tagged CTPS1 and CTPS2 proteins, the tag is separated from the enzyme by a hinge of 18 residues, to minimize possible steric hindrance caused by the large size of the GFP tag. In contrast, the DYK tag was directly added at the N-terminal end of CTPS1 and CTPS2 without a hinge sequence (Figure 40).

To be adequately compared, untagged, GFP-tagged and DYK-tagged forms of CTPS1 and CTPS2 were cloned in the same pLVX vector containing a mCherry reporter gene, packaged in lentiviral particules and expressed in CTPS1-KO (CTPS-null) Jurkat cells. 48h post-infection, without prior washing to avoid unnecessary stress to the cells, the cells were split in complete medium with or without cytidine and transduction efficiencies evaluated by cytometry (Figure 41). While the initial transduction rates were very high for the untagged and DYK-tagged proteins, lower transduction rates were observed for GFP-tagged CTPS1 and CTPS2. However, the lower efficiency observed for the GFP-tagged vectors can be explained by the larger size of the constructs owing to the addition of the large tag and was also reflected by lower production efficiencies of the viral particles (data not shown).

While non-transduced cells were present during the first days of culture for untagged and GFP-tagged CTPS1 and CTPS2, these cells progressively disappeared, owing to the consumption of the leftover cytidine from the original culture medium, and all mCherry-positive cell populations expanded in absence of cytidine. The maintenance of part of the transduced cells in presence of 200µM of cytidine confirmed that the selection observed was due to cytidine deprivation. Therefore, all constructs are active and restore the proliferation of CTPS1-KO Jurkat cells.

As we previously showed for both GFP-tagged and untagged CTPS2 expression in complemented CTPS1-KO and double KO HEK cells, we observed a selection towards higher levels of reporter for the GFP-CTPS2 construct compared to GFP-CTPS1 (Minet et al. draft; Figure 28; Figure 35). Surprisingly, this was not the case for DYK CTPS2, although transduction rates were also higher compared to the untagged protein, potentially nullifying the need for a further selection. Conversely, we also observed after a few passages a decrease in the reporter levels for the CTPS1 and GFP-CTPS1 populations (Figure 41, day 19, 37), as previously reported (Figure 28), suggesting that cells expressing very high levels of CTPS1

were counterselected, probably owing to a deleterious impact of by the overexpression of the protein.

Intriguingly, we did not observe such a drastic effect on cells complemented with the DYK constructs, potentially highlighting an effect of the tag on the enzymes.

N-terminal tags as tools to quantify the relative expression levels of both enzymes

We then verified the expression of the various constructs by western blot. Cell lysates were prepared at day 37 and the detection of CTPS1/CTPS2 and of the DYK/GFP tags realized in parallel on independent western blots using the same lysates. Unlike GFP-tagged proteins, DYK-tagged proteins did not show signs of degradation, confirming the lower impact of this tag on protein stability (Figure 42). The signal intensities for specific antibodies were relatively proportional to the mean fluorescence intensities (MeanFI) of the various populations, excepted for the CTPS2 staining on DYK-CTPS2 and for the GFP staining on GFP-CTPS1 (Figure 42B). The difference for GFP-CTPS1 might be explained by a better stability of the construct compared to GFP-CTPS2, similarly to what we observed in the HEK cells.

However, the source of the discrepancy for DYK-CTPS2 is unclear. As expression levels of these constructs are lower compared to the other CTPS2 constructs, the signal might be below the linear detection range of our antibody.

As previously observed and mentioned earlier, multiple bands are detected by the anti-CTPS1 and anti-CTPS2 antibodies in lysates of cells overexpressing GFP-tagged CTPS1 and CTPS2. Interestingly, the GFP-CTPS2 degradation bands are detected by the anti-CTPS2 antibody. According to the manufacturer, this polyclonal antibody is raised against the C-terminal part of CTPS2, and our data shows that it is specific for CTPS2. This suggests that its target region might be at the very end of the CTPS2 C-terminus and therefore that the N-terminus of the fusion protein, including the GFP tag, is preferentially degraded.

Our observations suggest that the DYK tag does not induce protein degradation, and the proportionality of the DYK antibody signal compared to the MFI of the DYK-CTPS2 cells (Figure 42B) indicates that the tag itself does not seem to be degraded either. The proliferation of the complemented populations in presence of cytidine or of a range of doses of the non-selective CTPS inhibitor 3-deazauridine (3-DU) was assessed to evaluate the impact of the various tags on the activity of the constructs. All of the constructs fully complemented the proliferation of the CTPS1-KO cells, as evidenced by the similar proliferation rates in presence or in absence of cytidine (Figure 44), while CTPS1-KO failed to proliferate in absence of cytidine, as previously described (Minet

et al. draft).
As previously observed (Minet et al. draft), all the populations depending on the activity of CTPS2 (and tagged forms of CTPS2) were more sensitive to a treatment with 3-DU. As our intracellular staining validated the use of mCherry levels as a surrogate marker for protein expression for this experiment (Figure 43), we compared the response levels of the cells to 40µM of 3-DU to the median fluorescence intensities of the respective populations. The response of CTPS1-KO Jurkat cells expressing the various CTPS1 constructs was proportional to their mCherry MeanFI. However, despite globally higher MeanFI compared to the CTPS1-expressing cells, all the CTPS2-expressing cells were significantly more sensitive to 3-DU. This indicates that CTPS2 is more sensitive to inhibition by 3-DU.

Conclusion and discussion

The GFP tag destabilizes the fusion proteins

Although GFP tags have provided numerous insights on the biology of proteins over the years, and in particular tremendous amounts of information on the behaviour of CTP synthases in a wide range of species, they are also unsurprisingly prone to have an impact on the functions of the proteins. This has already been illustrated by the fact that GFP self-dimerization favours filament formation [START_REF] Chang | Interfilament Interaction between IMPDH and CTPS Cytoophidia[END_REF] and by a study on the five A. thaliana CTP synthases that showed that the size and shape of filaments could be modified by the localization (N-terminus or C-terminus) of the tag (YFP) [START_REF] Daumann | Characterization of Filament-Forming CTP Synthases from Arabidopsis Thaliana[END_REF] . While the N-terminal GFP tag does not disrupt filament formation on the human enzymes as both GFP-CTPS1 and GFP-CTPS2 assemble as filaments in absence of any treatment, it is clear that overexpression of both fusion proteins modifies their behaviour as we did not observe detectable filaments with the endogenous proteins in absence of an inducing treatment with 3-DU (Minet et al. draft). Additionally, we have observed that all of our GFPtagged CTPS constructs present degradation bands after western blot analyses, and that the CTPS activity levels in stably complemented cells are highly variable. It is probable that both the GFP tag and the overexpression of the fusion protein are involved in the spontaneous filament formation, which in turn probably impacts the activity of the proteins.

It would therefore be interesting to compare the relative activities of GFP-CTPS1 and GFP-CTPS2 to GFP-tagged variants unable to assemble as filaments such as H355A to evaluate the impact of filament formation on the activity of these fusion proteins, although this would not prevent the degradation of the fusion proteins. The antibody used to detect CTPS2 targets the C-terminus of CTPS2, which is the least homologous part of both proteins, detects a large amount of degradation bands whose molecular weight is superior to that of the wild-type protein. It is therefore possible that these bands result from degradation products that preserve this domain of the protein, and that the GFP tag is preferentially degraded. Some of these products may or may not be active, which renders the comparison of protein levels on western blot to enzymatic activity levels difficult. It is possible that the degradation profiles for GFP-CTPS1 are similar, although we lack information regarding the specific region targeted by the monoclonal antibody.

Of note, we initially tried to complement these cells using linearized C1 EGFP vectors coding for GFP-CTPS1 and GFP-CTPS2 on 3 separate experiments, as previously done on CTPS1-KO and double KO HEK (Figure 30), but failed (data not shown). Even though these failures could be attributed to the low efficiency of the transfections and to a decreased ability of Jurkat cells to integrate the linearized constructs, they may also be explained by the lower expression levels of the constructs (driven by the CMV promoter, while an EF1α promoter is present in the pLVX) in conjunction with the lower stability of the constructs and potentially lower activity (Minet et al. draft).

Protein tags have no major impact on the sensitivity of CTPS1 and CTPS2 to 3-DU

Intriguingly, while the proliferation assays on CTPS1-KO and double KO HEK complemented with GFP-CTPS1 and GFP-CTPS2 showed that populations complemented with both fusion proteins were highly sensitive to inhibition by 3-DU (Minet et al. draft), we observed a different behaviour in complemented Jurkat cells (Figure 44). While, as expected, GFP-CTPS2 Jurkat cells were highly sensitive to 3-DU, GFP-CTPS1 cells appeared to have a comparable sensitivity to that of the cells complemented with CTPS1 and DYK-CTPS1 relative to the expression levels of the constructs. As the complementation approaches as well as the vectors used in the HEK and Jurkat models were different, it could be interesting to explore this discrepancy by complementing HEK cells using the same approach to understand if the variability comes from the complementation or from cell-specific factors.

The DYK tag may have an impact on the activity of CTPS1

Our results (Minet et al. draft) suggest that CTPS2 is less active than CTPS1. Indeed, it has recently been shown that human CTPS2 is five times more sensitive to CTP feedback inhibition than CTPS1 121 . To adequately compare the relative expression and activity levels of CTPS1 and CTPS2 in complemented cells, we have generated DYK-tagged forms of both proteins and transduced CTPS1-KO Jurkat cells with these constructs in parallel with wild-type and GFP-tagged forms of the proteins using concentrated viral particles. As the complementation of CTPS1-KO Jurkat cells has only been realized one time yet, these interpretations are very preliminary. All CTPS2 populations exhibited high levels of mCherry reporter that maintained over time as previously observed. However, while both wild-type and GFP-tagged CTPS1 decreased over time to stabilize at lower expression levels, suggesting that overexpression of CTPS1 might decrease the growth of the cells, DYK-CTPS1 levels remained elevated and stable over time. This suggests that the overexpression of this construct might be less deleterious than that of the wild-type protein, potentially because the activity or regulation of the protein is affected. We also had to remove the start codon of CTPS1 and CTPS2 during the addition of the tag to avoid a suspected skipping of the DYK tag start codon, which could also impact the activity of both proteins (Figure 40). However, as we used concentrated particles and a high titer of infection (MOI of 10), we cannot exclude that the lack of selection over the course of the experiment could be due to the absence of cells with lower integration levels which could be positively selected. Transducing the populations with a lower MOI would allow us to generate heterogenous populations whose evolution could be more accurately followed over time.

Use of the mCherry reporter as a surrogate for protein expression

The EF1α promoter is a strong promoter that is less prone to silencing, unlike the CMV and SV40 promoters 341 . The results presented in this chapter suggest that the mCherry reporter levels could be an adequate surrogate marker for the expression of CTPS1 and CTPS2 notwithstanding eventual stability issues of the various constructs. However, we have previously observed in some cases an unexplainable extinction of the mCherry expression (see chapter 4), which cannot be attributed to the silencing of the promoter. Indeed, the IRES (internal ribosome entry site) present between the CTPS1/CTPS2 and mCherry genes allows their expression to be driven by the same promoter and our CTPS-null Jurkat are strictly dependent on the expression of CTPS1 and CTPS2 to survive and proliferate in absence of cytidine supplementation. It is possible that spontaneous mutations in the IRES that precedes the mCherry gene could result in a loss of expression of the transgene. Even so, such mutations are expected to be rare events and the loss of the mCherry gene expression is not expected to have a significant impact on cell proliferation and to impact their selection. Still, we observed relatively large mCherry-negative cell populations in some cases (see chapter 4), which suggests that other regulatory events might be involved.

Further validation of the relationship between mCherry and CTPS1/CTPS2 expression levels would require the analysis of the expression of the transcripts by quantitative PCR.

We did not observe signs of degradation of the DYK-tagged CTPS1 and CTPS2, and this small tag is by design meant to have a minimal impact on the fusion proteins. A degradation of this tag would therefore be difficult to identify by gel migration and cannot be excluded. This question may be answered by the comparison of an intracellular staining targeting the DYK tag to the mCherry levels.

Using protein tags to understand the biology of CTPS1 and CTPS2

Our attention has been focused on the GFP and DYK protein tags. There are of course several fluorescent (DsRed, mCherry, GFP derivatives such as RFP and YFP…) and non-fluorescent (polyhistidine-tag, Strep-tag…) alternatives. The localization (C-or N-terminus), the distance between the tag and the protein and the eventual linker that separates them could all have an impact on the functionality of the protein.

For this reason, we also tried to use the signal from GFP and DYK-tagged forms of CTPS1 and CTPS2 to quantitate the relative levels of expression of endogenous CTPS1 and CTPS2 in HEK cells but failed to get a linear response (data not shown). With the appropriate antibodies, this approach could allow us to evaluate the relative contribution of CTPS1 and CTPS2 activities in single knock-out cell lines, like the HEK cell line, and to study the activity levels of each construct in complemented cell lines relative to their expression levels. Still, our results (Minet et al. draft) showing that knocking out CTPS2 has little to no effect on the global CTPS activity levels in HEK (despite the absence of compensation by overexpression of the other enzyme) suggest that the analysis of the activity of isolated enzymes does not necessarily represent the effect both of them would have in the same cell. It is possible that the relative levels of expression of each enzyme can modify the global regulation of the CTPS activity through their potential association in tetramers and filaments. One way to quantify the impact of their association would be to generate CTPS1 and CTPS2 proteins carrying the same tag for quantification purposes, but expressed in inducible expression vectors that would allow an independent, dose-dependent induction of the expression of CTPS1 and CTPS2. However, such an approach requires an extensive validation of the absence of effect of the protein tag on both enzymes and may not fully predict an eventual impact of the tag on the interaction between the two enzymes, especially in a cellular context Finally, we have confirmed the association between CTPS1 and CTPS2 by immunoprecipitation studies using GFP-tagged CTPS1 GFP-tagged CTPS2 as well as endogenous CTPS2. We currently lack an antibody that efficiently and specifically immunoprecipitates endogenous CTPS1. In the meantime, DYKtagged CTPS1 could be used, should further validation of this interaction be required. It could also allow us to improve the study of the impact of targeted mutations on the interaction between CTPS1 and potential protein partners that may be disturbed by the presence of a bulky tag such as GFP.

Chapter 4 -Isoleucine 250 is a key residue for the selective inhibition of human CTPS1 by small chemical inhibitors Introduction

Although CTP synthase-targeting compounds have been identified or generated over the years, the severe adverse effects associated with their use have limited their clinical uses. These adverse effects were attributed to the lack of specificity of these inhibitors. The recent identification and characterization of human patients with a CTPS1 deficiency has renewed the interest in targeting CTPS enzymes, notably by developing specific inhibitors for CTPS1. CTPS1 deficiency in humans causes an immunodeficiency characterized by the impaired expansion of T lymphocytes that fail to mount a protective immune response, in particular to viral infections. Thus, we hypothesised that CTPS1 may represent a target for new immunosuppressive drugs to dampen uncontrolled lymphocyte proliferation responsible of pathologies such as autoimmunity, graft versus host disease and malignant lymphoproliferation. Furthermore, recent data generated in our lab suggest that CTPS2 is involved in the basal maintenance of CTP levels, while CTPS1 may be used to meet increased CTP requirement, for instance in cells with high proliferation rates like activated T lymphocytes or lymphoma cells [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] . Therefore, a specific inhibition of CTPS1 could provide better therapeutic benefits as preserving the activity of CTPS2 necessary to preserve basal CTP pools, avoiding severe adverse effects.

Despite the high homology between human CTPS1 (hCTPS1) and CTPS2 (hCTPS2) (75,2% of identity), small inhibitory molecules exhibiting a high selectivity for human CTPS1 were recently identified by Step Pharma, a company that develops small molecules to inhibit CTPS1. Intriguingly, early data from Step Pharma with the murine enzymes showed that inhibitory compounds specific for hCTPS1 (and not for hCTPS2) were active on both murine CTPS1 (mCTPS1) and CTPS2 (mCTPS2) indicating that their selectivity was lost in mouse (unpublished data). This was rather surprising if we consider the very high degree of conservation between human and mouse CTPS1 and CTPS2 (respectively 97.8% and 90.4% of identity). Based on these preliminary observations, we have analysed in the present study the molecular determinants that drive the selectivity of these new hCTPS1-specific inhibitory compounds on in vitro enzymatic activity and cell proliferation. We have been able to demonstrate that the isoleucine residue at position 250 in hCTPS1, mCTPS1 and mCTPS2 enzymes is required for the inhibition by these compounds. Replacement of this residue by a threonine (like the one present in hCTPS2) renders the enzyme resistant to inhibition.

Results

STEP-SC1 is a selective inhibitor of CTPS1 in HEK cells

Inhibitory compounds pre-screened on purified recombinant human CTPS1 and CTPS2 enzymes for their selectivity were further validated by proliferation assays using cellular models of CTPS1 and CTPS2 deficiencies generated by CRISPR-Cas9 from HEK cells (unpublished data and Minet et al. draft). Two compounds were tested and used in the present study: STEP-STEP-SC1, a highly specific compound for CTPS1 (IC50 fold change CTPS1 versus CTPS2 >100 fold) and STEP-STEP-SS1, a compound with a less selective activity (Table 4). Wild-type HEK cells express both CTPS1 and CTPS2. Cell lines deficient for CTPS1 and CTPS2 are viable and able to proliferate in absence of CTP supplementation (Minet et al., submitted for publication).

While CTPS1-KO cells exhibit a slight proliferation defect that is rescued by cytidine supplementation, CTPS2-KO cells proliferate normally, suggesting that the activity of CTPS1 is sufficient to sustain HEK cell proliferation. We previously showed that CTPS1-KO cells, that rely on CTPS2 activity to sustain their metabolic CTP need, are more sensitive to a treatment with the specific CTPS inhibitor 3-deazauridine (3-DU), suggesting that CTPS2 might be less active, or more sensitive to this molecule, compared to CTPS1 (Minet et al., draft).

With these data in mind, we tested STEP-SC1 for its selectivity on the proliferation of control, CTPS1-KO and CTPS2-KO HEK cell lines (Figure 45). As expected from the moderate inhibition of human CTPS2 enzymatic activity (assessed on purified recombinant enzyme), the proliferation of CTPS1-KO cells was weakly inhibited by STEP-SC1, while the proliferation of CTPS2-KO cells was significantly impacted and rescued by addition of cytidine in the medium. Proliferation of control HEK, expressing both CTPS1 and CTPS2, was moderately inhibited by the compound (similarly to what is observed with CTPS1-KO cells).

These data show that the selectivity observed in vitro on the enzymatic activity of purified enzymes is translated in cells on proliferation, which is a CTPS-dependent cellular response.

Figure 45: Selective inhibition of CTPS2-KO cells by the STEP-SC1 compound

Confluency curves as percentages (%) showing the proliferation of control HEK or CTPS1 or CTPS2-deficient HEK cells (CTPS1-KO and CTPS2-KO). Confluency measurement using an IncuCyte Zoom system. Cells were seeded for 24h, then treated with the indicated concentrations of STEP-SC1 compound in presence or not of cytidine.

STEP-SC1 disrupts CTPS1 filament formation

CTP synthases have been reported to form metabolic filaments in all living reigns. We have generated CTPS1-KO HEK stably expressing N-terminal GFP-tagged CTPS1 (Minet et al. draft). Overexpressed GFP-CTPS1 spontaneously forms filaments in HEK cells, and we have shown that, in accordance with previously published data [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] , CTP supplementation through the salvage pathway using cytidine disrupts filaments formation, resulting in a diffuse localization of the fusion proteins. Conversely, treatment of the cells with 3-deazauridine (3-DU), a UTP analog and well-characterized inhibitor of CTPS activity, increases filament formation. Interestingly, we also observed that some of the selective compounds, including STEP-SC1, disrupt GFP-CTPS1 filament formation (Figure 46). This result was also observed when cells were pre-treated with 3-DU (data not shown). These results suggest that the mechanisms of CTPS1 inhibition of 3-DU and STEP-SC1 are different. We further validated the effect of STEP-SC1 on CTPS1 filament formation on control HEK. CTPS1 and CTPS2 filaments are not detectable in absence of treatment (Minet et al. draft and Figure 46B). Therefore, cells were incubated with 40µM 3-DU for 24h to induce filament formation, and then treated for an additional period of 24h with the indicated doses of cytidine or STEP-SC1. Unexpectedly, STEP-SC1 not only disrupted endogenous CTPS1 filaments, but also CTPS2 filament formation (Figure 46B). It is very likely that inhibition of CTPS2 filaments is not due to a direct effect of STEP-SC1 on CTPS2. Indeed, we have previously found that CTPS1 is required for CTPS2 to form filaments in our assay conditions. These results could suggest that STEP-SC1, when bound to CTPS1, blocks both CTPS1 and CTPS2 filament formation, which in the case of CTPS1 is expected to be the most active form of the enzyme [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] . In silico identification of a candidate residue in CTPS1 driving the selectivity of

STEP-SC1

Human CTPS1-selective molecules, including STEP-SC1 were further screened against recombinant CTPS1 and CTPS2 enzymes from commonly used model species including mouse, rat, cynomolgus monkey and dog. CTPS activity was measured using the RapidFire Mass spectrometry-based enzyme assay that detects ADP production resulting from ATP hydrolysis. Multiple CTPS1-selective compounds, including STEP-SC1, were found to have a lower selectivity against mouse and rat CTPS1 and CTPS2 compared to the human, cynomolgus monkey and dog enzymes in which they are specific for CTPS1 but not for CTPS2 (data not shown and Table 4). The sequence identities between CTP synthases from the same abovementioned species are very similar (around 75% between CTPS1 and CTPS2). However, CTPS2 appears to be less conserved between human and mouse/rat (resp. 90.4% and 90.6%) than with human and cynomolgus monkey/dog (resp. 99.3% and 96.4%) (Table 4).

As competition analyses suggested that CTPS1-selective compounds could bind near the ATP and UTP binding sites (data not shown), an in-silico modelling focused on residues within 8Å of the ATP or UTP binding domains was realized using the cryoEM structure of CTPS1 in filament form (5u03.pdb [START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] ) and the crystal structure of the synthetase domain of CTPS1 (2VO1.pdb 342 ). All non-conserved species differences were in the ATP binding site (Figure 47). Since several residues within this region could be involved in the selectivity between hCTPS1 and hCTPS2, we focused on residues that could explain the loss of selectivity in rodent enzymes. Similar

IC50s were obtained for most compounds for the CTPS1 (Table 4) but not for the CTPS2 from the tested species. Therefore, we hypothesized that a candidate residue would 1) have been conserved in CTPS1 across the species tested and 2) lost in CTPS2 in species in which the selectivity towards CTPS1 was conserved (Cynomolgus monkey and dog), but not in species in which there was no selectivity (mouse and rat), resulting in a gain of activity on CTPS2. Sequence analysis revealed that several residues around the binding sites for ATP and UTP were not conserved between CTPS1 and CTPS2 in a number of mammal species. Interestingly, the isoleucine at position 250 in CTPS1 isoleucine was only conserved in CTPS2 of rodent species, including mouse and rat, while it was replaced by a threonine in the CTPS2 of cynomolgus monkey, dog and human (Figure 47A, B and Table 5). Additional residues following the same pattern (altered in CTPS2 compared to the CTPS1 of the same species, conserved between CTPS1 and CTPS2 in rodent species) were also identified such as leucine 255, but deprioritized due to their distance with the ATP binding site (Figure 47C). Therefore, these observations suggested that the isoleucine at position 250 could be involved in the selective inhibition of CTPS1 by compound like STEP-SC1.

Table 5: List of the residues within the nucleotide binding region possibly involved in the selectivity of STEP-SC1 for hCTPS1 versus hCTPS2, mCTPS1 and mCTPS2 Only isoleucine 250 in hCTPS1 is lost both in rodent species and hCTPS2.

Obtention of Jurkat cell lines expressing the different human and murine CTPS1 and CTPS2 mutants

The CTP synthase activity is a crucial element of the cellular metabolism, and cellular models of complete CTP synthase activity deficiency like the leukemia Jurkat cell line depend on exogenous CTP supplementation to expand. We previously showed that CTPS1 is strictly required for proliferation of Jurkat cells with the obtention of CTPS1-deficient Jurkat cells (CTPS1-KO cells) by the CRISPR-Cas9 technology (Minet et al., draft). In absence of CTPS1, the proliferation of Jurkat cells is impeded and cells rapidly die. However, survival and proliferation of CTPS1-deficient Jurkat cells can be maintained by CTP supplementation in the culture medium or through gene complementation using active CTP synthase forms (Minet et al.,draft,[START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] ). Thus, we next used this model to test the effect of the isoleucine at position 250 on the selective inhibition by compounds with different selectivity. CTPS1-KO Jurkat cells were reconstituted with human or murine CTPS1 or CTPS2, in which the isoleucine 250 had been replaced or not by threonine or vice-versa (Table 6). First, human and mouse cDNAs coding for CTPS1 and CTPS2, modified or not by site-directed mutagenesis to introduce the desired mutations, were cloned into a lentiviral vector, the pLVX-EF1α-IRES-mCherry plasmid. The pLVX contains an internal ribosome entry site (IRES) that allows the expression of CTPS1 or CTPS2 along with the mCherry reporter gene to be driven by the same elongation factor 1 α (EF1α) promoter. CTPS1-KO Jurkat cells were transduced using these vectors packaged into lentiviral particles and cultured in absence of cytidine supplementation to validate the ability of the various constructs to restore cell survival and proliferation. The expression of the constructs was tested by western blot showing nearly equivalent expression of wild-type and mutant hCTPS1, mCTPS1 and hCTPS2 in the corresponding cell lines (Figure 48A). Unfortunately, we failed to detect murine CTPS2 by western blot using currently available commercially antibodies, thus the presence of murine CTPS2 in the corresponding cell lines was verified and validated by PCR and sequencing (data not shown). The list of the constructs and the name of the complemented cell lines is indicated in Table 6. Intracellular FACS staining for CTPS1 and CTPS2 confirmed the linear relationship between mCherry reporter and human and mouse CTPS1 expression (Figure 48B). However, a loss of mCherry expression over time was observed in cell lines reconstituted with human CTPS2 and CTPS2 T250I (Figure 48B andC). While the loss of mCherry expression in cells complemented with WT CTPS2 was solved by another complementation, this was not the case for CTPS2 I250T. However, the complete dependency of CTPS1-KO Jurkat on complementation with active CTPS constructs along with the western blot showing expression of the construct (Figure 48A) suggests that cells negative for mCherry expression in fact expressed CTPS2 I250T. Inhibition of the proliferation of CTPS1-KO Jurkat cells expressing CTPS1 or

Species

CTPS2 by 3-deazauridine

Many compounds targeting the activity of CTP synthases have been discovered and generated over the years. Among them, 3-deazauridine (3-DU) and its metabolite 3-DU-TP, a UTP analog 292 , was used in our lab to mimic CTPS deficiency as it specifically inhibits CTP synthases, as evidenced by the complete reversal of 3-DU induced inhibition by cytidine supplementation (Martin et al., 2014). The effect of 3-DU was assessed using the CellTiter-Blue cell viability reagent, after a 3-day exposure to the indicated concentrations of 3-DU of control Jurkat and the different complemented CTPS1-KO Jurkat cell lines cultured in complete medium in absence of cytidine. Specific inhibition of the CTPS activity was validated by cytidine supplementation. 3-DU inhibited all of our complemented cell lines at cytidine-reversible doses (Figure 49A). Interestingly, the differences in sensitivity between the cell lines complemented with CTPS1

(human and mouse, wild-type and mutant) were proportional to the mean fluorescence intensity (MFI) levels in these cells, suggesting that they were indeed proportional to the relative expression levels of CTPS1 (Figure 49B). In contrast, compared to CTPS1-expressing cell lines, all CTPS2-expressing cell lines were more sensitive to 3-DU despite higher MFI levels for CTPS2, with the exception of CTPS2

I250T-expressing cells that have lost mCherry expression (see above). This higher sensitivity of CTPS2expressing cell lines may be explained by the lower enzymatic of CTPS2 that we previously showed (Minet et al. draft). However, this could also suggest a higher selectivity of 3-DU for CTPS2 compared to CTPS1. Residue 250 is partially involved in the selectivity of the semi-selective compound

STEP-SS1

We next tested the effect of inhibitory compounds with different selectivity towards CTPS1 versus CTPS2 on the proliferation of these different cell lines. Several compounds with similar structures to that of the STEP-SC1, but with various selectivity profiles were identified. One of them, the STEP-SS1 was found to be semi-selective since it inhibited both hCTPS1 and hCTPS2 in vitro, albeit with a better selectivity for In accordance with its semi-selective profile, KO hCTPS1(WT) cells were found to be more sensitive to STEP-SS1 than KO hCTPS2(WT) (Figure 50), while both KO mCTPS1(WT) and KO mCTPS2(WT) cells exhibited similar sensitivity to STEP-SS1 and comparable to that of KO hCTPS1(WT) cells. Replacement of isoleucine 250 in hCTPS1 by a threonine decreased the sensitivity of KO hCTPS1(I250T) cells to STEP-SS1, while the change of the threonine 250 in hCTPS2 to isoleucine increased the sensitivity of KO hCTPS2(T250I) cells. Decreased sensitivity to STEP-SS1 was also observed on mCTPS1 and mCTPS2 when the isoleucine was replaced by a threonine. Thus, these data suggest that at least part of the selectivity of STEP-SS1 is mediated by the isoleucine 250 found in human CTPS1 and mouse CTPS1 and CTPS2. Isoleucine 250 when changed to threonine abolishes the specificity to the CTPS1selective inhibitor STEP-SC1

We then assessed the impact of residue 250 on the selectivity of STEP-SC1, which is highly selective for hCTPS1. STEP-SC1 was shown to be 113-fold more efficient to inhibit recombinant hCTPS1 than hCTPS2 as assessed by a RapidFire mass spectrometry measurement (Table 4). As expected, a strong inhibition of KO hCTPS1(WT) cell proliferation was observed when cells were treated with STEP-SC1, whereas, in contrast, no or a slight inhibition (at the higher doses) was observed on cell proliferation of KO hCTPS2(WT) cells (Figure 51). Similarly marked inhibition by STEP-SC1 was observed on the proliferation of KO mCTPS1(WT) and KO mCTPS2(WT) cells. The replacement of isoleucine 250 by threonine markedly decreased the sensitivity to STEP-SC1 of KO hCTPS1(I250T) , KO mCTPS1(I250T) and KO mCTPS2(I250T) cells.

However, KO mCTPS2(I250T) appeared to be more resistant than KO mCTPS1(I250T) cells to STEP-SC1. The higher mean fluorescence intensity level of KO mCTPS2(I250T) cells compared to KO mCTPS1(I250T) likely explain this increased resistance (Figure 49B). This switch effect associated with residue 250 was also observed with hCTPS2, when threonine 250 was changed to isoleucine, leading to a significant increase in sensitivity to STEP-SC1 of KO hCTPS2(T250I) cells. Therefore, these data confirm the tendency that was observed with semi-selective compound STEP-SS1 and demonstrate that the presence of an isoleucine residue at position 250 in CTPS enzymes is necessary for the inhibition by STEP-SC1. The presence of a threonine at position 250 in hCTPS2 decreased by more than 100-fold the sensitivity to STEP-SC1. Well-characterized murine models are readily available for the study of a wide range of pathologies that involved excessive or uncontrolled T-cell proliferation and responses. However, the observed lack of selectivity of STEP-SC1 for mCTPS1, that also inhibits mCTPS2 could complicate the interpretation of the effects of a such compound if tested in mice. Interestingly, structurally related compounds demonstrating an improved selectivity for recombinant murine enzymes measured with RapidFire Mass Spectrometry assay have been obtained (data not shown). When tested on cell proliferation, two of these compounds, STEP-SC2 and STEP-SC3 selectively inhibited the proliferation of KO hCTPS1(WT) and KO mCTPS1(WT) cells with no effect on the proliferation of KO hCTPS2(WT) (Figure 52, Figure 53). An inhibitory effect on proliferation of KO mCTPS2(WT) cells was only seen at higher concentrations. Intriguingly, the threonine to isoleucine residue switching in hCTPS2 moderately increased the sensitivity of KO hCTPS2(T250I) cells to STEP-SC2 compared to STEP-SC1, even though it led to a drastic resistance of KO mCTPS2(I250T) cells to inhibition by STEP-SC2 (Figure 52). Similar results were obtained with STEP-SC3 (Figure 53). Taken together, these results suggest that, while residue 250 is still an important player in the selectivity of human/murine-selective CTPS1 inhibitors, the structural modifications that led to the improved selectivity on murine enzymes may have created new interactions within the protein. Hypothetically, such a residue (or residues) would be located within the same area (although structural docking data is missing) and be different between mCTPS1 and mCTPS2, but unlike residue 250 would potentially be conserved between the CTPS2 enzymes among the tested species. Several candidates have already been identified (Table 5) and further exploration would be needed to assess the additional determinants of species selectivity between both enzymes.

Conclusion and discussion

The investigation of the precise mechanism of action of therapeutic compounds, which includes the identification of the binding sites, is an important part of the drug development process. CTPS1 appears to be an ideal target due to clinical data suggesting that its role in the organism consists in providing ample amounts of CTP in highly proliferating cells such as activated lymphocytes. In parallel, the activity of CTPS2 seems sufficient to ensure a normal development as long as the organism is not challenged by pathogens such as viruses [START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] . Although most of the data is not specifically linked to CTPS1 expression, increased CTP synthase activity has also been observed in cancer. However, the high identity (75.2%) and even higher similarity (90.6%) between CTPS1 and CTPS2 pose a significant challenge to the development of selective compounds.

Additionally, while the CTP synthases have been therapeutic targets for decades, the lack of knowledge regarding the relative contributions of CTPS1 and CTPS2 in physiology and pathology did not encourage the development of specific inhibitors for any of the two enzymes, and severe adverse effects have been associated with the use of such drugs.

Using 3-deazauridine as a CTPS-inhibiting control drug in our experiments led to the unexpected discovery that cells depending on CTPS2 expression were more sensitive to this compound than cells expressing CTPS1 (Minet et al. draft and Figure 49). Although our data regarding this specific point is limited, these results along with other, unpublished data suggest that CTPS2 might be more sensitive to 3-DU induced inhibition. Whether this is due to a lower activity of CTPS2 or to a higher selectivity of the compound due to structural specificities of this enzyme are unknown at thi<s time. Additionally, recently published data have identified a secondary CTP binding site in D. melanogaster CTPS that appears to be also present in human CTPS2 [START_REF] Zhou | Structural Basis for Ligand Binding Modes of CTP Synthase[END_REF]121 . Potentially because CTP is able to inhibit CTPS2 via both sites, CTPS2 is also five times more sensitive to CTP negative feedback. This also suggests that CTPS2 might also be more sensible to cytidine analogs such as CPEC and Gemcitabine. CTPS2 is suspected to be the housekeeping CTP synthase. If CTPS2 is more sensible to UTP, or CTP analogs, it is possible that many of the adverse effects that have been associated with the use of CTPS inhibitors could be explained by a complete or near-complete abrogation of the CTPS2 activity as higher doses were required to achieve a therapeutic efficacy on the pathology-driving enzyme, CTPS1.

Our in cellulo data on selective CTPS inhibitors is in agreement with the data recently obtained by the Kollman lab 121 . Our data were obtained using small molecule inhibitors that were different from the ones used in this study (personal communications). Enzymatic assays are invaluable tools for high-throughput screening of therapeutic compounds in the drug discovery process. However, our results in cellular models confirm that the impact of these compound on the enzymatic activity of CTPS1 as well as the modification of the selectivity profile by the modification of residue 250 is functionally translated by an impact on cell proliferation.

While cell-based assays are time-consuming and subject to higher biological variability, they can offer precious insights on the mechanism of action as well as on potential unforeseen effects of compounds of interest. For instance, while the data presented here confirms the results beforehand obtained by mass spectrometry approaches, they also highlight potential additional effects of the compounds through the disruption of CTPS1 filament formation and its impact on CTPS2 filaments in HEK cells. Although the biological relevance of this finding is unclear, this goes to show the unforeseen impact a compound can have, even those who display a high selectivity for their target, as evidenced by cytidine reversal in our model. In our case, it is possible that the disruption of CTPS2 filament formation through the disruption of CTPS1 filaments modifies the regulation of CTPS2. However, the loss of CTPS2 filaments is not expected to have a large impact on its enzymatic activity as CTPS1 and CTPS2 mutants deficient for filament formation are active both as purified enzymes [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] and functional in CTPS-null cells (Minet et al. draft).

Obviously, our model is limited by the fact that we cannot directly compare the relative proteins levels of the human and murine CTPS1 and CTPS2. While we observed a signal linearity with the detection of the human and murine enzymes relative to the level of expression of the fluorescent mCherry reporter, suggesting that the expression levels of the CTPS1 constructs could be estimated to be relatively proportional to the reporter levels, this was not the case for all CTPS2 constructs as a loss of reporter expression was observed with some constructs. Additionally, the expression of murine CTPS2 could not be assessed by western blot at the time due to the lack of adequate antibodies, even though the mCherry profiles suggest that there was no extinction of the reporter gene expression in this population. Addition of a tag could solve the quantification issue; still, we cannot exclude an impact of such a modification on the activity or on other properties of the proteins. For instance, multiple phosphorylation sites have been identified at the C-terminal domain of CTPS1 and the lack of stability of a mutant with an aberrant, alternative version of this domain coded by the exon 19 of human CTPS1 has been reported [START_REF] Martin | CTP Synthase 1 Deficiency in Humans Reveals Its Central Role in Lymphocyte Proliferation[END_REF][START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] . N-terminal tags are also susceptible to affect protein activity and regulation, as the P-loop residues 12-18

were shown to interact with the phosphate moieties of ATP and CTP/UTP [START_REF] Endrizzi | Crystal Structure of Escherichia Coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets † , ‡[END_REF][START_REF] Endrizzi | Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex † , ‡[END_REF]342 , and some conserved residues located early in the protein sequence (K2, Y3 and A20) play a crucial role in D. melanogaster CTPS filament formation 134 .

As previously reported (Minet et al. draft), we also observed different mCherry profiles between CTPS1 and CTPS2-complemented cells, suggesting that selective pressure drives the global levels of each enzyme in the cells by favouring cells with higher integration levels. As CTPS1-KO Jurkat cells depend on cytidine supplementation or complementation with such constructs, a selection of the cells over time appears to be unavoidable, until optimal enzyme levels are achieved. However, we observed similar protein expression and reporter levels in cells complemented with each enzyme and their corresponding mutant form, suggesting that the isoleucine to threonine change does not impact the activity of the protein or its ability to sustain cell proliferation. Comparing the activity levels of wild-type and mutant enzymes to further validate that the selectivity profiles that we observed cannot be attributed, at least in part, to differences in activity levels could strengthen these conclusions.

General discussion

CTPS1Δ18 is not stabilized by CTPS2 in patient cells

Recent data have suggested that filament formation might protect CTPS1 from degradation, preserving the protein pool during nutritional stress 119,125 . CTPS1 filaments are organized aggregates of thinner filaments [START_REF] Liu | Intracellular Compartmentation of CTP Synthase in Drosophila[END_REF] that might themselves result from the assembly of chains of tetramers ("fibres"). How these fibres assemble is still unclear, although multiple studies have suggested that cytoskeleton proteins might be involved 106,187 . While we have failed to detect CTPS1Δ18 filaments in complemented cells even after 3-DU induction, which could be impaired by the low levels of this mutant, expression of a N-terminal, GFPtagged form of this mutant confirmed its ability to assemble as filaments. However, these filaments presented with a distinct, more curved shape compared to CTPS1 filaments, suggesting that the structure of these filaments is affected. It is currently unknown whether this specific morphology stems from structural modifications at the tetramer level of affect the interaction between CTPS1Δ18 and potential other members of the filament structure.

As the expression of the stable, endogenous form of CTPS1 seems to stabilize this mutant in the cells of the heterozygous parents, one could assume that the overexpression of the mutant could allow the protein levels to reach an association threshold that could stabilize it as well. That did not seem to be the case with the strong overexpression of CTPS1Δ18 driven by the EF1-α promoter in CTPS1-KO Jurkat cells.

The strongly increased mCherry reporter levels in the CTPS1Δ18 population were not associated with increased CTPS1Δ18 protein levels compared to wild-type CTPS1 and CTPS1Δ19, suggesting that the protein was also unstable in this model, despite expression levels that were above that of CTPS1 in control cells (Figure 28).

Although we cannot exclude a new mode of interaction that would be independent of dimer and tetramer formation, our data strongly suggests that CTPS1 and CTPS2 might associate in the same tetramers. It remains to be seen if CTPS1Δ18 indeed associates with CTPS2 at the filament level, or even at the tetramer level (which could be evaluated using a CTPS1Δ18/H355A double mutant), and if overexpressing CTPS2 could stabilize the mutant.

However, while CTPS2 filaments were a rare occurrence in CTPS1-KO HEK as well as in HEK treated with a compound that disrupts CTPS1 filament formation (Minet et al. draft, chapter 4), we have observed that both N-terminal GFP-tagged CTPS1 (Minet et al. draft) and CTPS1Δ18 restore CTPS2 filament formation. This suggests that CTPS1Δ18 can associate with endogenous CTPS2 (interaction in which the presence of the GFP tag is not expected to play a role), although this interaction has not been verified by association studies. In spite of the fact that CTPS2 is expressed in the patient cells [START_REF] Martin | Impaired Lymphocyte Function and Differentiation in CTPS1-Deficient Patients Result from a Hypomorphic Homozygous Mutation[END_REF] , it does not seem to preserve the levels of CTPS1Δ18. Whether this is due to insufficient proteins levels of CTPS2 remains to be explored, for instance by studying the impact of a decrease or of an increase of CTPS2 levels in CTPS1Δ18-expressing cells or by complementing CTPS1-KO Jurkat cells expressing CTPS1Δ18 with CTPS2.

A minimal activity of CTPS1 seems to be required for a normal development Expression of CTPS1Δ18 in Jurkat cells also allowed us to confirm that the protein was active, both in terms of enzymatic activity and in its ability to restore the survival and proliferation of CTPS-null cells. We now know that this mutant is hypomorphic. Outside of the CTPS1Δ19 mutant reported here, no other CTPS1 deficiencies have been reported. Similarly, no CTPS2 deficiencies have been reported so far outside of an isolated case of "uncertain significance" with no experimental validation 343 . Despite their partially redundant roles and their ability to substitute for each other in our cellular models, this suggests that a minimal activity level of both proteins is essential for normal development. The high conservation of these enzymes could indicate that the slightest modifications of their sequence could have dramatic repercussions on their enzymatic activities and disrupt cell metabolism. Hence, it is possible that mutations that are restricted to a regulation domain such as the C-terminal domain of CTPS1 are the only viable ones. Even though CTPS2 can substitute for CTPS1 in our models, the expression of the transgenes here is driven by strong promoters that are not meant to be regulated by the cells. This is not the case for endogenous CTPS1 and CTPS2, and while it is possible that either protein could substitute for the other ones with appropriate expression levels, their expression is probably restrained by the regulatory mechanisms of the cells as well as by their respective regulations (see below).

Additionally, validating the activity, or lack thereof, of the CTPS1Δ18 mutant can help defining the minimal CTPS1 levels that have to be preserved to limit the occurrence of already known side effects of nucleotide inhibitors. Although the clinical data suggest that CTPS1-specific inhibitors might be a viable therapeutic approach since the immunodeficient patients have a normal development and no identified defects outside of the hematopoietic compartment, we also must consider the potential remaining CTPS1-related activity in patient cells. As we were unable to distinguish between the relative contribution of CTPS1 and CTPS2 in patient samples, our complementation approach in a cellular model devoid of any CTP synthase activity allowed us to validate that, while it is unstable, the hypomorphic mutant is active. It is therefore possible that the contribution of CTPS1Δ18 to cellular metabolism represents a baseline of minimal CTPS1 activity required to preserve most of the cellular functions. This suggests that even a highlyselective CTPS1 inhibitor should not aim for a complete extinction of the CTPS1-related activity.

Overexpression of CTPS1 and CTPS2 might be deleterious

We have observed in the case of the CTPS1, CTPS1Δ19 and to a lesser extent CTPS2 pLVX complementations that, while cells expressing high levels of the transgenes were initially selected, they were progressively counterselected towards more moderate levels (Minet et al. draft, Figure 5C). Even though we cannot exclude molecular mechanisms that would downregulate the expression of the reporter gene, this suggests that high levels of CTPS1, and of CTPS1Δ19, might decrease the growth of the cells, although we lack data regarding the evolution of the expression levels of the transgenes themselves over time. Overexpression of a GFP-tagged form of mouse CTPS has been reported to reduce cell proliferation and to impair corticogenesis in a murine model 132 , and overexpression of CTP synthases may also disrupt nucleotide pools despite the presence of regulatory mechanisms. It remains to be seen if the regulation of CTPS1Δ19 could be affected by the loss of part of its C-terminal domain, as five potentially phosphorylated residues are removed from this variant (S573, S574, S575, S578 and T581).

Post-translational modifications of CTPS1 and CTPS2

CTPS1 and CTPS2 are highly homologous, and calculated molecular weights (that do not take into account potential post-translational modifications) give a size difference of 1.01 kDa, which should result in a minute shift on our standard polyacrylamide gels (8% of polyacrylamide). The same could be said of CTPS1Δ18 (0.12 kDa) and CTPS1Δ19 (2.04 kDa). Of course, while this is a rare occurrence in bacteria 344 , we know that a significant part of proteins in the cells is subjected to post-translational modifications in eukaryotic cells and CTPS1 and CTPS2 are no exceptions.

Phosphorylation

While the results obtained during the early explorations of phosphorylation profiles of human CTPS1 and CTPS2 using yeast and bovine PKA and PKC [101][102][103] have not been confirmed by further studies in human cells (Higgins et al. 2007), multiple conserved (S574, S571, S568 although no phosphorylation of this residue has been reported on CTPS1 yet) and non-conserved residues (CTPS1 S575) are subject to phosphorylation [START_REF] Kassel | Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation[END_REF]104 . Even though the molecular weight added by the phosphorylation itself is negligible (80 Da, as assessed by mass spectrometry 345 ), it has been reported that the observed shifts of phosphorylated proteins on SDS-PAGE are due to decreased SDS binding around the phosphorylated sites 346 . The lower apparent molecular weight for CTPS1Δ18 and Δ19 could partially result from the loss of multiple candidate sites (Figure 22). Similarly, the poorly conserved C-terminal end of CTPS2 contains less phosphorylated sites, although several potentially phosphorylated candidates are present towards the N-terminal end of the protein (Table 2). As the overall sequence of CTPS1 is mostly unaffected in CTPS1Δ18 and Δ19, the loss of these phosphorylation sites is the most likely candidate to explain the size difference on SDS-PAGE. The instability of CTPS1Δ18 may be caused by the abnormal last 26

residues, that also introduce potential new phosphorylation sites (Figure 54), or to the loss of CTPS1 phosphorylation sites that could also impact its stability.

Glycosylation

We have not explored the potential glycosylation of CTPS1 and CTPS2. However, a predictive analysis of the sequences using NetNGlyc 1.0 and NetOGlyc 4.0 347 from the DTU (Technical University of Denmark) Health Tech shows that, even though there is no predicted signal peptide for both proteins, CTPS1 possesses more potential N-glycosylation sites (2 for CTPS1, 1 for CTPS2) and O-glycosylation sites (15 for CTPS1, 8 for CTPS2). As far as we know, no sites other than residue 420 N-glycosylation of CTPS1 have been experimentally verified 348 . Further exploration, including protein deglycosylation studies, would be required to validate the impact of glycosylation on both proteins. enzymes with a tag that would allow us to precisely quantitate their respective protein levels in cell extracts, an array of data suggests that CTPS2 might be also be less active than CTPS1 in a cellular context.

Even if some of our complementation approaches (transfection with linearized vectors, transduction with unconcentrated and untitered viral supernatants) may, and probably do induce biases, we have repeatedly observed that the selection of cells transduced with the CTPS2 constructs was occurring at a slower rate compared to the active CTPS1 constructs. Furthermore, they are selected towards higher expression levels of the reporter gene, suggesting that unlike CTPS1, there is a strong selective pressure that favours high levels of CTPS2 expression. While it is possible that CTPS2 is less stable than CTPS1, this suggests that the enzyme is less active, requiring higher levels of expression to fully restore the proliferation of CTPS1-KO Jurkat. Recent studies suggest that CTP feedback regulates CTPS2 activity levels more tightly than CTPS1 activity levels. This is explained by a lower IC50 for CTP that could be explained by the presence of a second CTP binding site on CTPS2 and by the propagation of conformational changes induced by CTP binding along the filament [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF]121 ,Minet et al. draft), further impeding its ability to substitute for CTPS1 in proliferating cells.

CTPS2 seems therefore less suited to the production of high amounts of CTP during proliferation spikes, and overexpression of this protein in a highly proliferating cellular model such as Jurkat cells might partially solve this issue as a "brute-force" approach.

We have also observed that cells depending on CTPS2 expression were more sensitive to a treatment with the non-selective inhibitor 3-deazauridine, even in cells in which reporter levels suggest that there is a high level of CTPS2 expression compared to their CTPS1 counterparts. Whether this is due to a better affinity of the compound for CTPS2 and/or reflects a lower enzymatic activity making it more sensitive to 3-DU is unknown. However, this data might also give us new insights. The use of CTPS inhibitors has been associated with severe adverse effects (CPEC 304 , 3-DU 337 ), but the clinical data for the CTPS1Δ18 patients suggest that low CTPS1 expression and activity levels may not have highly deleterious effects outside of the hematopoietic compartment. It is possible that CTPS2 is globally more accessible to 3-DU and CTP analogs, making it their prime target and explaining the severe adverse effects.

On one hand, CTPS1 can be overexpressed to drive CTP production in highly proliferating cells, including immune cells and cancer cells, while CTPS2 appears to be tightly regulated by CTP levels and is thus likely unable to drive a boost of CTP or high levels of CTP in highly proliferating cells. This suggests that CTPS2 acts as a housekeeper, while CTPS1 provides CTP to cells to meet increased metabolic needs, in particular during cell proliferation. Therefore, it is possible that, involuntarily, the prime target of most non-selective inhibitors is CTPS2. If CTPS1 is indeed the driver of most pathologies in which elevated CTP synthase activity levels have been reported, the high doses of inhibitor used to effectively inhibit CTPS1 probably fully abrogate the CTPS2-related activity, thereby leading to a host of deleterious effects on normal tissues and cell populations. The impact of CTPS1 and CTPS2 on cell proliferation can mostly be explained by differences in their enzymatic activities; indeed, cytidine supplementation restores the proliferation of CTPS-deficient cells and counters the effect of CTPS inhibitors. However, we cannot exclude further, undocumented effects of both proteins that may also impact cell metabolism.

Impact of the interaction between CTPS1 and CTPS2 in the regulation of both enzymes Exogenous CTPS1 and CTPS2 filaments model

While the colocalization of CTPS1 and CTPS2 at the filament level was previously observed, it was unknown whether this was mediated by a direct interaction between the enzymes or chains of tetramers (CTPS "fibres") or through partners involved in the assembly of the filaments. Our data suggests that CTPS1 and CTPS2 could be associated at the tetramer or dimer level as a mutant deficient for filament formation, H355A, failed to disrupt the interaction between CTPS1 and CTPS2. Additionally, we have observed that the co-expression of untagged CTPS1 H355A with GFP-tagged CTPS1 or CTPS2 decreased filament formation. Even though this was expected for CTPS1 as this mutant was shown to have a dominant negative effect [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF]124 , this is particularly interesting for CTPS2. As mentioned previously, GFP-CTPS2 consistently assembles as filaments in HEK cells, and the treatment of the cells with cytidine or 3-deazauridine had little to no effect. Co-expression of CTPS1 H355A with GFP-CTPS2 resulted in a clear decrease of CTPS2 filaments, with the apparition of a diffuse localization of CTPS2 which is a rare occurrence compared to CTPS1. This suggests that the interaction between CTPS1 and CTPS2 has a functional impact, at least at the filament level.

Intriguingly, while the overexpression of CTP synthases has been reported to increase filament formation, even on purified enzymes [START_REF] Ingerson-Mahar | The Metabolic Enzyme CTP Synthase Forms Cytoskeletal Filaments[END_REF][START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF] , we also observed a decrease in GFP-CTPS1 and GFP-CTPS2 filament in cells co-overexpressing untagged CTPS1 and CTPS2. However, in our assay conditions, our cells were cultured in normal culture medium (no cytidine or 3-deazauridine supplementation), as both GFP-CTPS1 and GFP-CTPS2 spontaneously assemble as filaments. As we have observed that endogenous CTPS1 and CTPS2 do not form filaments in this condition, it is possible that overexpression is not sufficient to induce filament formation of untagged proteins in this model. Untagged CTPS1 and CTPS2 expression is driven by the strong EF1-α promoter, while the expression of the GFP-tagged proteins is driven by the less efficient CMV promoter. Therefore, they are susceptible be the most expressed forms, to associate with the GFP-tagged enzymes and to dominantly drive their behaviour in co-transfected cells, decreasing filament formation. Treating co-transduced cells with 3-deazauridine to induce filament formation could allow us to validate this hypothesis.

Endogenous filament formation

While HEK cells express both CTPS1 and CTPS2, we did not detect filaments in normal culture conditions.

Although the UTP analog 3-DU consistently induced CTPS1 filament formation in control and CTPS2-KO HEK, CTPS2 filaments were a very rare occurrence in CTPS1-KO cells and only clearly detectable in control HEK. CTP and cytidine supplementation did not induce filament formation and disrupted 3-DUinduced CTPS1 and CTPS2 filaments. This suggests that, in HEK, CTPS1 drives filament formation.

Whether this is due to increased expression levels of CTPS1 compared to CTPS2 is unknown. It is possible that the expression of CTPS2 alone is under the threshold for self-polymerization, and that CTPS1 integrates CTPS2 subunits or multimers in its filament structure. It is also possible that CTPS2 has a different, potentially higher, concentration threshold for filament assembly. Still, we cannot exclude that other inducing factors such as nutritional stress could have a different impact on endogenous CTPS1 and CTPS2 filament formation.

Are CTPS2 filaments in the product-bound state physiologically relevant?

The determinants of the formation of chains of tetramers as fibres, which are expected to be the base unit of the large filaments structures that can be observed in cells, have been well-studied on the human enzymes [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] . CTPS1 and CTPS2 can form both active (substrate-bound) and inactive (product-bound) tetramers. Despite the fact that both purified CTPS1 and CTPS2 can independently assemble as filaments, CTPS1 filament formation requires active tetramers, while both active and inactive CTPS2 tetramers can form filaments. All these filaments rely on the same interaction surfaces, and CTPS2 has been demonstrated to be able to dynamically switch between active and inactive filaments depending on substrate and product availability. The rare assembly of inactive CTPS1 tetramers has been observed on the purified enzymes. However, the structural data suggests that the C-terminal domain of CTPS1 prevents the assembly of inactive tetramers into large-scale structures, while it could stabilize the assembly of active tetramers [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] .

Unlike GFP-CTPS1, GFP-CTPS2 is mostly assembled as filaments in stably transfected CTPS1-KO and double KO HEK cells in absence of any treatment. Though the GFP tag is probably involved in this behaviour, it is also highly possible that, unlike purified enzymes that can be studied in very controlled conditions, including in the total absence of nucleotides, the cellular nucleotide balance is sufficient to induce GFP-CTPS2 filaments, in particular as the enzyme is already overexpressed. Thus, the filaments we observe in the cells may have grossly similar morphologies, but may actually be different species depending on the metabolic state (in particular the UTP, ATP/CTP balance) of the cell they are in. We have yet to explore this aspect. For instance, it remains to be seen if GFP-CTPS2 filaments can induce the formation of endogenous CTPS1 filaments. If this is the case, the impact of a supplementation with cytidine/CTP on CTPS1 filaments would have to be verified. Since our results suggest that GFP-CTPS1 filaments drive the formation of endogenous CTPS2 filaments, it is conversely possible that overexpressed GFP-CTPS2, by associating with CTPS1, could induce CTPS1 filament formation. As purified CTPS2 fibres, and GFP-CTPS2 filaments can assemble in presence of CTP, would mixed CTPS1/CTPS2 filaments be maintained in presence of CTP? CTPS2 appears to be expressed along with CTPS1 in most if not all tissues (Figure 16). It is therefore probable that they also physiologically interact, and it is likely that CTPS1 tetramers integrate into the CTPS2 fibres. While this could facilitate filament formation in presence of substrates by increasing the global concentration of CTP synthases (1+2), it is possible that the conformational changes induced by product binding on CTPS1 could disrupt the fibres (Figure 56). In fact, it is suspected that the C-terminal domain is sterically incompatible with product-bound tetramer assembly as filaments, at least between CTPS1 tetramers [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] . We have observed that GFP-tagged CTPS1Δ18 assembles as filaments, which like GFP-CTPS2 do not appear to be sensitive to the addition of cytidine or 3-DU, unlike GFP-CTPS1. This suggests that the alternative C-terminal end of the protein does not prevent the formation of filaments in presence of CTP.

An association of this mutant with CTPS2 in a hypothetical hybrid filament structure could therefore result in a more stable structure that could, as reported for the CTPS2 filament, switch dynamically between the substrate and the product-bound state, also making this mutant more sensitive to CTP feedback due to the repercussion of conformational changes along the filament as previously reported [START_REF] Lynch | Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 Filaments[END_REF] .

The association between CTPS1 and CTPS2 may impact the activity of both enzymes Although we have shown that the association between CTPS1 and CTPS2 affects filament formation, the impact of their association on their regulation and activity still eludes us.

We have observed that CTPS2-KO HEK, that do not present proliferative defects, have a similar CTPS activity level compared to control HEK, while CTPS1-KO HEK have a strongly reduced activity level, yet sufficient to preserve most of their proliferation capacity. This suggests that, in presence of CTPS1, CTPS2 may have a low contribution to the total CTPS activity. However, it is also possible that, conversely, the activity of CTPS1 is reduced in control HEK. CTPS2 is highly sensitive to feedback inhibition by CTP ( 121 ,Minet et al. draft). The ratio of CTPS1 and CTPS2 in putative heterotetramers could therefore modulate the sensitivity of both enzymes to feedback inhibition. Despite the fact that the existence of such hybrid tetramers remains to be firmly established, mixing purified CTPS1 and CTPS2 proteins at various ratios to assess their activity and sensitivity to feedback inhibition could reinforce the hypothesis of a functional interaction.

Preliminary data generated by transfecting CTPS1-KO HEK (that express CTPS2) with linearized vectors (to generate stable cell lines) coding for an inactive mutant deficient for UTP binding, CTPS1 G148A along with CTPS1 E161K (no CTP binding) and CTPS1 R294D (reported to disrupt filament formation 125 ) suggest that the activity of one CTP synthase might modulate the activity of the other one. Indeed, although we did not anticipate G148A cells to be positively selected as this mutant was expected (and later confirmed in double KO HEK using a GFP-tagged form) to be inactive, we observed on two separate experiments a counter-selection of this mutant (In comparison, all of the active constructs were positively selected). This suggests that this mutant decreased the growth of the cells (data not shown). We cannot exclude a toxic effect of this mutant as, though CTP synthesis is impaired, the activity of the glutaminase domain is expected to be preserved. This could result in a decoupling of glutamine hydrolysis and to a toxic accumulation of NH3. It is also possible that the mutant associates with endogenous CTPS2, decreasing the global ability of the tetramer to bind UTP and to generate CTP, although a formal confirmation of this effect would require the comparison of CTP synthase activity levels in CTPS1-KO cells complemented or not with this mutant.

To what extent are filaments physiologically relevant in non-pathological contexts?

Self-assembly as filaments is not a unique feature of CTPS1, and it has recently been shown that many E. coli proteins are evolutively very close to developing new filament-forming properties. Whether there are bound to become filament-forming proteins, or have lost this ability is unknown, and it is possible that natural selection prevented the emergence of this new property 352 .

Unlike those proteins, the CTP synthases did not only evolve this ability, but also maintained it across all reigns despite the appearance of structural changes that affected the determinants of filament formation as well the filament interfaces, suggesting that the conservation of this ability is crucial.

Most studies rely on the exogenous overexpression of tagged forms of CTPS1 and CTPS2 that are known to induce biases. For instance, high concentrations of purified E. coli CTPS have been shown to favour filament formation [START_REF] Barry | Large-Scale Filament Formation Inhibits the Activity of CTP Synthetase[END_REF] . Overexpressed Hemagglutinin-tagged CTPS1 in HeLa cells forms filaments [START_REF] Keppeke | Assembly of IMPDH2-Based, CTPS-Based, and Mixed Rod/Ring Structures Is Dependent on Cell Type and Conditions of Induction[END_REF] .

However, no filaments could be detected in HeLa cells in normal culture conditions [START_REF] Carcamo | Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells[END_REF] . Additionally, most fluorescent tags are known for their capacity to dimerize, and the GFP tag in GFP-CTPS1 has been shown to increase spontaneous filament formation [START_REF] Chang | Interfilament Interaction between IMPDH and CTPS Cytoophidia[END_REF] .

Endogenous CTPS filaments have been observed in the drosophila larvae and their formation and localization in tissues appears to be tied to the development stage. However, no such data exist for the human enzymes. Additionally, D. melanogaster possesses a single filament-forming CTPS (dmCTPS) 129 , whose characteristics appear to be in between CTPS1 and CTPS2. dmCTPS tetramers can assemble as filaments in presence of substrates and products and possess a second noncanonical CTP binding site at the ATP site, like human CTPS2 [START_REF] Zhou | Structural Basis for Ligand Binding Modes of CTP Synthase[END_REF]121 . However, like human CTPS1, the disruption of the filament interface by the introduction of the H355A mutation decreases its activity [START_REF] Zhou | Drosophila CTP Synthase Can Form Distinct Substrate-and Product-Bound Filaments[END_REF][START_REF] Lynch | Human CTP Synthase Filament Structure Reveals the Active Enzyme Conformation[END_REF] . It would therefore be complicated to extrapolate this data to the human who possesses two CTP synthases with significant differences in their behaviour. CTPS1 filaments have been observed in tumors samples, notably ovarian, endometrial and prostate cancer and their presence was correlated with increased HSP90 (Heat-shock protein 90) levels.

Interestingly, filaments were also observed in some adjacent non-tumor tissues (bile duct in liver tissue, colon and prostate) and their presence was also correlated with increased HSP90 levels, indicative of stress 127 . However, as these tissue samples were in the vicinity of tumor samples, they may not be representative of normal tissues as we cannot exclude an impact of the transformed tissue on the surrounding "healthy" tissue due to their different metabolic needs as well as the potential secretion of factors. Nutritional stress has been reported to induce filament formation, for instance in S. cerevisiae [START_REF] Noree | Common Regulatory Control of CTP Synthase Enzyme Activity and Filament Formation[END_REF]353 , D. melanogaster 126 and human Hep-2 cells 125 . Filaments in normal culture conditions appear to be rare, even in cell lines, in absence of treatment with inhibitors. Filaments have also been observed on freshly defrosted cells, which could also reflect stress [START_REF] Carcamo | Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells[END_REF] . CTPS filaments therefore appear to be mostly an adaptation to various cellular stresses.

Additional materials and methods

Most methods can be found in the Minet et al. draft.

Primers used in this study

Mutagenesis

Site-directed mutagenesis was performed using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer instructions using the primers mentioned above.

Sequencing

The constructs were validated by Sanger sequencing using the BigDye™ Terminator v3.1 Cycle

Sequencing Kit (Life Technologies) and a 3500xL Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. Sequence analysis was performed using DNADynamo (BlueTractorSoftware).

Stable transfection with linearized C1 EGFP vectors

A single cut was introduced into the C1 EGFP vectors using the ApaLI restriction enzyme (New England Biolabs) to increase the integration rates. The vectors were then purified on High Pure PCR Product Purification Kit columns (Roche) and eluted in water. After transfection by electroporation (Gene Pulser Xcell, Bio-Rad) or lipofection (Lipofectamine 2000, Life technologies), the cells were cultured in absence of cytidine to induce selection.

List of figures

List of tables

  ................................................................................................................................................. A BIT OF HISTORY ON THE CTP SYNTHASES .......................................................................................................... IDENTIFICATION OF THE ENZYME AND THE MAIN COFACTORS AND SUBSTRATES .................................................... THE MULTIMERIC STRUCTURE OF THE CTP SYNTHASE ........................................................................................ REGULATION OF THE CTP SYNTHASE ACTIVITY ................................................................................................... STRUCTURAL STUDIES ....................................................................................................................................... THE "FILAMENT ERA" ......................................................................................................................................... CTPS1 AS A THERAPEUTIC TARGET? .................................................................................................................. STRUCTURE OF THE CTPS FAMILY OF ENZYMES ......................................................................................... DIMERIC AND TETRAMERIC STRUCTURES ............................................................................................................... FILAMENTS, CYTOOPHIDIA, OR ROD AND RINGS STRUCTURES ................................................................................. MECHANISMS OF REGULATION .............................................................................................................................. TRANSCRIPTIONAL REGULATION ......................................................................................................................... REGULATION BY PHOSPHORYLATION .................................................................................................................. REGULATION BY UBIQUITINATION ........................................................................................................................ FEEDBACK REGULATION ..................................................................................................................................... FILAMENT FORMATION ....................................................................................................................................... ASSOCIATION OF THE CTP SYNTHASES WITH PROTEINS AND PROTEIN COMPLEXES .......................... CTP AND CTP SYNTHASES IN PHYSIOLOGY .................................................................................................. CRUCIAL ROLE OF THE CTP SYNTHASE ACTIVITY DURING DEVELOPMENT ............................................................... IMPACT OF THE N-TERMINAL GFP TAG ON THE STABILITY AND THE ACTIVITY OF THE PROTEINS ........................... GENERATION OF DYK-TAGGED CTPS1 AND CTPS2 ......................................................................................... DYK-TAGGED AND GFP-TAGGED CTPS1 AND CTPS2 RESTORE THE PROLIFERATION OF CTPS1-KO JURKAT CELLS ....................................................................................................................................................................... N-TERMINAL TAGS AS TOOLS TO QUANTIFY THE RELATIVE EXPRESSION LEVELS OF BOTH ENZYMES ..................... MCHERRY REPORTER LEVELS CAN BE USED AS A SURROGATE MARKER TO EVALUATE PROTEIN EXPRESSION LEVELS ....................................................................................................................................................................... DYK AND GFP TAGS DO NOT MODIFY THE SENSITIVITY OF CTPS1 AND CTPS2 TO THE UTP ANALOG 3-DU ....... DESTABILIZES THE FUSION PROTEINS .......................................................................................... PROTEIN TAGS HAVE NO MAJOR IMPACT ON THE SENSITIVITY OF CTPS1 AND CTPS2 TO 3-DU .......................... THE DYK TAG MAY HAVE AN IMPACT ON THE ACTIVITY OF CTPS1 ...................................................................... USE OF THE MCHERRY REPORTER AS A SURROGATE FOR PROTEIN EXPRESSION ................................................ USING PROTEIN TAGS TO UNDERSTAND THE BIOLOGY OF CTPS1 AND CTPS2 ................................................... CHAPTER 4 -ISOLEUCINE 250 IS A KEY RESIDUE FOR THE SELECTIVE INHIBITION OF HUMAN CTPS1 BY SMALL CHEMICAL INHIBITORS ..............................................................................................................IS A SELECTIVE INHIBITOR OF CTPS1 IN HEK CELLS ....................................................................... STEP-SC1 DISRUPTS CTPS1 FILAMENT FORMATION ........................................................................................ IN SILICO IDENTIFICATION OF A CANDIDATE RESIDUE IN CTPS1 DRIVING THE SELECTIVITY OF STEP-SC1 ........... OBTENTION OF JURKAT CELL LINES EXPRESSING THE DIFFERENT HUMAN AND MURINE CTPS1 AND CTPS2 MUTANTS ....................................................................................................................................................................... INHIBITION OF THE PROLIFERATION OF CTPS1-KO JURKAT CELLS EXPRESSING CTPS1 OR CTPS2 BY 3-DEAZAURIDINE ................................................................................................................................................. RESIDUE 250 IS PARTIALLY INVOLVED IN THE SELECTIVITY OF THE SEMI-SELECTIVE COMPOUND STEP-SS1 ........ ISOLEUCINE 250 WHEN CHANGED TO THREONINE ABOLISHES THE SPECIFICITY TO THE CTPS1-SELECTIVE INHIBITOR STEP-SC1 ..................................................................................................................................................... MURINE CTPS1-SELECTIVE COMPOUNDS STEP-SC2 AND STEP-SC3 SELECTIVITY PARTIALLY INVOLVES RESIDUE 250 ................................................................................................................................................................. STABILIZED BY CTPS2 IN PATIENT CELLS .............................................................................. A MINIMAL ACTIVITY OF CTPS1 SEEMS TO BE REQUIRED FOR A NORMAL DEVELOPMENT ...................................... OVEREXPRESSION OF CTPS1 AND CTPS2 MIGHT BE DELETERIOUS ....................................................................

Figure 1 :

 1 Figure 1: Structure of the main nucleotidesAddition of a sugar (ribose/deoxyribose) to a nucleobase (purine or pyrimidine) results in a nucleoside/ deoxyribonucleoside. Further addition of up to three phosphate groups generates a nucleotide.

Figure 3 :

 3 Figure 3: CTPS filaments present in various shapes and thicknesses Endogenous CTPS1 filaments in HEK 293T cells induced by a 3-DU treatment: A) Curved and ring-shaped filaments. B) Forked filaments, which could indicate fusion or disassembly of larger structures. C) Filaments of various thicknesses.

Figure 4 :

 4 Figure 4: protein map of human CTPS1 Human CTPS2 shares the same structure, with a slightly shorter C-terminal domain.

Figure 6 :

 6 Figure 6: Overview of the structure and of the reactions catalyzed by CTPS1 and subsequent use of CTP for RNA and DNA synthesis Alternatively, CTP can be synthetized by the salvage pathway using cytidine generated during the degradation of nucleic acids.

Figure 7 :

 7 Figure 7: A model of large filament formationCTPS forms small aggregates, that assemble as small filaments that can fuse side-by-side or end-to-end to form massive structures. (Based onGou et al., 2014)

Figure 8 :

 8 Figure 8: Comparison of phosphorylation sites between the human, murine and yeast enzymes (Based onHiggins et al., 2007)

Figure 10 :

 10 Figure 10: CTPS2 filament formation is induced by substrate and product binding CTPS2 filaments can dynamically switch between the S-state (substrate) and the P-state (product) filaments, propagating conformational changes along the filament structure depending on substrate/product availability, allowing for a very sensitive mode of regulation. (Modified from Lynch and Kollman, 2019)

Figure 11 :Figure 12 :

 1112 Figure 11: Summary of the pyrimidine synthesis pathways (based onWang et al., 2021 and Zhou et al. 2021)

Figure 13 :

 13 Figure 13: Reactions catalyzed by some of the filament-forming enzymes found colocalized with CTPSGathering enzymes who share substrates and products might be beneficial to optimize enzymatic reactions and regulation of the enzymes.IMP:Inosine monophosphate XMP: Xanthosine monophosphate IMPDH: Inosine-5′-monophosphate dehydrogenase GMPS: Guanosine monophosphate synthetase P5CS: △1-pyrroline-5-carboxylate synthase PYRC: pyrroline-5-carboxylate reductase ASNS: Asparagine synthetase

Figure 14 :

 14 Figure 14: Pathways for the biosynthesis of phospholipids in S. cerevisiae The CDP-DG pathway is indicated in blue. (Based onOstrander et al., 1998)

  main component of most eukaryotic membranes 157 , and decreased CTPS activity levels have been reported to cause membrane defects in drosophila egg chambers 113 .

Figure 15 :

 15 Figure 15: Sialic acid conjugation requires CTP (Based onCastilho et al., 2008 168 )

  Uridine diphosphate N-acetylglucosamine ManNAc: N-Acetyl-D-mannosamine ManNAc-6-P: ManNAc-6-phosphate NeuAc-9-P: N-acetylneuraminic acid-9-phosphate Neu5Ac: N-acetylneuraminic acid PEP: Phosphoenolpyruvate GNE: UDP-GlcNAc 2-epimerase/ManNAc kinase NANS: Sialic acid synthase NANP: N-acylneuraminate-9-phosphatase CMAS: N-acylneuraminate cytidylyltransferase CST: CMP-Neu5Ac transporter ST: Sialyltransferase GalT

Figure 17 :

 17 Figure 17: Pathways for purine and pyrimidine metabolism in human cellsdCMP deaminase reaction, which represents the principal route of formation of dUMP, is not shown here. (Based onBofill et al., 1995)

Figure 18 :

 18 Figure 18: Human and CTPS2 expression data in immune cell populationsTop: expression levels of CTPS1 and CTPS2 in the different cell populations evaluated. Bottom: gating strategy used to define the assessed cell populations (Data extracted from Immgen/Immune Cell Atlas).

Figure 19 :

 19 Figure 19: Structure of ddhCTP, a naturally-ocurring antiviral CTP analogThe human enzyme Viperin consumes CTP to generate ddhCTP, an analog with antiviral properties

Figure 20 :

 20 Figure 20: Model of the organization of the filament structureThe colocalization between CTPS and IMPDH filaments suggests that they are connected through an unidentified component of the structure. (Based onChang et al., 2018)

Figure 21 :

 21 Figure 21: Observation of cytoplasmic and nuclear CTPS filaments in human cellsNuclear filaments are straight, while cytoplasmic filaments are curved.

  Through the generation, screening and optimization of banks of compounds, CTPS1-selective compounds were generated by Step Pharma. I obtained a CIFRE fellowship (Convention Industrielle de Formation par la Recherche) from the Association Nationale de la Recherche et de la Technologie (ANRT) to develop a PhD project in collaboration with Step Pharma. The first aim of the thesis project was to develop cell lines deficient for CTPS1 and/or CTPS2 to help for the screening of selective compounds for CTPS1 and use these models to better understand the biology and the relative function(s) of CTPS1 and CTPS2. The second aim of the project consisted in a structure/function study of CTPS1 by mutagenesis, which included an analysis of natural mutants of CTPS1 (associated with human CTPS1 deficiencies) and the use of mutants of CTPS1 and CTPS2 to characterize the molecular basis of the selectivity of CTPS1-selective compounds. our laboratory published the description of a cohort of 8 immunodeficient patients from the northwest of the UK suffering from recurrent Epstein-Barr virus (EBV) infections as first clinical presentation. However, some also developed some other viral and bacterial infections. A homozygous intronic mutation in the CTPS1 gene, modifying a splice donor site located at the junction of intron 17-18 and exon 17, was identified in three of these patients by whole exome sequencing, and its presence confirmed in the rest of the cohort. The consequence of this mutation is the creation of an alternative splice site that leads to the expression of an unstable form of CTPS1 in which the exon 18 is skipped and replaced by the exon 19, usually not expressed due to the presence of a stop codon in exon 18. The low protein levels of the mutant CTPS1, named CTPS1Δ18, were associated with a strong proliferation defect of activated T-blasts. Following T-cell receptor activation, normal T lymphocytes expand their CTP pool by upregulating CTPS1, dramatically increasing their global CTPS activity levels, to fuel their expansion.

Figure 22 :

 22 Figure 22: Two naturally occurring CTPS1 mutants identified in immunodeficient patients Sequence alignment of the 3 CTPS1 proteins (Coding regions). All proteins are identical up to exon 17 included. CTPS1Δ18 (c.1692-1G>C, p.T566Dfs26X) is a splicing variant while CTPS1Δ19 (c.1714_1724del, p(Gly572ProfsTer2) results from a 11bp deletion. Sequence alignment of the protein sequences. CTPS1Δ18 yields a protein with a similar length compared to WT CTPS1. The deletion present in the CTPS1Δ19 sequence results in the loss of 19 residues at the end of the protein. Both mutants result in the loss of identified (*) and predicted phosphorylation sites (°) Comparison of the structure of the 3 CTPS1 proteins. Both mutants result in the loss of the C-terminal domain containing previously described phosphorylation sites.

Figure 23 :

 23 Figure 23: CTPS1Δ19 can be expressed in HEK cells Double KO HEK were transiently transfected with pLVX vectors coding for WT CTPS1, WT CTPS2, Δ18CTPS1 or Δ19CTPS1. Verification of the transfection efficiency by cytometry. The transfection efficiencies were low, yet similar. Western blot. No differences in the expression levels of the three CTPS1 constructs were observed.

Figure 24 :

 24 Figure 24: Expression of CTPS1Δ18 mutant partially restores the proliferation of CTPS1-KO Jurkat cells Histograms representing flow cytometry analysis of CTPS1 expression in WT (gray) or CTPS1-KO (red) Jurkat cells. Isotype staining is represented by dotted lines. Immunoblots for CTPS1 and actin protein expression in lysates of WT, CTPS1-KO, or complemented CTPS1-KO Jurkat cell lines. CTPS1-KO Jurkat cells were complemented with lentivirus expressing GFP, CTPS1 plus GFP, CTPS1Δ18 plus GFP, or the empty lentivirus (shown with a slash). Black and red arrows indicate CTPS1 and CTPS1Δ18 proteins, respectively. Analysis of WT (gray histogram) and CTPS1-KO (red histogram) Jurkat cell proliferation using cell trace violet staining. Cells were treated or not with 200 μM of cytidine for 4 days. Data shown are representative of 2 independent experiments. Immunoblots for CTPS1, actin, and GFP protein expressions in lysates of CTPS1 and CTPS1Δ18 complemented CTPS1-KO Jurkat cell lines at different times of culture in the presence or not of cytidine. Same as D except that GFP protein expression was quantified by flow cytometry and represented in histograms. (cont. on next page)

Figure 25 :

 25 Figure 25: Preserved enzymatic activity of CTPS1Δ18 protein Immunoblots for CTPS1 and actin protein expression in CTPS1-KO Jurkat cell lines complemented with GFP, CTPS1Δ18 plus GFP, or CTPS1 plus GFP and maintained in culture with or without 200 μM of cytidine. Percentages of GFP+ cells of a coculture of CTPS1-KO Jurkat cells complemented with GFP (green symbols), CTPS1Δ18 plus GFP (red symbols), or CTPS1 plus GFP (gray symbols) with noncomplemented CTPS1-KO Jurkat cells at a 1:10 ratio for 16 days. Coculture was supplemented with 200 μM cytidine (triangle) or not (circle). Complemented cells were preselected for high CTPS1 expression by cytidine starvation before the coculture. Percentage values were obtained from FACS analysis.The values represent the mean ± SEM of 2-3 independent experiments.

Figure 26 :

 26 Figure 26: GFP-tagged CTPS1, CTPS1Δ18 and CTPS2 filament formation in stably transfected CTPS1-KO and Double-KO HEK cells A) Double-KO HEK cells stably expressing GFP-tagged CTPS1, CTPS2 or CTPS1Δ18 and treated 24h postplating with the indicated doses of compounds. Pictures taken using an IncuCyte live cell analysis system 24 hours post-treatment (60x IncuCyte images). B) CTPS1 or CTPS2 immunostaining on control and CTPS1-KO HEK cells stably expressing CTPS1Δ18 after 24h of culture with or without 40µM of 3-DU (40x images).

Figure 27 :

 27 Figure 27: Localization and morphological differences of filaments of GFP-tagged CTPS1, CTPS2 and CTPS1Δ18 expressed in double KO HEK cells 60x fluorescence (GFP) images of double KO HEK cells expressing GFP-CTPS1, GFP-CTPS2 and GFP-CTPS1Δ18 and cultured in complete medium in absence of cytidine. 3 fields/population.

Figure 28 :

 28 Figure 28: CTPS1Δ19 restores the proliferation of CTPS1-deficient Jurkat cells Low efficiency transduction of CTPS1-KO Jurkat with HEK viral supernatants using a pLVX vector (EF1α promoter, gene of interest and reporter linked by an Internal ribosome entry site (IRES)). A) Evolution of the viability of the cell populations over time in absence of cytidine supplementation. The viability of the cytidine-supplemented cells remained stable over time (data not shown). B) Evolution of the percentage of mCherry-positive cells supplemented with 200µM of cytidine over time. No selection occurs in absence of cytidine supplementation. (cont. on next page)

PY

  Wang et al. 2015 (Drosophila) KMKassel et al. 2015 (Human) C.Noree et al. 2014 (S. cerevisiae) McCluskey et al. 2018 (E. coli) Zhu et al. 2014 (C. glutamicum) EM Lynch et al. 2015 (Human) Aughey et al. 2014 (Drosophila) Ostrander et al. 1998 (Yeast) FA Lunn et al. 1998 (E. coli) synthesis (Mg 2+ /ATP binding site) E155K G149A/E162A (E. coli/C. glutamicum) prevents/lowers CTP binding and filament formation. K286E/E287R Could reduce filament formation without impacting activity? H355A Blockade of filament formation without affecting tetramerization filaments, no impact on activity expected (4-mers preserved) A20R Disrupts filament formation; forms foci, and filaments in presence of DON. G152E or R164H Tetramer interface mutant V114F or M157I Monomer-monomer interface mutant (disruption of dimer formation)WCLin et al. 2018 (Human)

  the filaments formed by E155K are not surprising since, unlike the bacteria, CTP binding disrupts human CTPS1 filament formation.

Figure 31 :

 31 Figure 31: Transient expression of the GFP-tagged CTPS1 mutants in double KO HEK Western blot, 48h post-transfection.

Figure 32 :

 32 Figure 32: Visualization of the various GFP-tagged CTPS mutants after transient transfection in doubleKO HEK 48h post-transfection. Cells were pre-treated with 400µm of 3-deazauridine for 4 hours to induce filament formation. 60x magnification.

Figure 33 :

 33 Figure 33: Generation of stable double KO HEK expressing the GFP-tagged CTPS1 mutants 48h post-transfection, the double-KO HEK transfected with linearized C1 EGFP vectors coding for the various GFP-tagged mutants were split and cultured with or without 200µM of cytidine. Each point represents a passage.

Figure 36 :

 36 Figure 36: CTPS1 mutants expected to decrease dimer and tetramer formation form filaments and associate with endogenous CTPS1 and CTPS2 A) Localization of the fusion proteins in control HEK, 48h post-transfection. 60x images. B) Anti-GFP immunoprecipitation of the GFP-tagged proteins followed by western blot. The black arrow marks the expected molecular weight of untagged CTPS1. C) Input and depleted fractions from B)

Figure 37 :

 37 Figure 37: SUGP1 is not associated with endogenous CTPS1 Immunoprecipitation of CTPS1 (Proteintech 15914-1-AP, Rabbit pAb, 4µg/IP) and CTPS2 (Abcam ab190462, 4µg/IP). SUGP1 is present as a faint band around 75kDa. No SUGP1 was detected in IP lanes, even with lower exposure levels.

Figure 38 :

 38 Figure 38: Expression of GFP-tagged CTP1 and CTPS2 in CTPS1-KO and Double-KO HEK cell lines A) Stable expression of GFP-or DsRed-tagged CTPS1 and CTPS2 in CTPS1-KO and double KO HEK cell lines. B) GFP levels in sorted cells after expansion. GFP-tagged CTPS2 levels are consistently higher. C) Comparison of the specific (CTPS1 or CTPS2) staining with the GFP staining in complemented CTPS1-KO cells. Multiple bands are detected for both fusion proteins, suggesting the existence of degradation products. 2 independent lysates loaded side-by-side for the GFP, GFP-CTPS1 and GFP-CTPS2 populations. D) Comparison of the mean fluorescence intensities (cytometry)/GFP signal (Western blot) from B) and C).

Figure 40 :

 40 Figure 40: Domains of the fusion proteins (CTPS1) A) Protein map of the WT and fusion proteins (to scale). B) Detail of the nucleic acid/predicted protein sequence of the region surrounded in A). The eGFP-tag is separated from the N-terminal end of the protein by a linker. The DYK tag is directly fused to the N-terminal part of the protein. DYK-tagged and GFP-tagged CTPS1 and CTPS2 restore the proliferation of CTPS1-KO Jurkat cells

Figure 41 :

 41 Figure 41: Evolution of the mCherry reporter levels in the complemented CTPS1-KO Jurkat cells cultured in presence or in absence of 200µM of cytidine CTPS1-KO Jurkat cells were infected with a MOI of 10 with pLVX vectors packaged in lentiviruses containing the indicated constructs in presence of 200µM of cytidine. At day 2, cells were diluted in medium with or without cytidine and the evolution of the population followed over time by cytometry. The DYK-tagged CTPS1 and CTPS2 are indicated by the red box.

Figure 42 :

 42 Figure 42: Expression levels of the various constructs by western blot at day 37 A) Equivalent amounts of cell lysates were loaded on two separate gels to independently assess CTPS1 and CTPS2 expression and DYK and GFP expression. Overexposed blot shown here to illustrate the presence of degradation products. B) Comparison of the signal intensity for each antibody against the mCherry geometric mean fluorescence intensity (MeanFI) for each population. MeanFI and antibody signal normalized to the highest value in each group. C) Sequence alignment of the region against which the specific anti-CTPS2 antibody is targeted. Residues 546 to the C-terminus of both proteins are poorly conserved.

Figure 43 :

 43 Figure 43: Correlation between mCherry reporter levels and CTPS1 and CTPS2 expressionAnalysis of the expression levels of CTPS1 and CTPS2 by cytometry on cell populations 37 days post-infection.

  of this figure is also present in Minet et al. draft.

Figure 44 :

 44 Figure 44: Proliferation of the control and CTPS1-KO Jurkat populations complemented with the WT andtagged constructs Cells cultured in complete RPMI in presence or in absence of the indicated doses of cytidine or 3-deazauridine (3DU). Data representative of 2 independent experiences. Proliferation after 4 days of culture in the indicated conditions. The amount of viable cells was assessed using the CellTiter-Blue reagent (Promega). Comparison of the proliferation of the various populations treated with 4µM of 3-DU at day 3 with the mCherry reporter mean fluorescence intensity.

Figure 46 :

 46 Figure 46: STEP-SC1 inhibits CTPS1 and CTPS2 filaments A) IncuCyte fluorescence images (60x) of CTPS1-KO HEK stably expressing GFP-CTPS1 and cultured with cytidine (200mM), 3-deazauridine (3-DU) (40 mM) or the indicated doses of STEP-SC1 for 72h. B) Control HEK treated for 24h with 40µM of 3-deazauridine to induce filament formation, then incubated for an additional 24h with cytidine (200mM) and the indicated concentrations of STEP-SC1. 60x images.

Figure 47 :

 47 Figure 47: Structural differences between human and murine CTP synthases at the predicted active siteA) CTPS1 and CTPS2 sequence alignments between mammals. Several differences between CTPS1 and CTPS2 are conserved among species. The human CTPS1 I250 to CTPS2 T250 change is lost in rodents. B) Additional residues not within 8Å of the ATP/UTP binding sites are also lost in rodents. C) Model of the region suspected of being involved in the different selectivity profiles between human and rodent enzymes.

Figure 48 :

 48 Figure 48: Expression of wild-type/mutant, human and murine CTPS1 and CTPS2 in reconstituted CTPS1-KO Jurkat cells A) Western blot of cell lysates from control Jurkat and CTPS1-KO Jurkat cells (CTPS1-KO) expressing or not human CTPS1 (hCTPS1) and CTPS2 (hCTPS2), murine CTPS1(mCTPS1) and CTPS2 (mCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (I250T or T250I). B) Dot-plots from FACS analyses of isotype (in red) and intracellular CTPS1 or CTPS2 (in blue) staining showing that the mCherry expression is proportional to CTPS1 or CTPS2. C) mCherry cytometry profiles for the different populations. Cells complemented with the human CTPS2 T250I have lost mCherry reporter gene expression over time but still express CTPS2 as shown in the western blot in panel A.

Figure 49 :

 49 Figure 49: Inhibition of the proliferation of cells expressing human and murine CTPS1 and CTPS2 with 3deazauridine Control Jurkat and CTPS1-KO Jurkat cells (CTPS1-KO) expressing human CTPS1 (hCTPS1) and CTPS2 (hCTPS2), murine CTPS1(mCTPS1) and CTPS2 (mCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (I250T or T250I) were treated with the indicated concentrations of 3-DU for 3 days. Proliferation assessed with the Celltiter-Blue reagent. Proliferation, shown as the percentage (%) of proliferation of untreated cells which corresponds to 100%. Values ±SEM of triplicates. Comparison of the proliferation of the various cell populations treated with 40µM of 3-DU of panel (A) with the mCherry reporter mean fluorescence intensity shown for each population from data of panel C of Figure 4. Data representative of 6 independent experiments.

Figure 50 :

 50 Figure 50: Switching the CTPS1 I250 and T250 residues modifies sensitivity to proliferation inhibition by the CTPS1 semi-selective molecule STEP-SS1 Upper panel, control Jurkat and CTPS1-KO Jurkat cells (CTPS1-KO) expressing human CTPS1 (hCTPS1) and CTPS2 (hCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 I250T or CTPS2 T250I ) were incubated with the indicated concentrations of STEP-SS1 for 3 days. Proliferation assessed with the Celltiter-Blue reagent. Triplicates values ±SEM. Middle panel, same as in the upper panel with CTPS1-KO Jurkat cells (CTPS1-KO) expressing murine CTPS1 (mCTPS1) and CTPS2 (mCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 T250I or CTPS2 T250I ) tested. Triplicates values ±SEM. Lower panel, same as in upper and middle panels, excepted that cell lines were incubated or not with the indicated concentrations of STEP-SS1 for 3 days in the presence or not (C200) of cytidine at 200mM. Proliferations shown as the percentage (%) of proliferation of untreated cells. Triplicates values ±SEM. Data representative of at least 3 independent experiments.

Figure 51 :

 51 Figure 51: Switching the CTPS1 I250 and CTPS2 T250 residues reverses the sensitivity to proliferation inhibition by the CTPS1 selective molecule STEP-SC1 Upper panel, control Jurkat and CTPS1-KO Jurkat cells (CTPS1-KO) expressing human CTPS1 (hCTPS1) and CTPS2 (hCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 I250T or CTPS2 T250I ) were incubated with the indicated concentrations of STEP-SC1 for 3 days. Proliferation assessed with the Celltiter-Blue reagent. Triplicates values ±SEM. Middle panel, same as in the upper panel with CTPS1-KO Jurkat cells (CTPS1-KO) expressing murine CTPS1 (mCTPS1) and CTPS2 (mCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 T250I or CTPS2 T250I ) tested. Triplicates values ±SEM. Lower panel, same as in upper and middle panels, excepted that cell lines were incubated or not with the indicated concentrations of STEP-SC1 for 3 days in the presence or not (C200) of cytidine at 200mM. Proliferations shown as the percentage (%) of proliferation of untreated cells. Triplicates values ±SEM. Data representative of at least 3 independent experiments.

Figure 52 :

 52 Figure 52: Switching the threonine 250 to serine reverses the sensitivity to proliferation inhibition by the murine selective molecule STEP-SC2 Upper panel, control Jurkat and CTPS1-KO Jurkat cells (CTPS1-KO) expressing human CTPS1 (hCTPS1) and CTPS2 (hCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 I250T or CTPS2 T250I ) were incubated with the indicated concentrations of STEP-SC2 for 3 days. Proliferation assessed with the Celltiter-Blue reagent. Triplicates values ±SEM. Middle panel, same as in the upper panel with CTPS1-KO Jurkat cells (CTPS1-KO) expressing murine CTPS1 (mCTPS1) and CTPS2 (mCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 T250I or CTPS2 T250I ) tested. Triplicates values ±SEM. Lower panel, same as in upper and middle panels, excepted that cell lines were incubated or not with the indicated concentrations of STEP-SC2 for 3 days in the presence or not (C200) of cytidine at 200mM. Proliferations shown as the percentage (%) of proliferation of untreated cells. Triplicates values ±SEM. Data representative of at least 3 independent experiments.

Figure 53 :

 53 Figure 53: Switching the threonine 250 to serine reverses the sensitivity to proliferation inhibition by the murine selective molecule STEP-SC3 Upper panel, control Jurkat and CTPS1-KO Jurkat cells (CTPS1-KO) expressing human CTPS1 (hCTPS1) and CTPS2 (hCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 I250T or CTPS2 T250I ) were incubated with the indicated concentrations of STEP-SC2 for 3 days. Proliferation assessed with the Celltiter-Blue reagent. Triplicates values ±SEM. Middle panel, same as in the upper panel with CTPS1-KO Jurkat cells (CTPS1-KO) expressing murine CTPS1 (mCTPS1) and CTPS2 (mCTPS2) or CTPS1 and CTPS2 mutants of residue 250 (CTPS1 T250I or CTPS2 T250I ) tested. Triplicates values ±SEM. Lower panel, same as in upper and middle panels, excepted that cell lines were incubated or not with the indicated concentrations of STEP-SC2 for 3 days in the presence or not (C200) of cytidine at 200mM. Proliferations shown as the percentage (%) of proliferation of untreated cells. Triplicates values ±SEM. Data representative of at least 3 independent experiments.

Figure 54 :

 54 Figure 54: NetPhos 3.1 analysis of potential phosphorylation sites of human CTPS1 and CTPS1Δ18 Grey/green: C-terminal domains of CTPS1/CTPS1Δ18. The sites in yellow/blue have been identified as potential phosphorylation sites by NetPhos 3.1 on CTPS1/CTPS1Δ18. The site in blue is a previously-identified phosphorylation site (T581) that was not detected by NetPhos 3.1.

Figure 55 :

 55 Figure 55: Analysis of potential N-and O-glycosylation sites of human CTPS1 and CTPS2 A) NetNGlyc 1.0 analysis. Asparagines predicted to be N-glycosylated are highlighted in red; Asn-Xaa-Ser/Thr sequons are highlighted in blue. No signal peptides were identified for either proteins. B) NetOGlyc 4.0 analysis of potential O-glycosylation sites of human CTPS1 and CTPS2.

Figure 56 :

 56 Figure 56: Integration of CTPS1 tetramers or monomers in the CTPS2 filament structure may impact the formation of product-bound filaments Unlike CTPS2, the conformational changes induced by CTP binding induce the dissociation of CTPS1 filaments. The presence of CTPS1 tetramers (left) or of CTPS1 subunits (right) might impede the dynamic switching between the substrate-bound and the product-bound CTPS2 filaments.

Figure 57 :

 57 Figure 57: The CTPS1/CTPS2 interaction may modulate CTP feedback regulation.

Figure 1 :

 1 Figure 1: Structure of the main nucleotides ............................................................................................

Figure 2 :

 2 Figure 2: Partial phylogenetic tree of CTPS from various species mentioned in this document .............

Figure 3 :

 3 Figure 3: CTPS filaments present in various shapes and thicknesses ...................................................

Figure 4 :

 4 Figure 4: protein map of human CTPS1 .................................................................................................

Figure 5 :

 5 Figure 5: Comparison of the C-terminal domains of CTPS1 and CTPS2 between different mammal species ...................................................................................................................................................

Figure 6 :

 6 Figure 6: Overview of the structure and of the reactions catalyzed by CTPS1 and subsequent use of CTP for RNA and DNA synthesis ...........................................................................................................

Figure 7 :

 7 Figure 7: A model of large filament formation ........................................................................................

Figure 8 :

 8 Figure 8: Comparison of phosphorylation sites between the human, murine and yeast enzymes .........

Figure 9 :

 9 Figure 9: CTPS filament formation is modulated by conformational changes induced by substrate and product binding ......................................................................................................................................

Figure 10 :

 10 Figure 10: CTPS2 filament formation is induced by substrate and product binding ...............................

Figure 11 :

 11 Figure 11: Summary of the pyrimidine synthesis pathways ...................................................................

Figure 12 :

 12 Figure 12: CTP needs to be converted to CDP and dCDP to be used as dCTP in DNA synthesis ........

Figure 13 :

 13 Figure 13: Reactions catalyzed by some of the filament-forming enzymes found colocalized with CTPS ...............................................................................................................................................................

Figure 14 :

 14 Figure 14: Pathways for the biosynthesis of phospholipids in S. cerevisiae ...........................................

Figure 15 :

 15 Figure 15: Sialic acid conjugation requires CTP.....................................................................................

Figure 16 :

 16 Figure 16: Human CTPS1 and CTPS2 expression data extracted from BioGPS ...................................

Figure 17 :

 17 Figure 17: Pathways for purine and pyrimidine metabolism in human cells ...........................................

Figure 18 :

 18 Figure 18: Human and CTPS2 expression data in immune cell populations ..........................................

Figure 19 :

 19 Figure 19: Structure of ddhCTP, a naturally-ocurring antiviral CTP analog ............................................

Figure 20 :

 20 Figure 20: Model of the organization of the filament structure................................................................

Figure 21 :

 21 Figure 21: Observation of cytoplasmic and nuclear CTPS filaments in human cells ..............................

Figure 22 :

 22 Figure 22: Two naturally occurring CTPS1 mutants identified in immunodeficient patients .................

Figure 23 :

 23 Figure 23: CTPS1Δ19 can be expressed in HEK cells .........................................................................

Figure 24 :

 24 Figure 24: Expression of CTPS1Δ18 mutant partially restores the proliferation of CTPS1-KO Jurkat cells .............................................................................................................................................................

Figure 25 :

 25 Figure 25: Preserved enzymatic activity of CTPS1Δ18 protein ............................................................

Figure 26 :

 26 Figure 26: GFP-tagged CTPS1, CTPS1Δ18 and CTPS2 filament formation in stably transfected CTPS1-KO and Double-KO HEK cells ..............................................................................................................

Figure 27 :

 27 Figure 27: Localization and morphological differences of filaments of GFP-tagged CTPS1, CTPS2 and CTPS1Δ18 expressed in double KO HEK cells ...................................................................................

Figure 28 :

 28 Figure 28: CTPS1Δ19 restores the proliferation of CTPS1-deficient Jurkat cells .................................

Figure 29 :

 29 Figure 29: Map of the mutations selected for this study .......................................................................

Figure 30 :

 30 Figure 30: Complementation of CTPS1-KO or double-KO HEK with GFP or N-terminal GFP-tagged CTPS1 or CTPS2 .................................................................................................................................

Figure 31 :

 31 Figure 31: Transient expression of the GFP-tagged CTPS1 mutants in double KO HEK ....................

Figure 32 :

 32 Figure 32: Visualization of the various GFP-tagged CTPS mutants after transient transfection in double KO HEK ...............................................................................................................................................

Figure 33 :

 33 Figure 33: Generation of stable double KO HEK expressing the GFP-tagged CTPS1 mutants ...........

Figure 34 :

 34 Figure 34: Transient expression of the second series of GFP-tagged CTPS1 mutants in double KO HEK .............................................................................................................................................................

Figure 35 :

 35 Figure 35: Generation of stable double KO HEK expressing the second series of GFP-tagged CTPS1 mutants ................................................................................................................................................

Figure 36 :

 36 Figure 36: CTPS1 mutants expected to decrease dimer and tetramer formation form filaments and associate with endogenous CTPS1 and CTPS2 ..................................................................................

Figure 37 :

 37 Figure 37: SUGP1 is not associated with endogenous CTPS1 ............................................................

Figure 38 :

 38 Figure 38: Expression of GFP-tagged CTP1 and CTPS2 in CTPS1-KO and Double-KO HEK cell lines .............................................................................................................................................................

Figure 39 :

 39 Figure 39: GFP-tagged CTPS1 and CTPS2 activity is highly variable in stable cell lines ....................

Figure 40 :

 40 Figure 40: Domains of the fusion proteins (CTPS1) .............................................................................

Figure 41 :

 41 Figure 41: Evolution of the mCherry reporter levels in the complemented CTPS1-KO Jurkat cells cultured in presence or in absence of 200µM of cytidine .....................................................................

Figure 42 :

 42 Figure 42: Expression levels of the various constructs by western blot at day 37 ................................

Figure 43 :

 43 Figure 43: Correlation between mCherry reporter levels and CTPS1 and CTPS2 expression .............

Figure 44 :

 44 Figure 44: Proliferation of the control and CTPS1-KO Jurkat populations complemented with the WT and tagged constructs ..........................................................................................................................

Figure 45 :

 45 Figure 45: Selective inhibition of CTPS2-KO cells by the STEP-SC1 compound .................................

Figure 46 :

 46 Figure 46: STEP-SC1 inhibits CTPS1 and CTPS2 filaments ...............................................................

Figure 47 :

 47 Figure 47: Structural differences between human and murine CTP synthases at the predicted active site .............................................................................................................................................................

Figure 48 :

 48 Figure 48: Expression of wild-type/mutant, human and murine CTPS1 and CTPS2 in reconstituted CTPS1-KO Jurkat cells ........................................................................................................................

Figure 49 :

 49 Figure 49: Inhibition of the proliferation of cells expressing human and murine CTPS1 and CTPS2 with 3-deazauridine .....................................................................................................................................

Figure 50 :

 50 Figure 50: Switching the CTPS1 I250 and T250 residues modifies sensitivity to proliferation inhibition by the CTPS1 semi-selective molecule STEP-SS1 ..................................................................................

Figure 51 :

 51 Figure 51: Switching the CTPS1 I250 and CTPS2 T250 residues reverses the sensitivity to proliferation inhibition by the CTPS1 selective molecule STEP-SC1 .......................................................................

Figure 52 :

 52 Figure 52: Switching the threonine 250 to serine reverses the sensitivity to proliferation inhibition by the murine selective molecule STEP-SC2 .................................................................................................

Figure 53 :

 53 Figure 53: Switching the threonine 250 to serine reverses the sensitivity to proliferation inhibition by the murine selective molecule STEP-SC3 .................................................................................................

Figure 54 :Figure 55 :

 5455 Figure 54: NetPhos 3.1 analysis of potential phosphorylation sites of human CTPS1 and CTPS1Δ18 Figure 55: Analysis of potential N-and O-glycosylation sites of human CTPS1 and CTPS2 ...............

Figure 56 :

 56 Figure 56: Integration of CTPS1 tetramers or monomers in the CTPS2 filament structure may impact the formation of product-bound filaments ...................................................................................................

Figure 57 :

 57 Figure 57: The CTPS1/CTPS2 interaction may modulate CTP feedback regulation. ...........................

Table 2 : Potential phosphorylation sites of human CTP synthases Brackets indicate lower probability sites. Data extracted from the Phosida database

 2

Table 4 : Inhibitory characteristics of the selective CTPS1 inhibitor STEP-SC1 analysed on enzymatic activity

 4 Activity measured by RapidFire MS assay on recombinant CTPS1 from different species.

Table 6 : List of CTPS1-KO Jurkat cell lines reconstituted with murine or human wild-type or mutant CTPS1 and CTPS2 used in the study

 6

Table 7 : Calculated amino acid lengths and predicted molecular weights for CTPS1, CTPS2 and CTPS1 mutants affecting the C-terminal domain

 7

Table 1 :

 1 Names commonly used for CTPS family members in different species ...................................

Table 2 :

 2 Potential phosphorylation sites of human CTP synthases ........................................................

Table 3 :

 3 List of the mutations selected for this study ............................................................................

Table 4 :

 4 Inhibitory characteristics of the selective CTPS1 inhibitor STEP-SC1 analyzed on enzymatic activity ..................................................................................................................................................

Table 5 :

 5 List of the residues within the nucleotide binding region possibly involved in the selectivity of STEP-SC1 for hCTPS1 versus hCTPS2, mCTPS1 and mCTPS2 .......................................................

Table 6 :

 6 List of CTPS1-KO Jurkat cell lines reconstituted with murine or human wild-type or mutant CTPS1 and CTPS2 used in the study ..................................................................................................

Table 7 :

 7 Calculated amino acid lengths and predicted molecular weights for CTPS1, CTPS2 and CTPS1 mutants affecting the C-terminal domain ..............................................................................................

The relationship between CTPS and cell cytoskeleton appears to be rather one-sided, with multiple components of the cytoskeleton likely involved in the assembly of CTPS filaments, potentially impacting their regulation and localization.

Article draft: Essential and differential roles of CTP synthetase 1 and 2 to promote cell proliferation Supplementary results mCherry reporter levels can be used as a surrogate marker to evaluate protein expression levels

The pLVX vector used for complementation contains an internal ribosome entry site (IRES) that allows the expression of the CTPS1 or CTPS2 and of the mCherry reporter gene to be driven by the same promoter. In theory, this could allow the use of reporter expression as a surrogate for protein expression as it is suggested by the analysis in Figure 43.

When analyzed by anti-CTPS1 or anti-CTPS2 intracellular staining, the cells complemented with CTPS1 and CTPS2 constructs show a linear relationship between the mCherry and the CTPS1 expression levels (Figure 43). However, the CTPS2 signal appeared less clearly defined than the CTPS1 signal; this might be explained by the nature of the antibodies as the anti-CTPS1 antibody is monoclonal, while the anti-CTPS2 is polyclonal. It is also possible that there is a variable accumulation of the exogenous proteins in the cells due to mechanisms leading to the stabilization of to the destabilization of CTPS1 or CTPS2 (like filament formation), yet this does not seem to be the case here. In this experiment, the presence of a CTPS1-positive population in the GFP-CTPS2-complemented cells appears to be a technical issue as this protein was not detected in cell lysates (Figure 42).

Ubiquitination

Ubiquitination of CTPS1 has been reported in multiple species including S. cerevisiae 114 , D. melanogaster 115,116 and H. sapiens 117 . Ubiquitin is an 8.5 kDa molecule that can be conjugated to proteins.

While it was initially thought that the role of ubiquitination was to address proteins for degradation in the proteasome, it is now known that this modification is also involved in the regulation of several cellular processes by modulating the activity of enzymes or their interaction with other proteins 349 . Mathematically, the addition of a single ubiquitin to CTPS1 would be sufficient to shift its molecular weight towards the ~75 kDa frequently observed on polyacrylamide gels. Yet, data on the impact of ubiquitination on CTP synthases are scarce. D. melanogaster CTPS was found to be ubiquitinated in Drosophila S2 cells (a macrophage-like lineage), but not in salivary glands and ovaries, suggesting that its ubiquitination profiles might be tissuedependent. It was suggested that the E3 ubiquitin ligase Cbl positively regulated dmCTPS filament formation and activity. However, the lack of ubiquitination of dmCTPS in some tissues in which filament formation was affected by the absence of Cbl suggested that at least part of this modulation happens through unidentified partners 115 . It appeared that some degree of protein ubiquitination was required for nutritional stress-induced CTPS filament formation in drosophila S2 and human HEp2 cells, a cell line often described as a laryngeal carcinoma cell line but suspected to be an HeLa derivative (cervical cancer cell line) 350 . Yet, filament formation was associated with a decreased ubiquitination of dmCTPS. Taken together, their results suggested that, in normal culture conditions, dmCTPS was monoubiquitinated 116 .

Nutritional stress induced the deubiquitination of dmCTPS but potentially induced the ubiquitination of potential partners, favouring filament formation. Interestingly, monoubiquitination is a form of ubiquitination mostly associated with the regulation of protein-protein interactions 351 , suggesting that ubiquitination might regulate the interaction between CTPS and its potential partners, or modulate its ability to assemble as filaments.

Conversely, it has also recently been reported that filament formation may protect murine CTPS1 from degradation through the ubiquitin-proteasome pathway by decreasing its ubiquitination 119 . Induction of filament formation was associated with increased protein levels, while filamentation-deficient mutants had a shorter half-life.

CTPS2 may be less active than CTPS1 in cells

Enzymatic activity measurements on purified enzymes suggest that CTPS2 is less active (Minet et al. draft,121 ). Although we do not have enzymatic measurements of the relative activities of both human

Final words

In many regards, CTP synthases are fascinating enzymes. Four of the main nucleotides (ATP, UTP as substrates, GTP as an allosteric effector and CTP as the final product) regulate their activity, to produce the most limiting nucleotide in the cell, CTP. The ubiquitous, yet still mysterious CTPS filaments have evolved in different ways to integrate the CTPS tetramers in various conformations, active and inactive, and potentially multiple CTP synthases from the same species. The potential interaction between CTP synthases in some species may result in a whole new level of regulation. CTPS1 may even have new, unexpected functions as recent data suggest that CTPS1 can deaminate IRF3 to modulate the innate immune response.

Thanks to the recently illustrated key differences between the behaviour of human CTPS1 and CTPS2, we now know that, while discrepant data obtained in other species do not necessarily indicate that these are unreliable, they have to be taken with care. Similarly, while most studies focus on CTPS1, the high homology between both enzymes along with data obtained in our lab may also mean that some of the results obtained using anti-CTPS1 antibodies may have involuntarily included CTPS2 in their findings.

The development of specific antibodies as well as studies taking into account the potential interaction of both enzymes may allow us to get a better understanding of the specific roles of CTPS1 and CTPS2 in the organism.

Annexes