Laurence Pibouin-Fragner

Anne Eichmann
email: anne.eichmann@inserm.fr

Luc Pardanaud
email: luc.pardanaud@inserm.fr

Environmental and intrinsic modulations of venous differentiation

Endothelial cells in veins differ in morphology, function and gene expression from those in arteries and lymphatics. Understanding how venous and arterial identities are induced during development is required to understand how arteriovenous malformations occur, and to improve the outcome of vein grafts in surgery by promoting arterialization of veins. To identify factors that promote venous endothelial cell fate in vivo, we isolated veins from quail embryos at different developmental stages that were grafted into the coelom of chick embryos. Endothelial cells migrated out from the grafted vein and their colonization of host veins and/or arteries was quantified. We show that venous fate is promoted by sympathetic vessel innervation at embryonic day 11. Removal of sympathetic innervation decreased vein colonization, while norepinephrine enhanced venous colonization. BMP treatment or inhibition of ERK enhanced venous fate, revealing environmental neurotransmitter and BMP signaling and intrinsic ERK inhibition as actors in venous fate acquisition.

We also identify the BMP antagonist Noggin as a potent mediator of venous arterialization.

Introduction

Endothelial cells (ECs) form the inner lining of all blood vessels in the vertebrate body. They constitute an essential and dynamic interface between the blood and the tissues. ECs develop phenotypic heterogeneity that is required for proper vascular function [START_REF] Aird | Endothelial cell heterogeneity[END_REF], as manifested already early in embryonic development when arteries, veins and capillaries differentiate to form the circulatory system. Lack of embryonic arterio-venous specification constitutes a lethal bottleneck in human pregnancies, while defective arterio-venous specification leads to debilitating arteriovenous malformations (AVMs) in human patients, underscoring the importance of understanding mechanisms of arterio-venous specification. Single cell RNA sequencing data are providing molecular insight into EC heterogeneity and arteriovenous differentiation, and available gene expression profiles clearly distinguish arteries, capillaries and veins from each other [START_REF] Kalluri | Single-Cell Analysis of the Normal Mouse Aorta Reveals Functionally Distinct Endothelial Cell Populations[END_REF][START_REF] Kalucka | Single-Cell Transcriptome Atlas of Murine Endothelial Cells[END_REF][START_REF] Vanlandewijck | A molecular atlas of cell types and zonation in the brain vasculature[END_REF][START_REF] Zarkada | Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation[END_REF]. However, it remains unknown how this information can be leveraged to promote EC specification towards a venous or an arterial fate.

The vascular system maintains tissue homeostasis while accommodating for tissue growth and repair, changing nutritional demands, and hemodynamic fluctuations. To adapt to such changes, ECs within fully perfused vessels rearrange and migrate, while maintaining vessel integrity, as shown by live imaging and lineage tracing experiments [START_REF] Xu | Arteries are formed by vein-derived endothelial tip cells[END_REF][START_REF] Franco | Dynamic endothelial cell rearrangements drive developmental vessel regression[END_REF][START_REF] Park | Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia[END_REF][START_REF] Lee | Role of Venous Endothelial Cells in Developmental and Pathologic Angiogenesis[END_REF][START_REF] Rosa | WASp controls oriented migration of endothelial cells to achieve functional vascular patterning[END_REF][START_REF] Yu | Single-cell analysis of endothelial morphogenesis in vivo[END_REF]. Blood flow drives arterio-venous specification together with biochemical and epigenetic changes, by promoting EC polarization and migration against the direction of blood flow, such that high flow vessels become stabilized whereas poorly perfused and redundant segments regress. The ability of ECs to sense and polarize against the direction of flow, and the resulting migratory behavior is termed flow-migration coupling.

Flow-migration coupling implies that ECs switch between venous, capillary and arterial gene expression profiles as they migrate against the direction of blood flow. Indeed, defective flow-migration coupling leads to AVMs in mice and zebrafish carrying deletions of the Acvrl1 gene [START_REF] Park | Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia[END_REF][START_REF] Rochon | Alk1 controls arterial endothelial cell migration in lumenized vessels[END_REF], indicating that flow-migration and arterio-venous specification are linked through ACVRL1 signaling. ACVRL1 mutations in human patients cause hereditary hemorrhagic telangiectasia (HHT), a debilitating disease characterized by AVMs that are fragile and prone to bleeding, suggesting implication of this mechanism in HHT patients. A conversion from venous to arterial ECs was induced experimentally by flow manipulations in chick embryos [START_REF] Noble | Flow regulates arterial-venous differentiation in the chick embryo yolk sac[END_REF]. Interestingly, flow-migration coupling and vein to artery conversion occurs during normal coronary vessel development in mice [START_REF] Su | Single-cell analyses of early progenitor cells that build coronary arteries[END_REF][START_REF] Luo | Arterialization requires the timely suppression of cell growth[END_REF], suggesting that this process could be promoted to improve coronary vessel function after myocardial infarction. However, venous bypass grafts subjected to arterial flow in animal models show loss of vein identity, but fail to induce artery gene expression, and coronary artery bypass grafts using saphenous veins are more likely to fail than those using radial arteries [START_REF] De Vries | Vein graft failure: from pathophysiology to clinical outcomes[END_REF][START_REF] Hashimoto | Membrane-mediated regulation of vascular identity[END_REF][START_REF] Isaji | Improving the Outcome of Vein Grafts: Should Vascular Surgeons Turn Veins into Arteries?[END_REF]. Taken together, a better understanding of the signals that determine artery and vein identity may allow manipulation of these fates in the adult circulatory system and lead to improved clinical outcomes.

The factors governing arterial specification have been extensively studied by many investigators [START_REF] Luo | Arterialization requires the timely suppression of cell growth[END_REF][START_REF] Chen | Developmental Perspectives on Arterial Fate Specification[END_REF][START_REF] Fernández-Chacón | Role of Notch in endothelial biology[END_REF][START_REF] Marziano | Vascular endothelial cell specification in health and disease[END_REF][START_REF] Simons | Molecular controls of arterial morphogenesis[END_REF][START_REF] Raftrey | Dach1 Extends Artery Networks and Protects Against Cardiac Injury[END_REF], revealing a pathway whereby secreted factors such as Vascular Endothelial Growth Factor (VEGF) promote arterial specification by activating the extracellular signal-regulated kinases (ERK) pathway, in concert with shear-stress induced Notch activation of Myc [START_REF] Luo | Arterialization requires the timely suppression of cell growth[END_REF]. However, factors inducing venous specification remain largely elusive. The COUPTFII transcription factor is considered a major determinant of venous fate, since mice lacking its expression lose expression of venous genes and acquire expression of arterial genes [START_REF] You | Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity[END_REF]. Furthermore, BMP signaling emerged as a potent inducer of venous angiogenesis [START_REF] Kashiwada | -catenin-dependent transcription is central to Bmp-mediated formation of venous vessels[END_REF][START_REF] Lee | Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis[END_REF][START_REF] Neal | Venous identity requires BMP signalling through ALK3[END_REF][START_REF] Tischfield | Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura[END_REF][START_REF] Wiley | Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein[END_REF].

Here we used quail-chick chimera grafts to identify factors driving venous differentiation. Veins from quail embryos at different developmental stages were grafted into the coelom of chick embryos at embryonic day 2 (E2), near the developing cardinal vein and dorsal aorta. ECs migrated out from the graft and colonized host veins or arteries, which was quantified by staining with an antibody that selectively labels quail ECs. We found that the sympathetic neurotransmitter norepinephrine (NE) and BMP pathways, as well as inhibition of ERK signaling affect venous differentiation.

Results

Venous differentiation in avian chimeras

We micro-dissected external jugular veins (JVs) from embryonic (E5-E15) and postnatal day (P) 15 quails and grafted them into the coelomic cavity of 2 day-old chick embryos (Fig. 1A). The grafted vessel fragments attached to the coelomic angle (Fig. 1B) and graft-derived ECs colonized chick vessels. ECs originating from the grafted vessel could be identified using the Quail hemopoietic 1 antibody (QH1) specific for quail ECs and additional markers such as Sambucus nigra lectin recognizing all avian ECs [START_REF] Hagedorn | Accessing key steps of human tumor progression in vivo by using an avian embryo model[END_REF], and connexin 40 (CX40) recognizing only arterial ECs (Fig. S1). We quantified QH1+ ECs colonizing host arteries and veins 48h after grafting on serial sections and 3D reconstitutions (Fig. 1C,D, [START_REF] Andrey | Free-D: an integrated environment for threedimensional reconstruction from serial sections[END_REF]). The total number of ECs emigrating from any given graft was set at 100%, and percentages of ECs found in host arteries and veins were calculated for each graft (see methods).

Interestingly, the majority (>80%) of QH1+ ECs emigrating from E15 or posthatching vein grafts was found in host veins, while only a small percentage of cells (<20%) colonized host arteries (Fig. 1C,E). QH1+ ECs were mainly found in the cardinal, umbical and omphalomesenteric veins (Fig. 1C). By contrast, when veins were isolated from embryos younger than E11 and implanted into E2 chick hosts, around 50% of the grafted cells also colonized host arteries (Fig. 1D,E). Triple staining with QH1, the Sambucus nigra lectin and an anti-CX40 antibody showed that quail ECs integrating host arteries maintained CX40 expression while those integrating veins lost CX40 expression (Fig. S1).

Because the overall number of ECs originating from grafted E15 or E8 veins was similar (Fig. 1F), it appeared that from E11 onwards, venous cells received signals driving them towards venous fate and prohibiting colonization of host arteries.

We reasoned that this assay could allow testing of factors that promote venous or arterial fate.

Sympathetic innervation regulates venous differentiation

E11 corresponds to the onset of innervation of the JV vessel wall by sympathetic nerves [START_REF] Rouwet | Hypoxia induces aortic hypertrophic growth, left ventricular dysfunction, and sympathetic hyperinnervation of peripheral arteries in the chick embryo[END_REF][START_REF] Ruijtenbeek | Chronic hypoxia stimulates periarterial sympathetic nerve development in chicken embryo[END_REF], suggesting potential implications of catecholamines in venous differentiation. We performed glyoxylic acid staining to detect catecholamine release by perivascular nerve terminals [START_REF] Noble | Contractile and relaxing reactivity in carotid and femoral arteries of chicken embryos[END_REF] and observed no staining of E8 veins, while E11 and E15 veins were clearly labeled with glyoxylic acid (Fig. 2A-C). Hence, venous innervation temporally correlated with acquisition of vein-specific colonization.

To test if innervation modulated venous identity, we performed a series of loss of function approaches. As opposed to E15 JVs, grafting of non-innervated E15 chorioallantoic membrane (CAM) veins into the coelom of an E2 chick embryo led to colonization of host arteries as well as veins (Fig. 2D,E). When E15 venous ECs were isolated from the vessel wall and cultured overnight, they failed to exhibit veinonly colonization and >40% colonized arteries (Fig. 2E). Likewise, when the innervated adventitia from E15 veins was surgically removed prior to grafting, venous colonization was reduced and arterial colonization was enhanced (Fig. 2E).

Next we tested effects of removing innervation by chemical treatment and by surgical ablation. Chemical denervation was achieved using 6HDOPA [START_REF] Lot | The consequences of loss followed by recovery of noradrenergic nerve function on muscarinic receptors in the chick expansor secundariorum muscle[END_REF], and a 3h30min treatment of isolated vein fragments efficiently depleted glyoxylic acid staining of sympathetic nerves on the JV wall (Fig. 2F,G). The absence of nerves induced a >30% reduction in venous colonization and enhanced colonization of arteries by the grafted denervated E15 and post-hatching JV ECs (Fig. 2J).

Surgically, we ablated neural crest derivatives including peripheral nerves by removing the neural tube and notochord from E2 quail embryos after the 5 th somite level [START_REF] Teillet | Consequences of neural tube and notochord excision on the development of the peripheral nervous system in the chick embryo[END_REF], (Fig. 2H). Vascular innervation was successfully removed, as attested by decreased glyoxylic acid staining of isolated E15 JVs (Fig. 2I), and we observed colonization of arteries by >35% of ECs from grafted denervated veins (Fig. 2J).

Sympathetic neurotransmitter regulation of venous fate

Since denervation decreased venous fate of EC JVs, we asked if sympathetic neurotransmitters could enhance venous colonization of grafted E8 JVs. We tested NE, which is produced by sympathetic nerve terminals and signals through 1 and 2 adrenoreceptors (ADR). NE significantly enhanced venous colonization of E8 jugular vein grafts (Fig. 3A). To identify ADRs involved in this process, we used QPCR analyses of JV fragments to determine changes in ADR transcripts between E8 and E15 veins. Among six ADR transcripts, ADR2A did not change at E15 and ADR1A and ADR2C expression decreased (not shown), while ADR1B, ADR1D, and ADR2B increased at E15 compared to E8 (Fig. 3B). We considered these receptors as potential mediators of venularization, and tested their implication using well characterized and specific pharmacological receptor agonists (see Table 1). We reasoned that treatment of E8 JVs with relevant agonists should mimic effects of NE on venularization (Fig. 3A). ADR2 agonists (BHT933 10 -6 M) indeed enhanced vein colonization, while ADR1 agonists (phenylephrin, PE, 150nM) did not (Fig. 3C,D).

The changes in vein colonization induced by BHT933 were observed at concentrations between 10 -8 and 10 -6 M and did not affect the number of ECs emigrating from grafted veins (Fig. S2), indicating that treatment affected cell fate rather than cell proliferation. Furthermore, 10 -6 M BHT933 treatment of isolated E15 JV ECs enhanced venous colonization, while PE had no effect (Fig. 3D), indicating that ADR2 activation in ECs was sufficient to induce venous fate.

ADR2 activates Gi and inhibits downstream adenylate cyclase (AC) and

Protein kinase A (PKA), suggesting that inhibition of AC and PKA should mimic venous colonization. Indeed, treatment of E8 jugular vein fragments with the pharmacological AC inhibitor SQ22536 or the PKA inhibitor KT5720 both enhanced venous colonization (Fig. 3D).

These data are consistent with a model whereby sympathetic nerve release of NE stimulates venous fate via ADR2 activation, and show that inhibition of AC and PKA downstream of adrenoreceptor signaling promotes venous fate (Fig. 3C).

ERK signaling antagonism and Bone Morphogenetic Proteins (BMPs) promote venous fate

ERK activation promotes arterialization of embryonic vessels [37], suggesting that ERK inhibition might be necessary to promote venous colonization. QPCR analysis of quail Mapk1 (ERK2), the only ERK isoform in the avian genome, showed that Mapk1 transcription was lower in E15 JVs than in E15 aortae (Fig. 4A).

Furthermore, examination of p-ERK levels in JVs showed decreased ERK activation at E15 when compared to E8 (Fig. 4B). Pharmacological ERK inhibition with U0126 had no effect on E15 JVs (Fig. 4C). As ERK phosphorylation is regulated by five known phosphatases (MKP3/DUSP6, MKP4, PP2A, PAC1 and HePTP), we sought to address whether enhancing ERK activation via phosphatase inhibition could modulate venous differentiation. Sodium Orthovanadate (SOV, pan phosphatase inhibitor), (E/Z)-BCI (MKP3/DUSP6 inhibitor), Fostriecin (PP2A inhibitor), Salubrinal (PAC1 inhibitor) and ML119 (HePTP inhibitor), all induced arterial colonization of grafted QH1+ ECs from E15 veins (Fig. 4D). Combined treatment with phosphatase inhibitors + U0126 reduced arterialization (Fig. 4D), suggesting that ERK contributed to arterialization of JVs. These data suggest that ERK inhibition enhanced vein colonization and acquisition of venous fate.

BMPs have recently been shown to induce venous differentiation in zebrafish and mouse embryos [START_REF] Kashiwada | -catenin-dependent transcription is central to Bmp-mediated formation of venous vessels[END_REF][START_REF] Neal | Venous identity requires BMP signalling through ALK3[END_REF][START_REF] Wiley | Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein[END_REF], prompting us to test effects of BMP stimulation of E8 JVs. Analysis of BMP receptor expression showed an increase of Alk2 in E15 JVs when compared to E8, a trend to increase for Alk1, a trend to decrease for Alk6, while Alk3 was dramatically downregulated (Fig. 5A-D). We next tested BMP2, 4, 6, 7, 9 and 10 (R&D Systems) on E8 JVs at different concentrations. High concentrations (10-100ng/ml) of BMPs did not dramatically change the distribution of QH1+ ECs in host vessels (Fig. S3). Low concentrations (0.1ng/ml) of BMP2, BMP4, BMP9 and BMP10 significantly enhanced venous colonization of E8 JV QH1+ ECs, with BMP10 exhibiting the most significant increase in venous colonization (Fig. 5E).

Furthermore, low concentrations of BMP4, BMP9 and BMP10 were able to enhance venous colonization by E15 aorta ECs (Fig. 5F).

Conversely, exposing E15 JVs to BMP signaling inhibitors, Dorsomorphin (DM, 10 -6 M), Dorsomorphin-Homolog-1 (DMH-1, 10 -7 M) or Noggin (100ng/ml), reduced venous colonization and enhanced arterial fate (Fig. 5G). Noggin treatment had the most dramatic effect and led to almost 100% colonization of host arteries (Fig. 5G). The arterialization of QH1+ ECs was mediated at least in part through ERK activation since a combined treatment Noggin plus the ERK inhibitor U0126 reduced the colonization of host arteries (Fig. 5G).

BMP pathway was shown to inhibit endothelial ERK transcription through histone deacetylase-1 (HDAC-1) recruitment [38], suggesting that HDACs could be involved in BMP repression of ERK. When E15 JVs were treated with the HDAC inhibitor, Trichostastin A 10 -8 M, ±60% of QH1+ ECs reached host arteries (Fig. 5H), implicating HDAC activation in venous differentiation.

Discussion

We here study venous endothelial fate decisions using a functional assay that monitors colonization of chick host arteries and veins by grafted quail ECs. We show that prior to E11, grafted JV ECs can colonize both host arteries and veins, whereas after E11, the majority of JV ECs colonized host veins but not arteries. This change in behavior occurs without changes in the number of emigrated ECs, and is therefore interpreted to reflect bias towards a venous fate. Any fate decisions that are made in this model occur in a non-perfused environment within the coelom, independently of shear stress. Using this assay, we here reveal environmental neurotransmitter and BMP signals and endothelial-intrinsic ERK signaling as modulators of arterio-venous specification (Fig. 6).

The capacity of ECs to switch between arterial and venous gene expression

was previously demonstrated experimentally in avian chimeras [38,[START_REF] Othman-Hassan | Arterial identity of endothelial cells is controlled by local cues[END_REF]. Grafted quail ECs that integrated chick arteries acquired arterial marker expression, while those that integrated veins acquired venous marker expression, irrespective of their arterial or venous origin [38,[START_REF] Othman-Hassan | Arterial identity of endothelial cells is controlled by local cues[END_REF]. We confirm here that grafted ECs colonizing host arteries maintain CX40 expression, while cells colonizing veins lose CX40 expression, demonstrating that marker expression and presumably fate decisions proceed according to the integration into a host vessel. In mice, genetic lineage tracing experiments showed that ECs move against the flow direction from veins to arteries, accompanied by a change in gene expression pattern [START_REF] Su | Single-cell analyses of early progenitor cells that build coronary arteries[END_REF][START_REF] Luo | Arterialization requires the timely suppression of cell growth[END_REF], underscoring that EC plasticity with respect to arterio-venous specification is a fundamental mechanism during vascular differentiation. As major signaling mechanisms implicated in vascular development are conserved between birds, mice and humans [START_REF] Marcelle | Molecular cloning of a family of protein kinase genes expressed in the avian embryo[END_REF][START_REF] Eichmann | Two molecules related to the VEGF receptor are expressed in early endothelial cells during avian embryonic development[END_REF][START_REF] Eichmann | Avian VEGF-C: cloning, embryonic expression pattern and stimulation of the differentiation of VEGFR2-expressing endothelial cell precursors[END_REF][START_REF] Moyon | Selective expression of angiopoietin 1 and 2 in mesenchymal cells surrounding veins and arteries of the avian embryo[END_REF][START_REF] Bouvrée | Netrin-1 inhibits sprouting angiogenesis in developing avian embryos[END_REF], mechanisms governing vein specification identified in this work may be relevant to mice and humans as well.

As E11 corresponds to the onset of sympathetic vessel innervation in quail embryos [37], we reasoned that catecholamines could contribute to enhance vein differentiation. Removal of sympathetic innervation indeed decreased vein colonization and enhanced artery colonization by grafted E15 JV ECs, while NE stimulation of ADR2B enhanced vein colonization and decreased artery colonization, supporting functional involvement of sympathetic neurotransmitter signaling in vein differentiation. As ECs are not innervated directly, they may experience signals derived from NE-activated smooth muscle cells that modulate their behavior. Alternatively, ECs could respond directly to circulating catecholamines, as suggested by our data that direct exposure of isolated JV ECs to ADR2 agonists enhanced venous colonization. Interestingly, non-innervated human umbilical vein ECs express the arterial marker ephrinB2 [START_REF] Diehl | Altered expression patterns of EphrinB2 and EphB2 in human umbilical vessels and congenital venous malformations[END_REF], therefore lack of innervation correlates with a mixed arterio-venous identity in this human vessel as well.

We had previously shown that sympathetic nerve-derived NE stimulated arterial fate via adrenergic 1 and 2 receptor activation of ERK signaling [37]. Since NE enhances both arterial and venous fate, this raises the question how the same environmental signal can trigger two opposing outcomes on EC fate. We identify two mechanistic explanations. First, sympathetic nerves target both ADR1 and ADR2 on arteries, but only ADR2 manipulation affected venous ECs, hence receptor activation may be a critical upstream element in the EC fate decision response.

Secondly, we find that ERK activation is markedly decreased in veins when compared to arteries. These data point to the central role of the ERK activation status in arterio-venous differentiation, and support a model whereby ERK must be activated for arterial differentiation to occur, while it must be antagonized in veins to allow venous differentiation. In support of this model, knockout studies using conditional ERK1 and 2 gene inactivation showed the critical role of ERK in mediating arteriogenesis in the ischemic hindlimb, with ERK1 and 2 playing isoformspecific roles in ECs and macrophages [START_REF] Ricard | Isoform-Specific Roles of ERK1 and ERK2 in Arteriogenesis[END_REF]. ERK overactivation was observed in sporadic brain AVMs in human patients carrying somatic activating KRAS mutations [START_REF] Fish | Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling[END_REF][START_REF] Nikolaev | Somatic activating KRAS mutations in arteriovenous malformations of the brain[END_REF], supporting functional involvement of ERK overactivation in AVMs, and suggesting that ERK inhibitors could be used therapeutically to prevent AVM formation in patients with such mutations. However, ERK plays additional roles in vascular development and homeostasis [START_REF] Ricard | The quiescent endothelium: signalling pathways regulating organ-specific endothelial normalcy[END_REF]. For example, high ERK activation is induced by VEGF in sprouting endothelial tip cells in the mouse retina or in zebrafish intersomitic vessels, and VEGF or ERK inhibition prevents tip cell sprouting. Despite being exposed to high mitogenic VEGF-ERK signaling, tip cells rarely proliferate, which presented a condundrum. A recent study showed that high ERK activation upregulates the cell cycle inhibitor p21 in tip cells to induce cell cycle arrest [START_REF] Pontes-Quero | High mitogenic stimulation arrests angiogenesis[END_REF].

Hence, precise regulation of ERK activation levels is critical for its function, and ERK must be regulated in a vascular bed-specific manner to promote tip cell formation or arteriogenesis. In our model, ERK transcripts were lower in E15 veins when compared to E15 arteries, and ERK phosphorylation decreased in E15 veins when compared to E8 veins, indicating that vein-only colonization was accompanied by a decreased ERK activation. Treatment with inhibitors of ERK-targeting phosphatases promoted colonization of arteries by vein-derived cells, and this effect was reduced by an ERK inhibitor.

In search of additional factors able to enhance vein differentiation, we tested various BMP family members. BMPs were previously shown to selectively affect development of veins but not arteries in zebrafish and mice. Overexpression of BMP2b during zebrafish development promoted sprouting of venous capillaries, while overexpression of the BMP inhibitor Noggin3 prevented spouting of the caudal vein plexus, without affecting arteries [START_REF] Wiley | Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein[END_REF]. In the brain, BMP2/4 production by osteoblasts regulated venous development in the dura mater, and inhibition of BMP expression caused cranial vein malformations [START_REF] Tischfield | Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura[END_REF]. The vein-specific action of BMPs may be due to suppression of BMP signaling in arteries via Notch-induced expression of the BMP antagonist Smad6 [START_REF] Ricard | Isoform-Specific Roles of ERK1 and ERK2 in Arteriogenesis[END_REF], and to signaling through vein-specific BMP receptors ALK2 and ALK3, which activate Smad1/5 proteins to induce EphB4 expression [9, 27,].

BMPs also mobilized -catenin to stimulate venous differentiation and survival in zebrafish [START_REF] Kashiwada | -catenin-dependent transcription is central to Bmp-mediated formation of venous vessels[END_REF]. Of note, vein or capillary-venous deletion of HHT causing Endoglin, ALK1 or SMAD4 was sufficient to cause AVMs in mice [START_REF] Park | Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia[END_REF][START_REF] Mouillesseaux | Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6[END_REF][START_REF] Snodgrass | Hereditary Haemorrhagic Telangiectasia, an Inherited Vascular Disorder in Need of Improved Evidence-Based Pharmaceutical Interventions[END_REF], demonstrating that defective BMP signaling in veins is at the origin of AVM lesions in HHT, even though Endoglin, ALK1 and SMAD4 are not restricted to veins but also expressed in arteries.

Several BMP family members promoted vein colonization in our model, including BMP2, 4, 9 and 10. Interestingly, the effect was dose-dependent and only observed at low BMP concentrations. BMPs affect lymphatic differentiation from mouse embryonic stem cells in a dose-dependent manner [START_REF] Subileau | Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation[END_REF]. In embryonic development, BMP gradients are essential for proper morphogenesis and high-or low-level BMPs induced different effects [START_REF] Bier | Embryo development. BMP gradients: A paradigm for morphogen-mediated developmental patterning[END_REF]. QPCR analyses showed a significant upregulation of the BMP receptor ALK2 and a trend for upregulation of Alk1 in E15 JVs compared to E8, suggesting that BMPs might induce venous fate via these BMP receptors. A recent study showed an important expression of Alk1 and Alk2 mRNAs in HUVEC as well as a low transcriptional activity for Alk3 [START_REF] Choi | Transcriptomic analysis identifies novel targets for individual bone morphogenetic protein type 1 receptors in endothelial cells[END_REF]. Nevertheless, Alk3 was shown to promote venous identity in the mouse embryo [START_REF] Neal | Venous identity requires BMP signalling through ALK3[END_REF]. During hematopoiesis, recruitments of Smad1/5 Alk effectors, and HDAC1 inhibited ERK transcription [38].

As HDAC inhibition with Trichostatin A promoted arterialization of quail ECs, this pathway could be involved in ERK downregulation and venous colonization.

Interestingly, we found that the BMP signaling inhibitor Noggin potently promoted arterialization of grafted veins and may be a good candidate to improve conversion of vein ECs to arterial ECs during coronary artery bypass grafts using saphenous veins.

Materials and Methods

Animals

Chick (Gallus gallus, JA57) and quail (Coturnix coturnix japonica) eggs were incubated at 38°C until they developed the appropriate stages (E=embryonic day; P=post hatching day). Aortae, jugular veins (JVs) and veins of CAM were dissected together with their wall and cut into rings (50-100m) before grafting or culture. To remove the adventitia from quail embryos, vessels were dipped in Phosphate Buffered Saline (PBS, Gibco) without Ca 2+ Mg 2+ , pinned at one end onto a black support and dissected using a fine pair of forceps and a sharpened micro-scalpel.

Following the dissection the tissues were placed in PBS+Ca 2+ Mg 2+ .

Coelomic grafts

Indian ink was diluted 1:1 in PBS+Ca 2+ Mg 2+ and injected beneath E2 chick host blastodiscs (18-24 somites). Coelomic grafts were performed as described previously [37]. The freshly prepared-or overnight cultured-explants were introduced into the coelom at the level of somite 15-20 (Fig. 1A,B), on the left side. The hosts were sacrificed two days later, fixed in Bouin's fluid or Paraformaldehyde 4% and processed for serial (7.5m) paraffin transverse sections. More than 1300 grafts (>200 000 sections) were analyzed in this study.

Immunohistochemistry

Graft analyses used QH1 monoclonal antibody [56, own production] and QCPN monoclonal antibody (Carlson and Carlson, Developmental Studies Hybridoma Bank, Dept. of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolf Street, Baltimore, MD21205) that recognized all quail cells. Immunohistochemistry was done as previously described and followed by a glychemalun nuclear staining [37].

Triple fluorescent QH1/Sambucus nigra/Connexin 40 stainings were performed on few grafts. QH1 was revealed using an alexa 647-coupled anti mouse IgM antibody. Biotinylated Sambucus nigra lectin (Vector, 1/200 in PBLEC buffer) that recognizes avian ECs (chick and quail), was revealed using Cy3 streptavidin. Connexin 40 was detected using the polyclonal anti-GJA5 antibody (Clinisciences, 1/50) and revealed using an alexa 488-coupled anti rabbit antibody.

Quantification of endothelial migration into host arteries and veins

The number of QH1 + ECs reaching host arteries and veins was counted manually on every fourth section (X25 objective, final magnification X110). A positive cell was scored when it showed a nucleus, visible by glychemalun, and membrane QH1 staining. The arterial or venous identity was obvious when the cells integrated great vessels (aorta, cardinal vein, omphalomesenteric artery and vein, subclavian and brachial arteries, umbilical vein, segmental arteries and veins). In the limb bud, we considered that all ECs present at the periphery belonged to the venous plexus while arterial cells were centrally located and connected to the brachial artery. In the perineural vascular plexus QH1 + ECs were only quantified if we could trace their connection with a known artery or a vein. If we could not classify the arterial or venous identity of a donor cell, it was not counted. The total number of cells per graft was set at 100%, and the percentage of grafted cells present in host veins or arteries was calculated.

Statistical analyses were carried out using unpaired two tailed t tests. P values were *P<0.05, **P<0.01, ***P<0.001 or ****P<0.0001.

Organotypic cultures

Vessel rings were placed onto the surface of a semi solid medium (50% semi-solid medium-for 100ml: agar 2g, NaCl 1,6g, KCl 74mg, CaCl 2 34mg, MgCl 2 42mg, Na 2 HPO 4 30mg, KH 2 PO 4 6mg, MgSO 4 14mg, C 6 H 12 O 6 , 200mg-, 40% DMEM, 5% fetal calf serum (FCS), Invitrogen-, 5% antibiotics -Gibco-) with or without drugs (Table 1) for one night at 37°C, 5% CO 2 . For associations, vessels and tissues were placed together on the semi-solid medium.

EC isolation

E15 JVs (16-25 per experiment) were longitudinally sectioned and treated with collagenase (Sigma) for 30 minutes at 37°C. ECs were then flushed out with a micropipette, placed in DMEM supplemented with 20% FCS, centrifuged and resuspended in 100l of the same medium ± drugs. Two aliquots of 10 l were cultured for 48 hours in a Petri dish to verify the presence of QH1+ ECs. Aliquots of 20 l were placed overnight in hanging drop cultures for aggregate formation and grafting (37°C, 5%CO 2 ).

Glyoxylic acid treatment

Freshly dissected quail JVs were longitudinally sectioned and incubated during 30 min. at room temperature in PBS with 2% of glyoxylic acid (Sigma) and 10% of sucrose (w/v), pH 7.5. The vessels were placed on a glass slide, the smooth muscle layer up, and dried 30 min. at room temperature. Vessels were then stretched during 4 min. at 100°C on a heated plate. Slides were mounted in Entellan (Merck) and imaged with a fluorescent microscope (Excitation wavelength 350nm, Emission wavelength 461nm).

Denervation

Quail embryonic JVs were placed in a solution of 6-hydroxy-dopamine (6HDOPA, 300M, Sigma) during 3h30 at 37°C to remove nerve fibers without affecting cell survival. After the treatment the vessels were rinsed in PBS+Ca 2+ Mg 2+ before grafting.

For surgical denervation, the neural tube and notochord were removed from E2 quail embryos as described [START_REF] Noble | Contractile and relaxing reactivity in carotid and femoral arteries of chicken embryos[END_REF]. The resulting embryos completely missed the peripheral nervous system. JVs were isolated from operated quail embryos at E15 (n=5) and grafted into E2 chick host coeloms.

Western-blotting

Aortae and JVs from E8 and E15 quail embryos were dissected and immediately frozen in liquid nitrogen. Next, samples were lysed in NP40 lysis buffer (Lifetechnologies) containing protease inhibitor set I (Millipore), 5mM orthovanadate and 2mM sodium pyrophosphate, then briefly sonicated, incubated for 15 minutes at 4°C and centrifuged 15 minutes at 4°C at 13.000 rpm. Supernatants were immediately frozen. Proteins (2.5 µg/sample) were separated using SDS-PAGE (NOVEX Nupage 4-12% LifeTechnologies) and transferred to a nitrocellulose membrane (Amersham Protran Premium 0.45 NC, GE Healthcare Lifesciences). All gels and transfers were run in double, allowing one membrane to be incubated with anti-Phospho-p44/42 MAPK (p-ERK, Cell Signaling, 4370), and the other with anti-p44/42 MAPK (total ERK, Cell Signaling, 9102). Membranes were blocked in TBST containing 5% non fat dry milk for 1hr at room temperature then incubated overnight at 4°C with primary antibody diluted in blocking buffer (1:1,000). Membranes were washed in TBST then incubated with HRP-conjugated secondary antibody (1:1,000 in blocking buffer, Pierce, 31460). Bands were revealed using Supersignal west Femto substrate (ThermoFisher). Membranes were imaged and bands quantified using the GE Healthcare ImageQuant LAS-4000 imaging system. After ERK detection, membranes were extensively washed in TTBS then incubated with anti-alpha-Tubulin (1:1.000; Abcam ab18251) and revealed as described above.

Quantitative real-time PCR analysis

E8 and E15 quail aortae and JVs were dissected and total RNA was extracted and purified using NucleoSpin RNA XS kit (Macherey-Nagel). RNA concentrations were measured on a Nanodrop and retrotranscribed by SuperScriptIII (Lifetechnologies).

We performed real-time Q-PCR reactions in duplicate on a T100 real-time thermal cycler (BIORAD). Primer sequences are shown in Table 2.

Figure S3

Quantification of BMP effects (high doses) on host venous colonization by QH1+ ECs from E8 JVs, each dot corresponds to one JV sample.*P<0.05, Mann-Whitney or

LegendsFigure 1 :

 1 Figure 1: Method to test venous fate in avian chimeras A) Dissection of an E15 quail jugular vein (JV, arrowhead) adjacent to the vagus nerve (arrow), the esophagus (E) and the trachea (Tr). A segment of JV (magnified box, A) is isolated and grafted in the coelomic cavity (C) of an E2 chick embryo (arrow, A-B). B) Transverse section of a chick host 3h after grafting shows QCPN+ quail tissue implanted in the coelom (arrow) on the left side (LS). Ao: aorta; NT: neural tube; S: somite; RS: right side. C and D) Representative 3D reconstructions [31] of QH1+ EC distribution in chick hosts 48h after the graft: as grafted cells never cross the midline, only the left half is illustrated. The neural tube (NT) and wing bud (W) are colored blue, the viscera in yellow, the graft (*) in black, the arterial plexus in red and the venous plexus in dark blue. C) the majority of QH1+ ECs originating from an E15 JV graft colonized host veins (green dots) but not arteries (white dots). D) QH1+ ECs emigrating from an E8 JV colonized host veins (green dots) and arteries (white dots). Insets show transverse sections that served to generate the 3D reconstructions in C and D respectively. The inset in C illustrates the colonization of the umbilical vein plexus (*) by QH1+ ECs (arrows). The inset in D shows QH1+ ECs around the graft (G), both in the aorta (Ao) and its dorsal and ventral branches (arrowheads) and in branches of the umbilical vein (arrow). C: caudal; R: rostral; CV: cardinal vein; UV: umbilical vein; W: wing bud. E) Histogram of venous colonization from JV grafts at different developmental stages. The columns show the percentage of QH1+ ECs that colonize host veins, each dot corresponds to one JV sample. *P<0.05, **P<0.01, ***P<0.001, by comparison with E8 column, Mann-Whitney or Unpaired 2-tailed nonparametric t tests. Top, Schematic illustration of the results: when isolated from E11 or older embryos, grafted JV ECs mainly colonize host veins (blue), but when isolated before E11, grafted JV ECs colonize host veins and arteries (blue and red). F) Quantification of the total number of emigrating QH1+ ECs in E15 and E8 grafts, each dot corresponds to one JV sample. Unpaired 2-tailed nonparametric t test. Error bars: SEM. n: number of grafts. The blue numbers correspond to the total number of cells counted in each condition.

Figure 2 :

 2 Figure 2: Denervation inhibits vein colonization A-D) Glyoxylic acid staining of sympathetic nerves in the JV vessel wall. No staining was detected at E8 (A); the first fibers were visible at E11 (B, arrowhead) and formed a network at E15 (C). D) CAM veins lack innervation. E) Quantification of host venous colonization by QH1+ ECs from CAM veins, isolated E15 JV ECs and E15 JV without adventitia, each dot corresponds to one JV sample. ***P<0.001, ****P<0.0001 by comparison with E15 column, Unpaired 2-tailed nonparametric t tests. F, G) 6HDOPA treatment abolished innervation of E15 (F) and P5 (G) JVs. H) Surgical denervation of a 15 somite-stage quail embryo by resection (*) of the neural tube and notochord between the fifth somite (arrow) and the caudal region (black arrowhead). Rostrally, the neural tube is intact (white arrowhead). I) Post-surgery glyoxylic acid staining shows the absence of nerve fibers at the surface of an E15 JV. J) Quantification of host venous colonization by QH1+ ECs from denervated JVs, each dot corresponds to one JV sample. *P<0.05, ***P<0.001, by comparison with E15 or post-hatching vessels, Unpaired 2-tailed nonparametric t tests. Error bars: SEM, n: number of grafts. The blue numbers correspond to the total number of cells counted in each condition.

Figure 3 :

 3 Figure 3: Sympathetic pathways regulating venous fate A) Quantification of host venous colonization by QH1+ ECs from E8 JVs treated or not with NE overnight on semisolid medium prior to grafting into E2 chick hosts, each dot corresponds to one JV sample. ****P<0.0001, by comparison with untreated E8 JVs, Unpaired 2-tailed nonparametric t test. B) QPCR analyses of ADR expression in E8 versus E15 JVs. Expression levels at E8 are set as 1, each dot corresponds to one JV sample. *P<0.05, ***P<0.001, Mann-Whitney tests. C) Schematic of NE signaling via ADR2B, including pharmacological agents used against downstream effectors that affect venous colonization. D) Quantification of host venous colonization by QH1+ ECs from E8 JVs treated or not with the indicated molecules, each dot corresponds to one JV sample. E) Quantification of host venous colonization by QH1+ cells from isolated ECs from E15 JVs. **P<0.01, ***P<0.001, ****P<0.0001, by comparison with control columns, Unpaired 2-tailed nonparametric t

Figure 4 :

 4 Figure 4: ERK inhibition enhances vein colonization A) QPCR analysis of ERK expression on E15 aortae and JVs samples, each dot corresponds to one vessel graft. Expression levels in JVs are set as 1. **P<0.01 by comparison with E15 JV, Mann-Whitney test. B) Top: Western blot of p-ERK, total ERK and tubulin in E8 and E15 JVs. Avian ERK migrates at 44 kD. Each lane represents one JV sample. Bottom: Western blot quantification, each dot corresponds to one JV sample. **P<0.01, Mann-Whitney tests. C) Quantification of effect of ERK inhibitor treatment on host venous colonization by QH1+ ECs from E15 JVs, each dot corresponds to one JV sample. Unpaired 2-tailed nonparametric t tests. D) Effects of treatment with phosphatase inhibitors (SOV, E/Z-BCI, Fostriecin, Salubrinal, ML119) effects with or without ERK inhibition on host venous colonization by QH1+ ECs from E15 JVs, each dot corresponds to one JV sample. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 Unpaired 2-tailed nonparametric t tests. Error bars: SEM, n: number of grafts. The blue numbers correspond to the total number of cells counted in each condition.

Figure 5 :

 5 Figure 5: BMP signaling promotes venous fate A-D) QPCR analyses of ALK2, ALK1, ALK3 and ALK6 expression in E8 and E15 JVs. Each dot corresponds to one sample. Expression levels in E8 JVs are set as 1. **P<0.01, ***P<0.001, Mann-Whitney tests. E) Quantification of BMP effects on host venous colonization by QH1+ ECs from E8 JVs, each dot corresponds to one JV sample. *P<0.05, **P<0.01, ****P<0.0001, Mann-Whitney or Unpaired 2-tailed nonparametric t tests. F) Quantification of BMP effects on host arterial colonization by QH1+ ECs from E15 aortae, each dot corresponds to one aorta sample. *P<0.05, **P<0.01, ***P<0.001, Unpaired 2-tailed nonparametric t tests. G) Quantification of BMP inhibitor effects on host venous colonization by QH1+ ECs from E15 JVs, each dot corresponds to one JV sample. **P<0.01, ***P<0.001, ****P<0.0001, Unpaired 2tailed nonparametric t tests. H) Quantification of HDAC 1 inhibitor effect on host venous colonization by QH1+ ECs from E15 JVs, each dot corresponds to one JV

Figure 6 :

 6 Figure 6: Summary model Venous colonization in quail chick embryo chimeras is enhanced by NE activation of ADR2B, ERK inhibition and BMP stimulation, while arterial differentiation is enhanced by ERK activation and BMP inhibition using Noggin.

Figure S1

 S1 Figure S1Transverse sections at the truncal level of an E4 chick host, 2 days after the graft of a quail E8 JV. Triple staining using the QH1 antibody (blue), an anti-CX 40 antibody that recognizes arteries (green) and the Sambucus nigra lectin that stains all avian vessels (red). The CX40+/Sambucus+ aortae (Ao) appear orange while cardinal vein (CV) and umbilical vein (UV) are only Sambucus+. A) The arrow indicates a quail EC (blue-green) that reaches the aortic endothelium. The arrowhead shows a QH1+ EC counted as an arterial EC because a ventral aortic branch is present close to this cell on a consecutive section. B) The arrow points to a grafted quail QH1+/Sambucus+ EC (purple) participating to the UV endothelium. Arrowheads indicate QH1+ EC close to chick veins and counted as venous EC because a venous branch is present close to this cell on a consecutive section. Blue QH1+ cells that were not counted are present near the graft (G). M: mesonephros.

Figure S2 Number

 S2 Figure S2 Number of emigrating QH1+ ECs from untreated E8 JVs and E8 JV treated by Phenylephrin (PE) and BHT933, each dot corresponds to one JV sample. Mann-Whitney or Unpaired 2-tailed nonparametric t tests. Error bars: SEM, n: number of grafts.

Funding

This work was supported by Institut national de la santé et de la recherche médicale (Inserm) and the European Research Council grant agreement No. 834161.

Availability of data and material

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Competing interests

The authors declare no competing or financial interests.

Author contributions

All authors contributed to the study conception and design.

Ethics approval and consent to participate

Non applicable

Unpaired 2-tailed nonparametric t tests. Error bars: SEM, n: number of grafts. The blue numbers correspond to the total number of cells counted in each condition.


Muhammad Salman Shahid

Benoît Delinchant

Béatrice Roussillon

Frédéric Wurtz

Daniel Llerena

The Formulation of a Reference Load Curve to Measure Energy Flexibility

Key Innovations

 Reference load curve for each residential consumer based on historical consumption data.

Practical Implications

For the days when energy flexibility is activated, the difference of reference load curve (RLC) and measured load curve (MLC) gives a measure of energy flexibility.

Introduction

An indirect feedback of energy flexibility is given to the consumer after the period of energy consumption. It is either historic (comparing the energy consumption with the past consumption of the same household), or normative (comparing household energy consumption with that of a group of similar households) [START_REF] Wang | Impact Analysis of Customized Feedback Interventions on Residential Electricity Load Consumption Behavior for Demand Response[END_REF]. Graphically, it is presented in the form of clustered baseline load curve of identical households (Abreu, Câmara Pereira, and Ferrão 2012) or average load curve of same household (Ozawa, Furusato, and Yoshida 2016). This extended abstract presents a statistical comparison of methods to formulate RLC of residential consumer. An RLC (superposed on MLC) is helpful in measuring energy flexibility of a residential consumer during a flexibility activated day 'D'. An example of RLC for load curtailment between 6 PM and 8 PM is given in figure 1.

Methods

The consumption data of 96 French households (spanning over a year) is taken. These households participated in an experiment of energy flexibility and indirect feedback is given in the form of images as illustrated in figure 1. Subjecting to the intended type of flexibility, RLC is used to measure flexibility on activation day 'D' for individual household to measure:  Load curtailment between 6 PM and 8 PM.  Load shifting from evening to 'noon and 3 PM'.

Kernel Density Estimation (KDE)

For each timestamp of alert period, KDE is implemented on the historical power consumption to choose the value with highest density (i.e. most probable value).

Mean [PD-1 & {Max or Min (PD-2 : PD-5)}]

For an alert day 'D', an RLC is created by taking average of measured load curve of previous day 'D-1' and the measured load curve having highest or lowest energy consumption between day 'D-2' and 'D-5', for load curtailment and load shifting respectively.

Max or Min (PD-1 : PD-5)

For an alert day 'D', the measured load curve having highest or lowest energy consumption between day 'D-1' and day 'D-5' is taken as reference load curve, for load curtailment and load shifting respectively. Random Forest (Sequence to Sequence) A sliding window of 5 alert periods between 'D-1' and 'D-5' is used to train a random forest regression model. This model predicts the RLC of alert day 'D'.

Random Forest (Point to Point)

For each timestamp of alert period, a sliding window of 5 same timestamps between 'D-1' and 'D-5' is used to train a random forest regression model. This model predicts the RLC of alert day 'D'.

Energy Difference Indicator

Using the historical data of 1 year, RLC is calculated for all the methods. An energy difference indicator is used to compare the methods using equations below. (2)

Discussion

An RLC is in fact an expected MLC if no flexibility activation is put in place. The difference of RLC (i.e. expected MLC) with flexibility driven MLC measures achieved energy flexibility on activation day 'D'. Hence, the difference between an ideal RLC and MLC is zero for non-activated days, and vice versa.

Practically, RLC can be formulated by using naïve methods or regression. An indirect feedback with an RLC that underestimates energy flexibility may discourage the consumer. On the other hand, an extreme overestimation is not helpful for distribution system operator (DSO) or energy aggregator to measure energy flexibility. Since an ideal RLC does not exist, therefore it is required to select an RLC that slightly overestimates energy consumption.

For the purpose of study, the difference between RLC and MLC of 96 households is calculated using EDI during non-activation days. EDI is calculated between 6 PM and 8 PM in the case of load curtailment. Ideally, the difference should be zero, however practically the difference should be small and positive for non-activated days. The statistical distribution of EDI is visualised in figure 2 for each method of RLC formulation. 2, that KDE method will mostly underestimate the prospective load curtailment on activated day. This is due to the fact that it takes historical consumption and therefore a most probable value is calculated for gross variation in energy consumption. The other 4 methods fulfil the criteria, however the method Max(PD-1 : PD-5) is widely dispersed and may extremely overestimates the prospective load curtailment on activated day, therefore it is not suitable. The random forest regression methods are less dispersed and the median value is closer to zero, therefore they create suitable RLC for load curtailment. In the case of load shifting, EDI is calculated between noon and 3 PM. Since it is expected that the consumer consumes more than usual during this time period, therefore the difference should be small and negative for non-activated days. Similar to figure 2, the statistical distribution of EDI is visualised in figure 3 in the case of load shifting.

Conclusion

Indirect feedback is given to the energy consumers in numerical form, as a clustered load curve of identical households or as average daily load curve. However a customized reference load curve for each consumer is required to measure individual energy flexibility. A simulated load curve can serve as a reference load curve. This simulated load curve is an expected MLC if no flexibility activation is put in place. The difference of this expected MLC with flexibility driven MLC measures energy flexibility on activation day 'D'. Ideally, the difference of reference load curve and measured load curve is zero. Practically, a good RLC should not underestimate or extremely overestimate the prospective energy flexibility and also it should nudge the consumer to carry on their efforts for energy flexibility. Random forest regression gives good formulation of RLC for load curtailment however it is not found effective for load shifting. Therefore, the naïve method with less dispersed EDI population and median close to zero is most suitable for load shifting. The visualization of RLC superposed on MLC gives the DSO or energy aggregator a measure of energy flexibility whereas on the other hand it may nudge the consumer through indirect feedback to continue practicing energy flexibility.

Figure 1 :

 1 Figure 1: Superposition of RLC over MLC to visualize load curtailment of a residential consumer on day 'D'

Figure 2 :

 2 Figure 2: Comparison of RLC formulation methods w.r.t energy difference indicator for load curtailment It can be observed by figure2, that KDE method will mostly underestimate the prospective load curtailment on activated day. This is due to the fact that it takes historical consumption and therefore a most probable value is calculated for gross variation in energy consumption. The other 4 methods fulfil the criteria, however the method Max(PD-1 : PD-5) is widely dispersed and may extremely overestimates the prospective load curtailment on activated day, therefore it is not suitable. The random forest regression methods are less dispersed and the median value is closer to zero, therefore they create suitable RLC for load curtailment. In the case of load shifting, EDI is calculated between noon and 3 PM. Since it is expected that the consumer consumes more than usual during this time period, therefore the difference should be small and negative for non-activated days. Similar to figure2, the statistical distribution of EDI is visualised in figure3in the case of load shifting.

Figure 3 :

 3 Figure 3: Comparison of RLC formulation methods w.r.t energy difference indicator for load shifting The random forest regression methods are not suitable for assessment of load shifting as they are widely dispersed

Acknowledgment

This work has been supported by the Auvergne Rhône-Alpes region, "Pack Ambition Recherche 2017 -Expesigno projet" (17 01110301). This work has also been partially supported by the CDP Eco-SESA receiving fund from the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02).

Nomenclature

 RLC : Reference Load Curve  MLC : Measured Load Curve  EDI : Energy Difference Indicator


Anna D Grabowska

Yoann Brison

Laurent Maveyraud

Sabine Gavalda

Alexandre Faille

Virginie Nahoum

Cécile Bon

Christophe Guilhot

Jean-Denis Pedelacq

Christian Chalut

Molecular Basis for Extender Unit Specificity of Mycobacterial Polyketide Synthases

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Molecular basis for extender unit specificity of mycobacterial polyketide synthases

Anna D. Grabowska, # Yoann Brison, # Laurent Maveyraud, # Sabine Gavalda, Alexandre Faille, Virginie Nahoum, Cécile Bon, Christophe Guilhot, Jean-Denis Pedelacq,* Christian Chalut,* and Lionel Mourey* ABSTRACT: Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g. mycolic acids, phthiocerol dimycocerosates and phenolic glycolipids, belong to the 'family' of polyketides, secondary metabolites synthesized by fascinating versatile enzymes -polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.

Mycobacterium tuberculosis, at the origin of tuberculosis, still constitutes a major threat for public health. About a quarter of the world's population has been infected by M. tuberculosis, the leading cause of death from a single infectious agent. In 2018, there were an estimated 1.5 million deaths and 10 million people developed tuberculosis. [START_REF] Who | Global Tuberculosis Report 2019[END_REF] Furthermore, the development of multiple types of resistance to anti-tuberculosis drugs represents a severe health emergency.

Among unique features of M. tuberculosis and related species is the multilayer structure of their cell envelope. [START_REF] Daffe | The cell envelope of tubercle bacilli[END_REF][START_REF] Daffé | Bacterial Membranes: Structural and Molecular Biology[END_REF] This thick hydrophobic layer has a multifaceted protective role, acting as a permeability barrier against toxic insults, enhancing pathogen adaptation to the hostile environment of infected humans, and makes the mycobacterial cell envelope a well-established drug target. [START_REF] Abrahams | Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target[END_REF] Important components, which contribute to the cell envelope impermeability and exert a key role as effector molecules in the host-pathogen interplay, are long chain lipids produced by polyketide synthases (PKSs). [START_REF] Quadri | Biosynthesis of mycobacterial lipids by polyketide synthases and beyond[END_REF] Among such lipids are mycolic acids, [START_REF] Marrakchi | Mycolic acids: structures, biosynthesis, and beyond[END_REF] phthiocerol dimycocerosates (DIMs/PDIMs) and structurally related phenolic glycolipids (PGLs) [START_REF] Arbues | Playing hide-and-seek with host macrophages through the use of mycobacterial cell envelope phthiocerol dimycocerosates and phenolic glycolipids[END_REF] (Figure 1a), which have been shown to play an important role in the virulence of M. tuberculosis, as well as M. leprae and M. ulcerans, the etiological agents of leprosy and Buruli ulcer, respectively.

Polyketide biosynthesis is highly related to that of fatty acids. It proceeds through successive decarboxylative condensations between the growing polyketide chain and coenzyme A (CoA)derived extender units. Similarly to fatty acid synthases (FASs), PKSs consist of catalytic domains, which may be present on a single high-molecular weight protein (type I PKSs and FASs) or are carried by different proteins (type II PKSs and FASs). Type I PKSs can be subdivided in two categories: those, like FASs, acting iteratively through several cycles of synthesis and modular ones where each module is responsible for a condensation reaction in an assembly line. In addition, modular PKSs can be further differentiated into cis or transacyltransferase (AT), depending on AT domains being embedded or not. [START_REF] Keatinge-Clay | The uncommon enzymology of cis-acyltransferase assembly lines[END_REF][START_REF] Musiol-Kroll | Acyltransferases as tools for polyketide synthase engineering[END_REF] . Thus, cis-AT modules minimally contain three conserved catalytic domains: an AT that selects and helps the transfer of the extender unit, an acyl carrier protein (ACP) to which the extender unit is transferred, and a ketosynthase domain (KS) that catalyzes the condensation between the intermediate and the extender unit resulting in a β-ketoacyl linked to ACP. PKSs may also contain additional domains displaying ketoreductase (KR), dehydratase (DH), or enoylreductase (ER) activity, which modify the β-keto group leading to a hydroxyl group, a double or a single bond, respectively. Most of the current knowledge about structure-function relationships of type I PKSs has been deduced from structures of the related FAS enzymes [START_REF] Maier | Structure and function of eukaryotic fatty acid synthases[END_REF] and individual domains or didomains derived from PKSs. [START_REF] Herbst | The architectures of iterative type I PKS and FAS[END_REF][START_REF] Robbins | Structure and mechanism of assembly line polyketide synthases[END_REF] Although there are no atomic resolution structures of intact PKSs solved so far, low-resolution models have been derived from single-particle electron cryo-microscopy in the case of the PikAIII pikromycin synthase [START_REF] Dutta | Structure of a modular polyketide synthase[END_REF] or by hybrid approach combining X-ray crystallography and small angle X-ray scattering for the M. smegmatis mycocerosic acid synthase-like PKS. [START_REF] Herbst | Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases[END_REF] Around 20 PKS-encoding genes are present in the M. tuberculosis genome. [START_REF] Cole | Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[END_REF] The corresponding PKSs mainly participate in the biosynthesis of complex lipid metabolites. [START_REF] Quadri | Biosynthesis of mycobacterial lipids by polyketide synthases and beyond[END_REF] The most studied mycobacterial PKS with respect to its structure, function and its potential use as a druggable target is Pks13, an essential enzyme responsible for the final condensation step in mycolic acid biosynthesis [START_REF] Aggarwal | Development of a novel lead that targets Mycobacterium tuberculosis polyketide synthase 13[END_REF][START_REF] Bergeret | Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13[END_REF][START_REF] Gavalda | The Pks13/FadD32 crosstalk for the biosynthesis of mycolic acids in Mycobacterium tuberculosis[END_REF][START_REF] Gavalda | The polyketide synthase Pks13 catalyzes a novel mechanism of lipid transfer in mycobacteria[END_REF][START_REF] Portevin | A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms[END_REF] (Supporting Information Figure 1a). Pks13 has a particular organization, which comprises the mandatory condensing units described above plus an additional ACP domain and a thioesterase (TE) domain involved in the release of the condensation product [START_REF] Gavalda | The polyketide synthase Pks13 catalyzes a novel mechanism of lipid transfer in mycobacteria[END_REF] (Figure 1b). Compared to other PKSs, the AT domain of Pks13 displays a very unusual specificity, selecting atypical substrates that are long carboxylated fatty acid chains. In a previous study, we determined the crystal structure of a 52 kDa AT-containing Pks13 fragment in apo and (carboxy)palmitoylated forms. [START_REF] Bergeret | Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13[END_REF] Given the importance of the AT domain in selecting elongation units, we decided to expand the characterization of this domain in three other mycobacterial PKSs: Mas, PpsA, and PpsC. All three enzymes are involved in the biosynthesis of DIMs and PGLs (Figure 1a), well documented lipid virulence factors. [START_REF] Arbues | Playing hide-and-seek with host macrophages through the use of mycobacterial cell envelope phthiocerol dimycocerosates and phenolic glycolipids[END_REF] DIMs were recently shown to be transferred from the mycobacterial envelope to macrophage membranes during infection and to promote phagocytosis. [START_REF] Augenstreich | The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages[END_REF] PpsA and PpsC (for phenolphthiocerol/phthiocerol synthase A and C) are encoded within the ppsABCDE modular PKS gene cluster and together with PpsB, PpsD and PpsE they participate in the formation of the C32-C34 phenolphthiocerol/phthiocerol chain (Supporting Information Figure 1b). The iterative Mas (for mycocerosic acid synthase) catalyzes the formation of the methyl-branched mycocerosic acids containing 24 to 30 carbon atoms (Supporting Information Figure 1c).

During biosynthesis of DIMs and PGLs, mycocerosates are esterified to the two hydroxyl groups of the (phenol)phthiocerol chain. Our selected PKSs differ in domain organization and in the number and type of auxiliary catalytic domains: DH, ER, and KR for Mas and PpsC; DH and KR for PpsA; none for Pks13 (Figure 1b). They also differ with respect to their substrate and extender unit specificity: methylmalonyl-CoA (MM-CoA) for Mas, malonyl-CoA (M-CoA) for PpsA and PpsC, and long acyl-CoA for Pks13. Finally, they carry out various number of elongation/condensation cycles: 2 to 5 iterations for Mas and only one for PpsA, PpsC and Pks13. In the present work, we describe the X-ray structures of AT-containing PpsA, PpsC and Mas fragments. We identified residues of the AT Mas domain responsible for extender unit specificity and subjected them to site-directed mutagenesis, providing biochemical and structural evidence for molecular-based reprogramming of mycobacterial PKS specificity towards extender units.

RESULTS AND DISCUSSION

Structure determination and overall analysis. The initial choice of construct boundaries for the AT domains of Mas (residues P447-W871) and PpsA (residues V542-P959) relied on a structure-based sequence comparison with the 52 kDa AT-containing fragment of Pks13, [START_REF] Bergeret | Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13[END_REF] hitherto called AT52 and renamed here L-AT-Pks13 (L standing for the KS-to-AT linker).

Those for the AT domain of PpsC (residues G546-L876) were identified using the domain trapping technique. [START_REF] Pedelacq | Experimental mapping of soluble protein domains using a hierarchical approach[END_REF] Mas and PpsA constructs also contain the L linker whereas that of PpsC only included a 28-residue long peptide stretch from the upstream linker. After recombinant expression, all proteins could be purified to homogeneity and in sufficient quantity for structural analysis. While L-AT-Mas and AT-PpsC gave crystalline materials that allowed solving the corresponding high-resolution structures, L-AT-PpsA remained reluctant to crystallization.

Small-angle X-ray scattering (SAXS) analysis of the purified protein revealed some floppy regions (Supporting Information Figure 2). A shorter construct was therefore designed, which included only the core AT domain (i.e. residues N621-H952) of PpsA. This construct allowed to obtain crystals that diffracted X-rays to a resolution of 1.6 Å. Crystals of AT-PpsC and L-AT-Mas diffracted X-rays to 1.9 and 2.4 Å, respectively. All structures were solved using the molecular replacement method (Supporting Information Table 1).

In terms of sequences, Pks13 and Mas are quite distant as they also are with respect to PpsA and PpsC (29-35% sequence identity), while PpsA and PpsC are much closely related (73% sequence identity). Nevertheless, their AT domains adopt the canonical fold found in individual acyltransferases or in AT domains associated with FASs and PKSs, which comprises the large / hydrolase subdomain and a smaller ferredoxin-like subdomain (Figure 2a). They also share a high degree of conservation with respect to both secondary and tertiary structure elements (Supporting Information Figure 3). This is also true for the upstream KS-to-AT L domain that precedes the AT domain in the resolved structures for Pks13 and Mas. Indeed, despite a low level of sequence identity (22%), the L domains of Pks13 and Mas have remarkably similar secondary structure elements with different orientations with respect to the AT domain (Figure 2a and Supporting Information Figure 3). A rotation of about 60° is necessary to superpose the L domains once having superimposed the AT domains. A search against the Protein Data Bank (PDB) allowed identifying 16 unique AT structures incorporating a L domain, which are exclusively found in PKSs and in FASs. No correlation could be established between the position of the L domain and factors such as PKS organization, the fragments used for structure determination, extender unit specificity, etc (Supporting Information Table 2). On the other hand, this variability does not seem to be due to the fact that these L-AT systems have been excised from the whole proteins since the linker of L-AT-Mas is found in the same orientation as that observed in the KS-L-AT structure of the Mas-like PKS. [START_REF] Herbst | Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases[END_REF] The orientation and dynamics of the linker might then be a trait specific to each protein with respect to intra-or inter-domain interfaces. This is in line with the finding that the flanking linkers of AT domains contribute to both efficiency and specificity of the transacylation reaction on the ACP domain. [START_REF] Wong | Protein-protein recognition between acyltransferases and acyl carrier proteins in multimodular polyketide synthases[END_REF] Description of the active sites and catalytic cavities. AT domains are characterized by a Ser/His catalytic dyad located at the interface between the hydrolase and ferredoxin subdomains. This dyad is found in Pks13 (S801/H909), Mas (S623/H727), PpsA (S720/H822) and PpsC (S660/H762) (Figure 2b and Supporting Information Figure 2) and the catalytic serine is part of a consensus sequence: G-X1-S-X2-G, with (X1=Q, X2=L) in Pks13 and (X1=H, X2=M) in Mas, PpsA and PpsC. In L-AT-Mas, the side chain of the catalytic serine adopts a very unusual conformation where it flips back to the main-chain of the consensus motif, making polar interaction with the side-chain of H622 at position X1 (Figure 2b). Histidine, the other catalytic residue, is at hydrogen bond distance of the hydroxyl group of the serine in all structures except that of L-AT-Mas. The histidine optimal position for increasing the reactivity of the catalytic serine 17 occurs through conserved hydrogen bonds with the main-chain carbonyl groups of two residues (Gly962 and His965 in Pks13; Asn776 and Asn779 in Mas; Asn871 and Asn874 in PpsA; and Ans815 and Asn818 in PpsC) (Figure 2b). The arginine residue involved in the selection of carboxylate substrates 17 is also conserved. However, whereas the corresponding Arg826 makes hydrogen bonds with the hydroxyl group of the catalytic serine in L-AT-Pks13, the side chain of its counterpart in L-AT-Mas (Arg648) is displaced by more than 1.0 Å farther resulting in (or because of) hydrogen bonding to the side chain of Asn776 (Figure 2b). In AT-PpsA and AT-PpsC, the corresponding arginines (Arg745 and Arg685, respectively) are even further displaced because of the side chain of the methionine residue found at position X2 (Met721 and Met661, respectively). Opposite to the hydrolase subdomains, the active sites are lined by a stretch of four consecutive residues from the ferredoxin-like subdomain, including the catalytic histidine at the fourth position, which is supposed to play a role in extender unit specificity, i.e. HAFH and YASH for M-CoA and MM-CoA, respectively. [START_REF] Reeves | Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations[END_REF] As expected for Pks13 ( 906 GASH) but not anticipated for Mas ( 724 VASH), PpsA ( 819 VAPH) and PpsC ( 759 VAPH), the residues found at the corresponding positions of these mycobacterial PKSs are quite different from the canonical HAFH or YASH residues (Figure 2b). From a topographical point of view, the active sites are accessible via a large opening on the surface of the proteins at the frontier between the two subdomains (Supporting Information Figure 4). An electropositive potential area lines the floor of the active site cavities due to the presence of the arginine residue and catalytic histidine described above (Figure 3).

While the electropositive character of the active site floors can be noted, the molecular surfaces as a whole are more electronegative, in accordance with the calculated pI of the proteins studied (between 5.0 and 5.1). However, the distribution of charges varies from one protein to another, probably in relation to the intra-or inter-domain interactions involved in their function (Figure 3).

In Mycobacterium tuberculosis, preferred Pks13 AT substrates are (C24-C26) 2carboxyacyl-CoA, and it was previously shown that long-chain (carboxy)palmitoylated states could be functionally and structurally characterized for L-AT-Pks13. [START_REF] Bergeret | Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13[END_REF] Ester formation did not induce significant structural changes except for R826, whose side chain moves back to establish a bidentate salt bridge with the carboxylate group in the case of carboxy-C16-CoA. The acyl chain adopts two conformations, one of which fits inside a long open preformed hydrophobic channel while the other runs perpendicularly in a deep, solvent-exposed cleft (Supporting Information Figure 5a). In L-AT-Mas, the two channels are blocked due to the presence of the side chains of residues Ile655 and Val724 in the major tunnel and of Gln540 in the exposed cleft (Supporting Information Figure 5b). The same situation occurs in AT-PpsA where the side chain of Val819 (resp. Arg638, Ile691, Glu818) would make steric clash with the acyl chain in the major (resp. minor) conformation (Supporting Information Figure 5c). In contrast, only the major cleft is blocked in PpsC through the side chains of Ile757, Val759, and Leu692 (Supporting Information Figure 5d). Thus, there is no hydrophobic tunnel in L-AT-Mas, AT-PpsA and AT-PpsC like those identified in L-AT-Pks13. As mentioned above, a very important change occurs in the position of the methionine residue from the active site G-H-S-M-G consensus motif in both PpsA (Met721) and PpsC (Met661). Such positioning is responsible for the displacement of the arginine side chain involved in bidendate salt bridge formation with the substrate. Furthermore, in the observed conformation, the methionine side chains would make steric clash with the carboxyl group of any carboxylated substrates (Supporting Information Figures 5c,d).

Mutational analysis of L-AT-Mas substrate specificity. In order to better understand the impact of the active site environment on substrate specificity, mutants of the L-AT domain of Mas were generated and their binding properties to M-CoA and MM-CoA were evaluated.

Previous studies have shown that the Ser726 residue of the Mas full-length protein plays a major role in substrate recognition and specificity. Indeed, in contrast to the wild-type Mas protein, which specifically incorporates MM-CoA, a S726F Mas mutant protein was shown to preferentially accept M-CoA as extender unit. [START_REF] Trivedi | Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid[END_REF] First using molecular modeling, then based on the crystal structure of L-AT-Mas, we identified additional amino acid residues, not involved in the catalytic machinery but potentially involved in substrate recognition: W544, V593, H622, M624, I655, V687, V722, D723, V724, A757 and N776 (Figure 2b). To confirm these predictions, we generated single-amino acid substitutions of the selected residues (Table 1).

Mutations were chosen according to a previous analysis of active site residues and additional conserved residues for the malonate specific AT domains. [START_REF] Yadav | Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases[END_REF] The generated mutants, as well as the S726F mutant and the wild-type L-AT-Mas proteins, were overexpressed in E. coli and purified to homogeneity to compare their ability to bind either MM-CoA or M-CoA. Three mutated proteins (W544R, H622Y, and A757N), which formed insoluble aggregates upon overexpression, could not be analyzed for substrate binding. As expected, wild-type L-AT-Mas exhibited a marked preference for MM-CoA over M-CoA (Table 1 and Figure 4a). On the other hand, whereas most mutations did not change the specificity towards M-CoA, two mutant proteins (S726F and N776H) exhibited a marked change in substrate preference. Indeed, these two proteins could bind MM-CoA and M-CoA with almost equal affinities (Table 1 and Figure 4a). Of note, mutated residues Val724 and Ser726 both belong to the 724 VASH sequence discussed above and their mutation led to sequences (HASH and VAFH) which are hybrids of the HAFH or YASH motifs involved in M-CoA and MM-CoA specificity, respectively. Only the transformation of VASH into VAFH resulted in a switch of specificity, but in contrast to what was previously shown, the single mutation S726F did not allow, at least in our assay, a complete inversion of specificity in L-AT-Mas. [START_REF] Trivedi | Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid[END_REF] It is noteworthy that, besides the specificity switch observed for N776H, the introduced mutation also drastically increased the affinity of L-AT-Mas for the natural substrate. To compare the relative affinity of the S726F and N776H mutant proteins for each substrate, we performed competitive binding assays by incubating the corresponding molecular species with fixed concentrations of radiolabeled M-CoA or MM-CoA and increasing concentrations of unlabeled competitive ligands. Analysis of radiolabeled proteins by SDS-PAGE and autoradiography indicated that the S726F mutant protein has a higher affinity for M-CoA than for MM-CoA whereas the N776H mutant protein has preference for MM-CoA (Figure 4b).

In an attempt to further improve the selectivity of L-AT-Mas for M-CoA, we constructed a series of double mutant proteins containing the S726F mutation in combination with substitutions that confer slightly or markedly enhanced selectivity to this substrate, i.e. V593T, M624V, V687A, D723S, V724H, and N776H (Table 1). Among these double mutants, M624V-S726F and V687A-S726F demonstrated substantially improved specificity for M-CoA compared with the S726F L-AT-Mas single mutant protein (Table 1 and Figure 4a). However, we noticed that M624V-S726F had reduced capacity to bind M-CoA and MM-CoA at 30 °C (Figure 4a). Substrate-binding capacities of this variant could greatly be improved for both substrates when assays were performed at lower temperatures (4, 12 and 20 °C) over extended periods, suggesting that the double mutation M624V-S726F may affect the ternary structure and/or binding capacity of the protein at elevated temperatures. Finally, single-residue substitutions S726F and N776H and the M624V-S726F double mutation were introduced into the Mas protein to investigate the influence of these mutations on substrate specificity in the context of the full-length protein. In good agreement with results obtained with the L-AT-Mas domain, the S726F and N776H Mas protein mutants demonstrated similar affinities for M-CoA and MM-CoA whereas the M624V-S726F double mutant protein exhibited higher selectivity toward M-CoA (Figure 4c). As observed for L-AT-Mas, the full-length M624V-S726F variant protein also displayed a weaker affinity for MM-CoA and M-CoA than the corresponding wildtype, S726F and N776H Mas proteins at 30 °C, confirming the possible effects of the double mutation on protein stability and/or ligand-binding capacity. Our analysis revealed that an almost complete inversion of specificity in Mas require secondary mutation such as that of methionine 624 (i.e. mutation M624V) that follows the catalytic serine. Overall, our study shows that the two combined mutations are located in two important sequence patterns of acyltransferases: the consensus active site sequence, which includes the catalytic serine, and the sequence previously identified as the selectivity switch. [START_REF] Reeves | Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations[END_REF][START_REF] Del Vecchio | Active-site residue, domain and module swaps in modular polyketide synthases[END_REF] As demonstrated in several studies, a mutation in only the second pattern seems to result in proteins with relaxed specificity whereas a complete switch of specificity would require secondary mutations. [START_REF] Reeves | Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations[END_REF][START_REF] Del Vecchio | Active-site residue, domain and module swaps in modular polyketide synthases[END_REF][START_REF] Sundermann | Enzyme-directed mutasynthesis: a combined experimental and theoretical approach to substrate recognition of a polyketide synthase[END_REF][START_REF] Zhang | Structural insights into the substrate specificity of acyltransferases from salinomycin polyketide synthase[END_REF] Structural dissection of L-AT-Mas extender unit specificity. To get further insight into L-AT-Mas extender unit specificity, important efforts were devoted to solve structures of complexes with M-and MM-CoA. Thus, we solved the structures of the wild-type L-AT-Mas enzyme acylated by the natural substrate MM-CoA as well as the S726F mutant protein alone and acylated with M-CoA or MM-CoA (Supporting Information Table 1). In addition, we have been able to solve the structure of the M624V-S726F doubly-mutated protein after soaking with MM-CoA (Supporting Information Table 1) but attempts to solve the structure of the double mutant after soaking with M-CoA were unfortunately unsuccessful. It is noteworthy that mutations, crystal handling and/or the strong concentrations of extender units used for soaking (final concentration 4.5 mM) had adverse effects on diffraction, leading to resolution around 3.0 Å compared to 2.4 Å for the structure of native L-AT-Mas whose crystals were already fragile. Decrease in resolution correlated very well with an increase in Wilson B factors (Supporting Information Table 1) and somehow with the diminished thermal stability of mutated proteins when assayed for substrate binding. Soaking of crystals of L-AT-Mas and of the S726F mutant protein with the natural substrate MM-CoA induced moderate structural changes in specific locations (Figure 5a). These changes were found exacerbated in the case of S726F L-AT-Mas after soaking with M-CoA (Figure 5b) and for the M624V-S726F double mutant soaked with MM-CoA (Figure 5c). Taken altogether, the most important structural changes were found in loop 11-6, at the junction between the hydrolase and ferredoxin domains, and in loop 12-7, helix 13, strand 9, and loop 9-4 (Figure 5c). The latter are all located in the ferredoxin domain, which alone seems to absorb all the variations linked to mutations and/or soaking and complex formation. L-AT-Mas crystallized in space group C2221 with two molecules in the asymmetric unit. For consistency reason, we will only focus on chain A whose active site is occupied in the different crystallographic structures solved, which is not the case for chain B (Supporting Information Table 1). Upon soaking with MM or M-CoA, the electron density maps clearly indicate that the side chain of the catalytic serine Ser623 is reoriented towards the catalytic histidine His727 whether the acylation has occurred (i.e. methylmalonylated wild type L-AT-Mas and (methyl)malonylated S726F L-AT-Mas) or not (M624V-S726F L-AT-Mas soaked with MM-CoA) (Figure 6 and Supporting Information Figure 6). It is noteworthy that such a switch of the catalytic serine upon acylation has also been described for the AT domain in the third module of ansamitocin PKS (AsmAT3). [START_REF] Zhang | Structural and biochemical insight into the recruitment of acyl carrier protein-linked extender units in ansamitocin biosynthesis[END_REF] By searching through the whole PDB, a similar conformation was also found in the structure of the KS-AT di-domain from module CurL of the curacin A polyketide synthase (4MZ0), and in the structure of mammalian FAS (2VZ8 and 2VZ9). When acylation occurs, the side chain of R648 makes polar interactions with the carboxylate group of the ligands (Figures 6a-c and Supporting Information Figures 6a-c). It is also apparent from the electron density that mutation of Ser726 to a phenylalanine does not impede complex formation. This is probably due to the discrete concerted motions observed in the ferredoxin domain, which allow steric adaption and accomodation of both malonate and methylmalonate substrates. These discrete motions accompany the movement of Phe726 to maintain the hydrophobic core of the ferredoxin domain (Figures 6b,c and Supporting Information Figures 6b,c). In contrast, the M624V-S726F double mutation has drastic structural effects, which completely abolished complex formation with MM-CoA. Indeed, no electron density was observed in the active site after soaking M624V-S726F L-AT-Mas, (Figure 6d and Supporting Information Figure 6d). In fact, the replacement of methionine by valine at position 624 leads to flipping of the guanidinium group of Arg648 to avoid sterical clash. Furthermore, Phe726 is displaced and reoreinted towards the active site so that the phenyl group is found at the position occupied by the methyl branch of the ligand.

The movement of Val624 and Phe726 has drastic effect on the positions of several residues from the ferredoxin domain (Figure 6d and Supporting Information Figure 6d).

CONCLUSIONS

PKSs are essential players in fundamental biological processes contributing to the chemical diversity observed in life, the most obvious example being the biosynthesis of molecules of pharmaceutical interest. Human and animal pathogens also use this macromolecular arsenal extremely efficiently. This is the case of mycobacteria, for which the biogenesis of the highly complex cell envelope involves a large number of PKSs. One key component of PKSs are AT domains, which control the incorporation of building blocks. Thus, strategies used for engineering of PKSs may rely on AT domain swapping/substitution or AT-site directed mutagenesis. [START_REF] Musiol-Kroll | Acyltransferases as tools for polyketide synthase engineering[END_REF] With that respect, essential information have been brought by molecular and structural studies of AT domains. However, a search through the whole PDB identified only a very few structures of AT domains trapped in the acyl-bound state, which correspond in all to six publications, of which four are related to PKSs. [START_REF] Bergeret | Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13[END_REF][START_REF] Zhang | Structural and biochemical insight into the recruitment of acyl carrier protein-linked extender units in ansamitocin biosynthesis[END_REF][START_REF] Li | Structural basis of a broadly selective acyltransferase from the polyketide synthase of splenocin[END_REF][START_REF] Liew | Crystal structure of the acyltransferase domain of the iterative polyketide synthase in enediyne biosynthesis[END_REF] Our work on three new mycobacterial PKSs and, in particular, the resolution of structures of mutants and complexes for one of them, reinforces the numerous efforts undertaken for the molecular understanding of AT domains.

They also show that a double mutation on the central units containing the catalytic dyad can reverse the specificity and that plasticity in the AT ferredoxin subdomain plays an essential role for substrate specificity, as evidenced by X-ray crystallography. The observed plasticity exquisitely extends to the PKS family of megasynthases a major conclusion drawn on type II FAS ACP-AT interactions. [START_REF] Misson | Interfacial plasticity facilitates high reaction rate of Escherichia coli FAS malonyl-CoA:ACP transacylase, FabD[END_REF] The information obtained will help better predict the specificity of polyketide synthases, opening the way to the rational manipulation of these systems to obtain new polyketide skeletons and the rational design of inhibitors, a key step in the development of new antibiotics against mycobacterial infections.

Supporting Information Available: Detailed information about methods are available free of charge via the internet at http://pubs.acs.org. superimposed with native L-AT-Mas (color-coded as in Figure 2). The ligands are in green and the mutated residues in red. The side chains of mostly affected regions, i.e. displacement (d) greater than 2  rmsd after superimposition of C carbon atoms (as in Figure 5), are depicted in magenta and labeled. Feature-enhanced map (FEM) 34 contoured at 1.0  is shown in blue for residues from the active site.

Figure 1 .

 1 Figure 1. Mycolic acids, phthiocerol dimycocerosates (DIMs) and phenolic glycolipids (PGLs). (a) Chemical structures. The motifs synthesized by Pks13, PpsA, PpsC and Mas are boxed in gray. (b) Domain organization with corresponding residue boundaries (taken from 5 )and related PDB entries, including those from this work (7AGP, 7AHB, and 7AKC). PDB entries 5BP1, 5BP2 and 5BP3 are from the M. smegmatis Mas-like PKS.[START_REF] Herbst | Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases[END_REF] Abbreviations: ACP, acyl carrier protein; AT, acyltransferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KS, ketosynthase; L, KS-to-AT linker domain. Note: mycolic acids of mycobacteria display a large diversity of chain lengths and different chemical modifications.[START_REF] Marrakchi | Mycolic acids: structures, biosynthesis, and beyond[END_REF]

Figure 2 .

 2 Figure 2. Crystal structures of L-AT-Pks13 (PDB code 3TZX), L-AT-Mas, AT-PpsA and AT-PpsC. (a) Overall structures in ribbon representation. The /-hydrolase subdomain is in orange, the ferredoxin-like subdomain is in yellow, the KS-AT L linker is in cyan, and the AT-ACP linker is in red. Helices and -strands of Pks13 are numbered as in 17 . The histidine and serine residues forming the catalytic dyad and the arginine involved in substrate binding are shown as sticks and boxed. The same color scheme has been applied throughout all the figures. (b) Detailed description of the active sites. Important residues defining the active sites are shown and labeled. Hydrogen bonds are represented by black dotted lines. Water molecules in proximity of displayed residues are shown as red spheres. L-AT-Mas residues that were mutated in the course of this study are depicted in gray with their labels underlined.

Figure 3 .

 3 Figure 3. Topography of the active site. (a) Molecular surface with electrostatic potential colorcoded from red (-6 kBT/e) to blue (6 kBT/e); white is neutral. Residues of L-AT-Pks13 delineating the groove overhanging the active site and the exit of the channel are highlighted in black as in Supporting Information Figure 4. Corresponding areas of L-AT-Mas, AT-PpsA and AT-PpsC are also shown. (b) Semi-perpendicular view.

Figure 4 .

 4 Figure 4. Substrate specificity of the L-AT-Mas and full-length Mas mutants. (a) Ligand binding assays with L-AT-Mas mutants. Purified proteins were incubated with 14 C-radiolabeled M-CoA or MM-CoA. Samples were then subjected to SDS-PAGE followed by staining with Coomassie blue and autoradiography. The bar graph (bottom) summarizes results from at least three independent experiments for each L-AT-Mas variant. Data shown are the means ± SD of

Figure 5 .

 5 Figure 5. Effects of mutations and substrate binding on the L-AT-Mas structure. (a) Comparison of native (color-coded as in Figure 2) and methylmalonylated (black) L-AT-Mas. (b) Comparison of native L-AT-Mas with S726F L-AT-Mas alone (light gray) and after malonylation (middle gray) or methylmalonylation (black). (c) Comparison of native L-AT-Mas with M624V-S726F L-AT-Mas soaked in MM-CoA (black). Top raw, overall structures depicted as ribbon with mostly affected regions, i.e. displacement (d) greater than 2  rmsd after superimposition of C carbon atoms, depicted in magenta on the corresponding MM-CoA structure and labeled. Bottom raw, displacement (d) analysis after superimposition of C carbon atoms of native L-AT-Mas with structures obtained after soaking with MM-CoA: left, methylmalonylated L-AT-Mas; middle, methylmalonylated S726F L-AT-Mas; right, unbound M624V-S726F L-AT-Mas. Continuous and dashed lines are for chain A and chain B,

Figure 6 .

 6 Figure 6. Crystallographic analysis of extender unit specificity for wild type and mutant L-AT-Mas proteins. (a) Structure of (2S)-methylmalonylated wild type L-AT-Mas. (b) Structure of (2S)-methylmalonylated S726F L-AT-Mas. (c) Structure of malonylated S726F L-AT-Mas. (d) Structure of unbound L-AT-Mas M624V-S726F. Structures are shown as black ribbon and were

Figure 3 a

 3 Figure1

Figure 4 A

 4 Figure 4

Table 1 . Substrate specificity of L-AT-Mas mutants.

 1

	Mutation(s)	M vs MM (%) a
	WT	4.3 ± 1.7 (11) b
	V593T	20.1 ± 3.4 (3)
	M624V	7.8 ± 1.8 (5)
	I655L	3.2 ± 2.0 (3)
	V687A	8.3 ± 5.4 (3)
	V722I	5.1 ± 2.6 (5)
	D723S	9.4 ± 1.7 (3)
	V724H	5.8 ± 3.6 (3)
	S726F	125 ± 17.5 (8)
	N776H	95.9 ± 5.4 (4)
	V593T-S726F	117.0 ± 12.2 (5)
	M624V-S726F	225.0 ± 44.7 (6)
	V687A-S726F	216.0 ± 9.9 (3)
	D723S-S726F	130.0 ± 12.5 (5)
	V724H-S726F	103.0 ± 6.2 (4)
	N776H-S726F	91.8 ± 10.8 (4)

a For each protein, data shown are the mean ± SD of percentage of M fixation relative to MM fixation, arbitrarily set at 100%. b Values in parentheses represent the number of independent experiments for each mutant.

ACKNOWLEDGMENTS

We thank the scientific staff of the European Synchrotron Radiation Facility (Grenoble, France) and SOLEIL (Gif sur Yvette, France). We particularly thank the staff of beamlines SWING and PROXIMA-1 at SOLEIL and ID23-1 and 2 at the European Synchrotron Radiation Facility, where the SAXS and crystallographic experiments were conducted. We thank F. Bergeret for his help with the design of protein constructs and V. Guillet for managing synchrotron radiation trips. The crystallization and macromolecular crystallography equipment used in this study are part of the Integrated Screening Platform of Toulouse (PICT, IPBS, IBiSA).

denis.pedelacq@ipbs.fr Christian Chalut -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France; Email: christian.chalut@ipbs.fr Lionel Mourey -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France; orcid.org/0000-0002-8259-1259; Email: lionel.mourey@ipbs.fr Authors Anna D. Grabowska -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France Present address: Department of Biophysics and Human Physiology, Faculty of Health Sciences, Medical University of Warsaw, Poland Yoann Brison -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France Present address: Toulouse White Biotechnology, TWB, INRAE, INSA, CNRS, 31520 Ramonville Saint-Agne, France Laurent Maveyraud -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France Sabine Gavalda -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France Present address: Toulouse Biotechnology Institute, TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France Alexandre Faille -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France Present address: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK Virginie Nahoum -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France Cécile Bon -Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France Funding The authors gratefully acknowledge financial support from the Agence Nationale de la Recherche grant ANR-09-BLAN-0298-01 (to C.G. and L.Mo.).

Notes

The authors declare no competing financial interest.