Claire Debarnot

Yoan R Monneau

Veronique Roig-Zamboni

Vincent Delauzun

Christine Le Narvor

Emeline Richard

Jérôme Hénault

Adeline Goulet

Firas Fadel

Romain R Vivès

Véronique Roig-Zamboni

Bernard Priem

David Bonnaffé

Hugues Lortat-Jacob

Yves Bourne
email: yves.bourne@afmb.univ-mrs.fr.

Y B Wrote

Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase

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

H eparan sulfate (HS) belongs to a family of linear anionic polysaccharides, the glycosaminoglycans, which are present at the cell surface and within the extracellular matrix [START_REF] Lindahl | Proteoglycans and Sulfated Glycosaminoglycans[END_REF]. Through their ability to bind a wide array of proteins and modulate their local concentration, stability, structure, and/or activity, HS participates in most major cellular processes, including cell-cell signaling [START_REF] Poulain | Heparan sulfate proteoglycans: A sugar code for vertebrate development?[END_REF][START_REF] Sarrazin | Heparan sulfate proteoglycans[END_REF]. From a structural standpoint, the HS-mediated binding activities, which are closely linked to the extensive structural variability of the polymer chain, are generated during a multistep biosynthesis process occurring predominantly in the Golgi apparatus. This process comprises the polymerization of a disaccharide repeat consisting of alternate GlcA and GlcNAc, followed by a series of modification reactions that include (i) GlcNAc N-deacetylation and N-sulfation, yielding N-sulfated glucosamine (GlcNS); (ii) C5-epimerization of GlcA into IdoA; and (iii) variable sulfation at O2 of IdoA and GlcA units, O6 of GlcNAc and GlcNS units, and occasionally O3 of GlcNS units [START_REF] Esko | Order out of chaos: Assembly of ligand binding sites in heparan sulfate[END_REF][START_REF] Kreuger | Heparan sulfate biosynthesis: Regulation and variability[END_REF]. Remarkably, these modifications occur not uniformly along the polymer, but rather within relatively short segments of usually three to six disaccharides (known as S-domains) that display tremendous variations in epimerization degree and sulfation patterning (ref. [START_REF] Xu | Demystifying heparan sulfate-protein interactions[END_REF] and references therein). It is commonly known that such structural variability may confer critical and biologically relevant docking sites for protein ligands [START_REF] Xu | Demystifying heparan sulfate-protein interactions[END_REF][START_REF] Imberty | Structural view of glycosaminoglycanprotein interactions[END_REF][START_REF] Kjellén | Specificity of glycosaminoglycan-protein interactions[END_REF]), yet the mechanisms by which the sulfation and epimerization patterns are defined and regulated remain unclear.

To better understand how HS functional motifs are built, we investigated structures of Glce, the key enzyme involved in reversible epimerization of GlcA into IdoA, the ratio of which greatly contributes to generate functional protein-binding motifs. Indeed, unlike most monosaccharides, IdoA can adopt two distinct ring conformations, 1 C 4 or 2 S O , conferring to the polymer local plasticity with enhanced binding properties that are commonly associated with the biological properties of the HS chains [START_REF] Casu | Structure and biological interactions of heparin and heparan sulfate[END_REF][START_REF] Capila | Heparin-protein interactions[END_REF]. In addition, IdoA is 2-O sulfated more efficiently than GlcA [START_REF] Rong | Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase[END_REF], a modification that prevents reverse epimerization into GlcA and strongly facilitates interaction with most HSbinding proteins. Not surprisingly, Glce-deficient mice (Glce -/-) display severe developmental defects resulting in a lethal phenotype, emphasizing the essential interplay between Glce and Significance Heparan sulfate (HS) belongs to the glycosaminoglycan family of carbohydrates anchored to core proteins and regulate many cell-cell signaling events. The HS large structural variability, orchestrated by sequential enzymatic modifications of the polymer, greatly regulates HS binding activities; however, the molecular mechanisms associated with these modification reactions are poorly documented. To decipher the mode of substrate binding and catalytic mechanism of the enzyme Glce, which converts the HS component GlcA into IdoA and dictates the epimerization profile for polymer activity, we combined glycoengineering, glycochemistry, biochemistry, and structural biology approaches. Our data unveil selective conformational distortions of the HS precursor chain on binding and document how a mechanistic strategy shared with mandelate racemase and HS lyases evolved toward a specific activity.

IdoA residues for polymer function [START_REF] Li | Targeted disruption of a murine glucuronyl C5-epimerase gene results in heparan sulfate lacking L-iduronic acid and in neonatal lethality[END_REF][START_REF] Feyerabend | Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells[END_REF]. In Caenorhabditis elegans, various HS-modifying enzymes have very specific effects in nervous system development, and a deficiency in Glce severely affects axonal development [START_REF] Bülow | Differential sulfations and epimerization define heparan sulfate specificity in nervous system development[END_REF]. In humans, the missense polymorphism SNP rs3865014 (A > G, Val597Ile) in the glce gene is associated with breast cancer susceptibility in Siberian women [START_REF] Grigorieva | Decreased expression of human D-glucuronyl C5-epimerase in breast cancer[END_REF][START_REF] Prudnikova | Antiproliferative effect of D-glucuronyl C5-epimerase in human breast cancer cells[END_REF][START_REF] Belyavskaya | GLCE rs3865014 (Val597Ile) polymorphism is associated with breast cancer susceptibility and triple-negative breast cancer in Siberian population[END_REF], consistent with the tumor-suppressor function of Glce in breast and lung carcinogenesis.

Crystal structures of zebrafish Glce (zGlce), unliganded and bound to a heparin-derived hexasaccharide, were recently reported [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF]. However, heparin is an inhibitor, not a substrate, of Glce, and the high level of 2-and 6-O sulfation might have impaired correct positioning of the hexasaccharide in the catalytic cleft. To decipher the mode of substrate binding and the catalytic mechanism of the epimerization reaction, we chemoenzymatically generated a series of GlcA-GlcNS disaccharide oligomers of various degrees of polymerization (dp6, dp8, and dp10) as natural substrates, and chemically synthesized the (IdoA-GlcNS) 4 reaction product [START_REF] Dilhas | Mixture synthesis and "charge tagging"-based demixing: An efficient strategy for the preparation of heparan sulfate libraries[END_REF][START_REF] Lubineau | Synthesis of tailor-made glycoconjugate mimetics of heparan sulfate that bind IFN-gamma in the nanomolar range[END_REF]. To document the mode of substrate binding and key residues involved in the epimerization mechanism, we solved crystal structures of human Glce (hGlce) in the unliganded form and of an inactive mutant as complexes with the dp10 substrate and the product, respectively, and recorded real-time NMR-based kinetics for hGlce and three active site mutants. We show that the O,3 B/B 1,4 constrained conformations of the GlcA/IdoA rings at the epimerization site (i) favor in-line abstraction/reprotonation on the axially oriented C5 hydrogen by two suitably poised acid/base residues; (ii) limit motion of the hexuronic acid carboxylic group; and (iii) facilitate the reaction by stabilizing high-energy boat conformers closely located on a conformational itinerary. Our data also reveal that Glce stabilizes a neutral enol intermediate, reminiscent of mandelate racemase, and preserves the glycosidic linkage while sharing part of its catalytic mechanism with heparin-sulfate lyases.

Results and Discussion

Overall Structures of hGlce. A recombinant, soluble form of hGlce (Val99-Asn617) was produced from a stabilized mammalian cell line and purified to homogeneity using complementary chromatography procedures (Materials and Methods). Real-time monitoring of the enzymatic activity of this recombinant hGlce by NMR spectroscopy using a 13 C-labeled N-sulfated heparosan substrate revealed efficient conversion of GlcA to IdoA in the first 15 min to rapidly reach an equilibrium with a final IdoA content of ∼38% (Fig. 1A and SI Appendix, Table S1), a value consistent with the 1:3 IdoA/GlcA molar ratio reported for natural Glce from bovine liver or mouse mastocytoma [START_REF] Hagner-Mcwhirter | Biosynthesis of heparin/heparan sulphate: Mechanism of epimerization of glucuronyl C-5[END_REF][START_REF] Malmström | Biosynthesis of heparin: Partial purification of the uronosyl C-5 epimerase[END_REF].

The 2.5-Å resolution structure of hGlce in the apo form shows an overall dimeric assembly and well-defined electron densities for most of the protein regions, along with well-ordered glycan chains harboring up to six units at all three potential Nglycosylation sites (Asn225, Asn303, and Asn393) (Fig. 1B). Each subunit consists of three intertwined domains of a predominantly basic character, as observed for zGlce [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF]. The Nterminal 54-residue β-hairpin domain is composed of two upsidedown/antiparallel β-hairpin motifs connected by a short helix. The 225-residue classical β-sandwich domain consists predominantly of a convex four-stranded β-sheet and a concave seven-stranded β-sheet. The dominant 285-residue C-terminal (α/α) 4 -barrel domain, which comprises the active site cleft, is composed of two noncontiguous regions and consists of four internal, mutually parallel α-helices interconnected by four external helices (Fig. 1B and SI Appendix, Fig. S1). The large β-sandwich domain is inserted between helices α1 and α2 from the α-barrel domain. The stable hGlce dimer forms by intersubunit swapping of the N-terminal β-hairpin and C-terminal α-helical barrel regions from each subunit. As a result, the subunit interface buries, to a 1.4-Å probe radius, a surface area of ∼3,100 Å 2 on each subunit, a value consistent with the stable dimer of ∼130 kDa observed by gel filtration. The N-glycans are clustered at the distal ends of the dimer near the convex and concave faces of the β-sandwich domain. Opposite to the active site cleft, the β-sandwich and α-barrel domains are separated by a deep, narrow channel, 10 Å long and 3 Å wide, which could favor interdomain motions and provide functional plasticity to the overall assembly.

In each subunit, a Ca 2+ is bound at the periphery of the β-sandwich domain, remote from the catalytic cleft but close to the Asn393-linked glycan, with a characteristic pentagonal bipyramidal geometry tightly coordinated by the invariant Glu240, Thr268, and Asp392 (bidentate ligation) side chains; the Thr238 and Asn269 main chain carbonyls; and a water molecule (Fig. 1B). Compared with zGlce, in which a water molecule occupies the same position as the bound Ca 2+ in hGlce, large conformational adaptations of the β1-β2 loop region in the β-sandwich domain, with movements up to 9 Å, ideally position the hGlce Thr238 and Glu240 residues to satisfy the Ca 2+ coordination geometry.

Structures of the hGlce Inactive Mutant Tyr578Phe Bound with a (GlcA-GlcNS) 5 Substrate and a (IdoA-GlcNS) 4 Product. Previous structure-guided mutagenesis of active site residues in zGlce identified the three invariant residues Tyr468, Tyr528, and Tyr546, corresponding to hGlce residues Tyr500, Tyr560, and Tyr578, as crucial for the enzyme activity (Fig. 1B) [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF]. Consistently, no catalytic activity was detected by real-time NMR spectroscopy for the hGlce Tyr578Phe mutant assayed at up to 400 nM, compared with hGlce at 40 nM (Fig. 1A). We used this inactive mutant to solve complementary structures of a complex with the chemoenzymatically prepared (GlcA-GlcNS) 5 substrate and a complex with the chemically synthesized (IdoA-GlcNS) 4 product (SI Appendix, Figs. S2 andS3). The 2.1-Å structure of the Tyr578Phe mutant bound with the dp10 substrate shows well-defined electron density maps for up to seven monosaccharide units (labeled -4 to +3 from the nonreducing end to the reducing end), indicative of a high occupancy for each of the two substrate molecules bound within the two active site clefts in the hGlce dimer (Fig. 2A). In each subunit, the negatively charged substrate is bound within a positively charged, elongated, and curved cleft that spans ∼25-30 Å in length (Figs. 1B and2A). The cleft is formed at the N-terminal ends of the central helices from the α-barrel domain with strong participation of the sparsely structured Tyr158-Tyr210 linker that connects the β-hairpin to the α-barrel and helix α1. The composite nature of the catalytic cleft composed of residues from both subunits and intricate domains is illustrated by the absolute requirement of the C-terminal Asn617 residue at the end of helix α8 for substrate binding. In addition, an extended β-hairpin, which connects α2 and α3 from the α-barrel domain, projects over the active site cleft near the reducing end of the substrate (Fig. 2A). A similar overall conformation of the bound dp10 was observed in a 3.1-Å structure of hGlce also solved during this study, indicating that a lack of the hydroxyl group in the Phe578 mutant does not alter substrate binding.

At the epimerization site, the shape of the active site cleft imposes a sharply bent conformation that spans the central GlcNS-GlcA-GlcNS trisaccharide at the -1 to +2 subsites and constrains the GlcA unit at subsite +1 into a boat conformation in the vicinity of O,3 B, as opposed to a predominant and more stable 4 C 1 chair conformation observed for the other GlcA rings. A dense network of interactions involving the GlcA endocyclic oxygen atom, the two transdiaxially oriented exocyclic O2-O3 hydroxyl groups, and the surrounding Gln202, Tyr210, Glu499, and Tyr500 side chains stabilize the distorted GlcA ring and bent trisaccharide conformation (Fig. 2A). The GlcA carboxyl group establishes short H bonds with the Asn510 Oδ and Tyr560 hydroxyl and faces the Asn510 side chain in a parallel arrangement. At subsite -1, the GlcNS moiety engages extensive polar interactions with a constellation of residues (Tyr180, Arg185, Arg187, Gln216, and Tyr514). Similarly, GlcNS at subsite +2 establishes numerous interactions with the Tyr500, Tyr560, mutated Phe578, and Arg575 side chains. As for subsite -1, the N-sulfate is anchored firmly to the Arg563 and Asn572 side chains and the Leu430 backbone carbonyl, highlighting the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the reducing end, the carboxyl group of GlcA at subsite +3 interacts with the Gln202-Trp203 residue pair, and the O3 hydroxyl is tightly bound to the swapped Asn617 carboxyl terminus arising from the second subunit. The remaining three GlcA-GlcNS-GlcA units, which occupy the -2 to -4 subsites, respectively, at the nonreducing end, moderately interact with hGlce. At subsite-2, the GlcA ring stacks against the Tyr180 phenol ring and the O2 and carboxyl group interact with the Thr581 hydroxyl, Arg185 side chain, and Asn181 backbone amide. At subsite -3, only the N-sulfate group of GlcNS is bound to the backbone carbonyl of Gly179, leaving sufficient space to accommodate a GlcNAc unit, a feature consistent with an irreversible versus reversible mode of epimerization if a GlcNAc or GlcNS unit occupies site -3 [START_REF] Sheng | Uncovering biphasic catalytic mode of C 5 -epimerase in heparan sulfate biosynthesis[END_REF]. The distal GlcA residue at subsite -4 only establishes a weak van der Waals interaction with Val184. Overall, water-mediated contacts occur mainly near the substrate ends, highlighting the complementary shapes of the enzyme and the GlcNS-GlcA-GlcNS trisaccharide motif at the epimerization site. The 2.45-Å structure of the Tyr578Phe mutant bound to the dp8 product shows well-defined electron density maps at the -4 to +4 subsites strikingly overlapping the corresponding units in the substrate complex, including the bent conformation of the central GlcNS-IdoA-GlcNS trisaccharide motif (Fig. 2B). Apart from small displacements of the ring atoms at both ends, the GlcA-to-IdoA epimerization process is not associated with significant conformational adaptation of the interacting residues. At the epimerization site, IdoA adopts a B 1,4 boat conformation, close to the O,3 B boat conformation of the corresponding GlcA in the hGlce-substrate complex (Figs. 2B and3A). In contrast, the other IdoA rings adopt the standard and less strained 2 S O skew-boat conformation. The short distance (∼3.0 Å) between the IdoA O2 hydroxyl and the adjacent GlcNS sulfamido group at subsite -1, along with the nearby Arg187 and Gln202 side chains, is incompatible with binding of a 2-O-sulfated IdoAcontaining substrate, as reported previously [START_REF] Jacobsson | Biosynthesis of heparin: Substrate specificity of heparosan Nsulfate D-glucuronosyl 5-epimerase[END_REF]. Similarly, the IdoA mode of binding at subsite -2 clearly prevents accommodation of a 2-O sulfate group, while it appears to be sterically tolerated at subsite +3. Consequently, the corresponding IdoA-2S in the zGlce-heparin structure is shifted by ∼7 Å away from the active site, associated with large movement of the loop tip harboring the Gln171-Trp172 residue pair (hGlce Gln202-Trp203) along with conformational adaptation of the nearby Arg156 guanidinium group (hGlce Arg187) (Fig. 2C) [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF].

Catalytic Mechanism and Mutational Analysis. Several C5-epimerases have been cloned and biochemically characterized [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF][START_REF] Li | Biosynthesis of heparin/heparan sulfate: cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung[END_REF][START_REF] Li | Characterization of the Dglucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate[END_REF][START_REF] Crawford | Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase[END_REF][START_REF] Préchoux | C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-Osulfated heparan sulfate domains[END_REF], and a reaction mechanism occurring via reversible abstraction and readdition of a proton at the GlcA/IdoA C5 position/atom, through an α-carbanion or enolate intermediate, has been proposed [START_REF] Prihar | Biosynthesis of heparin. Hydrogen exchange at carbon 5 of the glucuronosyl residues[END_REF]. Identification of the catalytic residues of Glce and of unique structural features in the substrate/product binding mode provides insight into the C5-epimerization mechanism marked by low-barrier H bonds [START_REF] Cleland | Low-barrier hydrogen bonds and low fractionation factor bases in enzymatic reactions[END_REF]. Moreover, stabilization of a neutral enol intermediate coupled with selective distortion of carbohydrate ring geometry along a particular catalytic puckering itinerary provide driving forces for the epimerization process. This study also reveals that the hGlce Tyr168 and Tyr222 residues, both of which are reportedly involved in catalysis [START_REF] Li | Using engineered 2-O-sulfotransferase to determine the activity of heparan sulfate C5-epimerase and its mutants[END_REF], do not participate in substrate binding, but substitutions at these positions might affect protein folding.

The structures indicate that the Tyr578 phenolic oxygen is poised (OH-C5 distance, ∼2.7 Å) to function as a general base for abstracting the C5 proton of the GlcA unit (Fig. 3A), consistent with inactivity of the Tyr578Phe mutant (Fig. 1A). The epimerization mechanism requires neutralization of the negative charge on the GlcA carboxylate anion to reduce the pK a value of the C5 hydrogen (α-proton). In this context, hGlce uses a strategy similar to that of the chondroitin AC lyase (32) and heparin lyase I (33), with the Asn510 side chain and Tyr560 hydroxyl nearly coplanar to the GlcA carboxylic group (Fig. 3A). Such a topology requires a protonated acid group from which a neutral enol intermediate can efficiently form (Fig. 3B). Thus, the Tyr560 hydroxyl, which is not ideally positioned to act as a general base, is poised to provide a proton relay path to the solvent, consistent with inactivity of the corresponding Tyr528-Phe mutant in zGlce [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF]. This mechanistic pathway resembles the Mg 2+ -dependent α-proton transfer reaction of carboxylic acid substrate catalyzed by mandelate racemase [START_REF] Bearne | A paradigm for CH bond cleavage: Structural and functional aspects of transition state stabilization by mandelate racemase[END_REF] and is kinetically more competent than the previously proposed reaction pathway involving formation of a α-carbanion or enolate intermediate [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF][START_REF] Hagner-Mcwhirter | Biosynthesis of heparin/heparan sulphate: Mechanism of epimerization of glucuronyl C-5[END_REF]. On the opposite side of the GlcA/IdoA pyranose ring, the glutamate of the Glu499-Tyr500 residue pair, with an Oe-C5 distance of ∼2.8 Å compared with the OH-C5 distance of ∼3.8 Å in the product-bound structure, is the best candidate to serve as a general acid (Fig. 3A). The nearby Tyr500, Arg428, and possibly Tyr210 side chains could provide a proton relay with the solvent. This active site topology is consistent with the inactivity of the Glu499Gln mutant relative to hGlce, compared with a decreased activity of only ∼29% for the Tyr500Phe mutant (Fig. 1A and SI Appendix, Table S1). Most of the hGlce residues interacting with the substrate/product oligosaccharide, as well as the two catalytic glutamate and tyrosine residues, are conserved in eukaryotic D-glucuronyl C5-epimerase family and in the bacterial D-glucuronyl C5-epimerase (RED65) and the heparosan-glucuronate 5-epimerase (HG-5epi) (SI Appendix, Fig. S4) [START_REF] Raedts | A novel bacterial enzyme with D-glucuronyl C5-epimerase activity[END_REF][START_REF] Mochizuki | Heparosan-glucuronate 5-epimerase: Molecular cloning and characterization of a novel enzyme[END_REF], arguing for a similar C5 proton abstraction/ readdition mechanism. Structural comparison of the substrate-and product-bound mutant reveals a nearly identical conformation of the two GlcA/ IdoA rings at the epimerization site, besides the two C5 atoms, distant by 0.8 Å. Thus, the active site of Glce has evolved to enable selective motion of the C5-epimerized center through the improper C5-O5-C6-C4 dihedral angle (Fig. 3A) while maintaining tighter conformation of the other GlcA/IdoA ring atoms and the two flanking GlcNS units. Most notably, ring distortion appears to act as an additional driving force for Glce activity. Indeed, the Cremer-Pople puckering parameters (37) of the substrate and product rings reveal unusual conformations in the vicinity of O,3 B and B 1,4 for the respective GlcA and IdoA units at the catalytic site, while the other GlcA/IdoA units fall within the most stable and populated 4 C 1 / 2 S O conformations. The O,3 Band B 1,4 -constrained conformations of GlcA/IdoA not only destabilize the ground states, but also preactivate the substrate/ product rings at subsite +1 by bringing them into closely related conformations while maintaining the collinearity and axial conformation of the C5-H5 bond. Indeed, the O,3 B and B 1,4 ring conformations lie on a conformational itinerary interconverting 4 C 1 GlcA to 2 S O IdoA through the less-constrained skew boat 3 S 1 conformation suitable for a neutral enol intermediate (Fig. 3B and SI Appendix, Fig. S5A). Taken together, these data support previous kinetic isotope effects showing that readdition of a proton to the intermediate is the rate-limiting step of the epimerization reaction [START_REF] Hagner-Mcwhirter | Biosynthesis of heparin/heparan sulphate: Mechanism of epimerization of glucuronyl C-5[END_REF]. Indeed, the higher-energy O,3 B/B 1,4 conformations of the GlcA/IdoA rings should permit a decrease in the activation energy needed for carboxylic acid tautomerization into a neutral enol intermediate that could adopt a more relaxed 3 S 1 conformation stabilized by low-barrier H bonds (SI Appendix, Fig. S5B). In fact, several oligosaccharide-degrading enzymes (e.g., lyases such as chondroitin AC lyase) are known to force the substrate to bind with the key uronic acid in an energetically less favorable nonchair conformation to improve catalytic efficiency.

The unified mechanism of action proposed for the HS lyases and epimerases [START_REF] Gacesa | Alginate-modifying enzymes: A proposed unified mechanism of action for the lyases and epimerases[END_REF], which involves a similar C5 proton abstraction, raises an intriguing question as to how Glce prevents glycosidic bond cleavage through a β-elimination reaction [START_REF] Li | Using engineered 2-O-sulfotransferase to determine the activity of heparan sulfate C5-epimerase and its mutants[END_REF][START_REF] Shaya | Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product[END_REF]. In the lytic reaction, the proton must be donated to the leaving group, while for the epimerase, it should occur on the C5 of the intermediate, emphasizing the need for accurate positioning of the general acid for proton donation (Fig. 3B). In Glce, the optimal position of the two catalytic residues on both sides of the GlcA/IdoA C5 atom and the lack of a general acid residue proximal to the otherwise "scissile" glycosidic bond support the concept of aborted β-elimination for uronate epimerases by preventing protonation of the glycosidic bond oxygen (Fig. 3B). Moreover, the dense intramolecular and intermolecular interaction network stabilizes the conformation of the target uronic acid along the O,3 B-3 S 1 -B 1,4 conformational itinerary (Fig. 2A). Thus, the tetrahedral geometry of the C4 carbon is preserved, which prevents this carbon from converting into the flat form that would be required for the anionic E1cB elimination mechanism of lyases. However, Glce shows no significant sequence homology with Mg 2+ -dependent dermatan sulfate epimerase and Ca 2+ -dependent alginate epimerase, the other two types of epimerases acting on HexA residues in polysaccharides, suggesting that these epimerases have acquired a similar function by convergent evolution [START_REF] Valla | Hexuronyl C5-epimerases in alginate and glycosaminoglycan biosynthesis[END_REF][START_REF] Maccarana | Biosynthesis of dermatan sulfate: Chondroitin-glucuronate C5-epimerase is identical to SART2[END_REF][START_REF] Thelin | Biological functions of iduronic acid in chondroitin/dermatan sulfate[END_REF].

Molecular Bases of the Nand O-Sulfation Pattern Specificity of Glce.

The structure of the hGlce-substrate complex is consistent with an octasaccharide being a substrate of minimum size for the epimerization reaction, since no activity was detected for a tetrasaccharide or hexasaccharide [START_REF] Jacobsson | Biosynthesis of heparin: Substrate specificity of heparosan Nsulfate D-glucuronosyl 5-epimerase[END_REF][START_REF] Préchoux | C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-Osulfated heparan sulfate domains[END_REF]. Moreover, the topology and distribution of polar and aromatic residues in the active site cleft impose unique structural constraints on the substrate/ product conformations at the epimerization site, emphasizing the stringent substrate specificity of Glce relative to GlcN N-/6-O-sulfation and GlcA 2-O-sulfation on either side of the GlcA/ IdoA units at subsite +1 (Fig. 4). Except for the GlcNS units located at the distal ends of the substrate/product, the N-sulfate groups of the two GlcNS units adjacent to the epimerization site are deeply anchored in the binding cleft, consistent with the absolute requirement for an N-sulfate group at subsite -1 for the epimerization reaction to occur [START_REF] Sheng | Uncovering biphasic catalytic mode of C 5 -epimerase in heparan sulfate biosynthesis[END_REF][START_REF] Hagner-Mcwhirter | Irreversible glucuronyl C5epimerization in the biosynthesis of heparan sulfate[END_REF][START_REF] Hagner-Mcwhirter | Biosynthesis of heparin/heparan sulfate: Kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates[END_REF]. In contrast, O-sulfation at O2 of IdoA and or O6 or O3 of GlcN units at subsite -2 to +2 lead to HS ligand binding in a nonproductive, extended conformation only partially overlapping with substrate binding, as has been reported for the structure of the crystalline zGlce-heparin hexasaccharide complex [START_REF] Qin | Structural and functional study of D-glucuronyl C5-epimerase[END_REF], illustrating the molecular basis for substrate specificity and heparin inhibition by Glce (Fig. 2C).

In summary, our structural analysis of apo hGlce and its substrate/product-bound inactive mutant has unveiled the catalytic mechanism of the HS C5 epimerases shared by other members of the family and clarified the structural bases by which Glce regulates HS structure and function. Although Glce appears to use many of the strategies used by polysaccharide lyases and mandelate racemases, including a distorted GlcA/IdoA ring geometry, we note subtle differences that likely evolved in response to unique chemical structures of the HS polymer. The structural variability observed for the HS precursor chain highlights the importance of conformational plasticity for HS structure-function activities. In addition, these structures provide a detailed atomic framework for future development of Glce inhibitors or chemoenzymatically synthesized heparin/HS analogs. Given the convergent mechanistic strategy between Glce and lyases, converting lyases into novel enzymes with epimerase activities could be considered, thereby extending current possibilities to alter uronate residues at the polymer level.

Materials and Methods

A soluble form of hGlce (Val99-Asn617; National Center for Biotechnology Information Gene ID 26035) and its Tyr500Phe and Tyr578Phe mutants were stably expressed in HEK-EBNA cells (from the National Research Center Canada, Biotechnology Research Institute), while the Glu499Gln mutant was transiently expressed from Expi293F cells. All proteins were purified to homogeneity using conventional procedures. The (GlcA-GlcNS) 3-5 oligosaccharides were prepared from a mixture of dp6, dp8, and dp10 heparosan (GlcA-GlcAc) as described previously [START_REF] Barreteau | Production of intracellular heparosan and derived oligosaccharides by lyase expression in metabolically engineered E. coli K-12[END_REF] and N-sulfated as described previously [START_REF] Préchoux | C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-Osulfated heparan sulfate domains[END_REF]. The (IdoA-GlcNS) 4 octasaccharide was prepared by chemical synthesis as described previously [START_REF] Lubineau | Synthesis of tailor-made glycoconjugate mimetics of heparan sulfate that bind IFN-gamma in the nanomolar range[END_REF]. Enzymatic activities of hGlce and its three mutants were recorded by real-time NMR spectroscopy using a 13 Clabeled N-sulfated heparosan substrate as described previously [START_REF] Préchoux | C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-Osulfated heparan sulfate domains[END_REF]. hGlce in the apo form and the Tyr578Phe mutant bound with the dp10 substrate or the dp8 product were crystallized by vapor diffusion. The structures were solved by molecular replacement or rigid-body motions and refined to 2.5 Å, 2.1 Å, and 2.45 Å, respectively.

Detailed descriptions of the procedures for protein expression and purification, preparation of oligosaccharides, recording of enzyme activity, and crystallization and X-ray crystallography are provided in SI Appendix, Materials and Methods. Statistics are summarized in SI Appendix, Table S2. The atomic structures and structure factors have been deposited in the Protein Data Bank (PDB ID codes 6HZZ, 6I01, and 6I02) [START_REF] Debarnot | Structure of human D-glucuronyl C5 epimerase[END_REF][START_REF] Debarnot | Structure of human D-glucuronyl C5 epimerase in complex with substrate[END_REF][START_REF] Debarnot | Structure of human D-glucuronyl C5 epimerase in complex with product[END_REF].

ACKNOWLEDGMENTS

Fig. 1 .

 1 Fig. 1. Enzymatic activity and overall structure of the substrate-free hGlce dimer. (A) 13 C-1 H correlation NMR assays of hGlce (40 nM; circles) and mutants Tyr500Phe (50 nM; squares), Tyr578Phe (40 nM; open triangles), and Glu499Gln (40 nM; open diamonds) incubated with 1 mg/mL of 13 C-labeled N-sulfated heparosan (1 mg/mL). The GlcA-to-IdoA conversion, calculated by nonlinear fitting of the data to Equation S1 (SI Appendix, Supplementary Materials and Methods), reaches ∼38% for hGlce and ∼29% for the Tyr500Phe mutant (solid lines). No activity was detected for the Tyr578Phe and Glu499Gln mutants at 400 nM (filled triangle and diamond). (B, Top) Linear representation of the polypeptidic chain and three domains of soluble hGlce. The three N-glycosylation sites and the Ca 2+ binding site are indicated by gray triangles and a black diamond, respectively. (B, Bottom) Overall view of the hGlce dimer with each subunit comprising a β-hairpin domain (yellow), a dominant (α/α) 4 -barrel domain (green), and a β-sandwich domain (pink) with a bound Ca 2+ (violet sphere) and three N-glycans (white sticks, transparent surface). The three invariant Tyr residues possibly involved in catalysis are shown as orange sticks. The cleft is indicated by a black arrow. (Right) Close-up view of the pentagonal bipyramidal coordination geometry around the Ca 2+ bound to the β-sandwich domain. (Top Right) Electrostatic potential distribution at the hGlce surface from -3 kT/e (red) to +3 kT/e (blue), showing the electropositive potential of the catalytic cleft.

Fig. 2 .

 2 Fig. 2. Structures of hGlce bound to a substrate or a product and comparison with zGlce. (A, Left) Closeup view of the catalytic cleft of the Tyr578Phe mutant bound to heptasaccharide substrate with the unbiased electron density map contoured at 3.0 σ and the molecular surface of hGlce, highlighting the shape complementarity. (A, Center) Close-up view of the catalytic cleft showing the bound substrate and surrounding secondary elements. The asterisk denotes the C-terminus position. (A, Right) Close-up view of the substrate distortion in the GlcNS-1-GlcA+1-GlcNS+2 trisaccharide motif at the epimerization site with intra-(gray), inter-(black), and short H bonds (orange) interactions shown as dashed lines. (B) Close-up view of the bound (IdoA-GlcNS) 4 product (light orange, violet sticks) (Left), same representation as in A, showing the conformational similarity with the substrate as shown in the overlay (Center). (C) Close-up view of the overlay of the hGlce-product complex, color-coded as in B, onto the zGlce-hexasaccharide heparin complex (pink cartoon and salmon heparin; PDB ID code 4PXQ), showing the large positional difference between the IdoA2S unit (blue) in zGlce and the corresponding IdoA unit (pink) at the hGlce epimerization site. The hGlce catalytic residues are shown as sticks.

Fig. 3 .

 3 Fig. 3. Proposed catalytic mechanism of Glce and comparison with lyases. (A) Structural comparison of the catalytic sites of hGlce (Left), heparinase I (Center; PDB ID code 3ILR) and chondroitin AC lyase (Right; PDB ID code 1RWG) viewed in similar orientation, showing the bound substrate/product and key active site residues. In all cases, the hGlce Asn510/Tyr560, heparinase I Gln149/Arg83/Lys353, and chondroitin AC lyase Asn183/His233 residues neutralize the uronic acid carboxyl group. (B) Comparison of the hGlce and heparinase I lyase catalytic mechanisms. (Top) hGlce catalyzes C5 epimerization of GlcA into IdoA (red arrows) by abstracting the GlcA C5 proton by the general base Tyr578 on one face of GlcA and donating a proton by the general acid Glu499 through a neutral enol intermediate on the opposite face. In the reverse reaction (blue arrows), the IdoA C5 proton is abstracted by deprotonated Glu499, while Tyr578 reprotonates the C5 atom on the opposite side of the uronic acid. (Bottom) Heparinase I catalyzes β-elimination of the glycosidic linkage between the -1 and +1 positions of the heparin substrate, with His151 and Tyr357 acting as a general base/acid through an anionic enolate intermediate (adapted with permission from ref. 33; permission conveyed through Copyright Clearance Center, Inc.). The H-bond network that neutralizes the carboxylate anion at the epimerization site and lowers the pK a value of the C5 hydrogen is evident for the two enzymes. The two low-barrier H bonds (orange) in hGlce are hallmarks of a neutral enol intermediate, while regular H bonds (grey) in heparinase I support an anionic enolate intermediate.

Fig. 4 .

 4 Fig. 4. Structure-based recognition pattern of N-and O-sulfated groups by hGlce. The scheme was drawn according to the Glce substrate specificity (24). The arrow points to the GlcA/IdoA unit at the epimerization site.

.

  We thank R. Lebrun (Proteomics Facility, Mediterranean Institute of Microbiology) for protein analyses, G. Sulzenbacher (Architecture et Fonction des Macromolécules Biologiques) for assistance with data collection, B. Henrissat and P. Marchot (Architecture et Fonction des Macromolécules Biologiques) for fruitful discussions, S. Fushinobu for sharing his web tool for calculating Cremer-Pople puckering parameters, and the European Synchrotron Radiation Facility and SOLEIL synchrotrons for beam time allocation and their beam line staff for assistance with data collection. This work was supported in part by the CNRS in the framework of the Groupe de Recherche sur les Glycosaminoglycanes (GDR 3739), Aix-Marseille University, the French Infrastructure for Integrated Structural Biology (ANR-10-INBS-05), the LabEx Grenoble Alliance for Integrated Structural & Cell Biology (ANR-10-LABX-49-01), and the LabEx Research on Medication and Innovative Therapeutics (ANR-10-LABX-33) under the Idex Paris-Saclay (ANR-11-IDEX-0003-01).

Downloaded at SCDU Mediterranee onDecember 21, 2020


Typhanie Dumontet

Isabelle Sahut-Barnola

Damien Dufour

Anne-Marie Lefrançois-Martinez

Annabel Berthon

Nathanaëlle Montanier

Bruno Ragazzon

Cyril Djari

Jean-Christophe Pointud

Florence Roucher-Boulez

Marie Batisse-Lignier

Igor Tauveron

Jérôme Bertherat

Pierre Val

Antoine Martinez
email: antoine.martinez@uca.fr

Antoine Marinez

Zona Reticularis

Ca Zg Zf M Ca

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex

Keywords: Endocrine diseases, PKA, SENP1/2, PIAS3, b-catenin, mouse models

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly-and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa and fasciculata zones. Activation of PKA signalling by acute hormonal treatment, mouse genetic engineering or in Carney complex disease results in repression of SUMO conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical WNT signalling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signalling pathways that orchestrate adrenal zonation and diseases.

INTRODUCTION

The adrenal cortex is divided into structurally and functionally distinct concentric zones [START_REF] Vinson | Functional Zonation of the Adult Mammalian Adrenal Cortex[END_REF]. Under the capsule, the zona glomerulosa (zG) is formed by a narrow layer of cells arranged in rosettes that ensure production of aldosterone under the control of angiotensin II and potassium, mainly through calcium signalling [START_REF] Bollag | Regulation of aldosterone synthesis and secretion[END_REF]. The zona fasciculata (zF), located just below, forms a wide layer of cells arranged in radial columns responsible for the secretion of glucocorticoids, in response to ACTH, acting through cAMP/PKA signalling pathway [START_REF] Gorrigan | Localisation of the melanocortin-2-receptor and its accessory proteins in the developing and adult adrenal gland[END_REF]. The zona reticularis (zR), located in the inner part of the cortex close to the medulla, is composed of cells organized in networks. It forms during adrenarche (around 7 years in humans) and supports the secretion of adrenal androgen precursors in response to ACTH [START_REF] Auchus | Adrenarche -physiology, biochemistry and human disease[END_REF]. These zones constitute the definitive/adult cortex. In rodents, the adult cortex only consists of zG and zF [START_REF] Yates | Adrenocortical development, maintenance, and disease[END_REF]. However, in mouse, a third and transient zone termed the X-zone is located in the innermost part of the cortex, and is considered as a remnant of the fetal cortex [START_REF] Morohashi | The fetal and adult adrenal cortex[END_REF][START_REF] Zubair | Developmental links between the fetal and adult zones of the adrenal cortex revealed by lineage tracing[END_REF]. By its capacity to rapidly release hormones acting both on blood pressure control and glucido-lipidic metabolism, the adrenal gland is considered as the endocrine gland for stress response.

The zonal organization and cellular renewal of the adrenal cortex are maintained throughout life by a centripetal mechanisms involving the recruitment of progenitor cells from the periphery of the gland that migrate inward and differentiate successively into zG and zF cells [START_REF] Freedman | Adrenocortical zonation results from lineage conversion of differentiated zona glomerulosa cells[END_REF][START_REF] King | Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages[END_REF]. Hormonal signals that control adrenal function not only ensure homeostatic maintenance but also participate to the zonation of the cortex. The Rspo/Wnt/β-catenin signalling pathway contributes to the acquisition of zG identity [START_REF] Vidal | The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3[END_REF][START_REF] Berthon | Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development[END_REF] whereas ACTH/cAMP/PKA signalling is essential for zF and zR differentiation [START_REF] Chida | Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis[END_REF][START_REF] Dumontet | PKA signaling drives reticularis differentiation and sexually dimorphic adrenal cortex renewal[END_REF]. Maintaining adequate zonation during the unidirectional renewal of the cortex is ensured at least by the antagonistic interactions between these two signalling pathways and through epigenetic programming of newly recruited cells [START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF][START_REF] Mathieu | Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex[END_REF][START_REF] Walczak | Wnt signaling inhibits adrenal steroidogenesis by cell-autonomous and non-cell-autonomous mechanisms[END_REF].

PKA (Protein Kinase A) is a tetramer composed of two catalytic and two cAMP-binding regulatory subunits.

When intracellular levels of cAMP increase, following activation of the G protein coupled ACTH receptor, the regulatory subunits release the catalytic subunits which become active and phosphorylate downstream effectors [START_REF] Sunahara | Complexity and diversity of mammalian adenylyl cyclases[END_REF]. These targets include transcription factors stimulating the expression of steroidogenic genes. Inactivating mutations of the PRKAR1A gene encoding the regulatory subunit R1a lead to constitutive activation of PKA and development of Carney complex, a multiple endocrine neoplasia syndrome [START_REF] Carney | The complex of myxomas, spotty pigmentation, and endocrine overactivity[END_REF][START_REF] Kirschner | Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex[END_REF]. Primary Pigmented Nodular Adrenocortical Disease (PPNAD), the most frequent endocrine tumor in these patients is a benign adrenal hyperplasia, responsible for ACTH-independent hypercortisolism (Cushing's syndrome) [START_REF] Bertherat | Mutations in regulatory subunit type 1A of cyclic adenosine 5'-monophosphatedependent protein kinase (PRKAR1A): phenotype analysis in 353 patients and 80 different genotypes[END_REF]. We have shown that the adrenal inactivation of Prkar1a in developing mice is sufficient to induce ACTH-independent Cushing's syndrome and formation of apoptosis-resistant hyperplasia in the inner zF [START_REF] Sahut-Barnola | Cushing's syndrome and fetal features resurgence in adrenal cortex-specific Prkar1a knockout mice[END_REF][START_REF] De Joussineau | mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD)[END_REF]. Moreover, by tracing the lineage of Prkar1a mutant cells in the adult gland, we have established that PKA signalling is a driving force for sexually dimorphic cortex replenishment and for the conversion of zF into zR identity [START_REF] Dumontet | PKA signaling drives reticularis differentiation and sexually dimorphic adrenal cortex renewal[END_REF].

SUMOylation has emerged as a critical mechanism for modulating fundamental cellular and developmental processes, whose dysregulation can lead to severe diseases. Its involvement in tissue morphogenesis [START_REF] Deyrieux | Sumoylation in Development and Differentiation[END_REF] has been demonstrated in enterocytes [START_REF] Demarque | Sumoylation by Ubc9 regulates the stem cell compartment and structure and function of the intestinal epithelium in mice[END_REF], keratinocytes [START_REF] Deyrieux | Sumoylation dynamics during keratinocyte differentiation[END_REF] and adipocytes [START_REF] Liu | Small ubiquitin-like modifier (SUMO) protein-specific protease 1 de-SUMOylates Sharp-1 protein and controls adipocyte differentiation[END_REF][START_REF] Liu | Protein inhibitor of activated STAT 1 (PIAS1) is identified as the SUMO E3 ligase of CCAAT/enhancer-binding protein β (C/EBPβ) during adipogenesis[END_REF]. However, it also participates in human malignancy by favouring cancer cell stemness [START_REF] Zhang | OTUB2 Promotes Cancer Metastasis via Hippo-Independent Activation of YAP and TAZ[END_REF][START_REF] Cui | SENP1 promotes hypoxia-induced cancer stemness by HIF-1α deSUMOylation and SENP1/HIF-1α positive feedback loop[END_REF]. Post-translational modification of proteins by the covalent addition of SUMO1-3 (Small Ubiquitin-like Modifier) peptides requires an enzymatic cascade involving the E1 Sae1/Sae2 heterodimer for activation step, the Ube2i (Ubc9) E2 enzyme for conjugation step, and E3 ligation enzymes including Pias1-4, RanBP2, Nse2 and Cbx4 proteins [START_REF] Geiss-Friedlander | Concepts in sumoylation: a decade on[END_REF]. The dynamics and reversibility of the reaction are ensured by Senp1-7 and Desi1 proteases that cleave SUMO peptides from their substrates [START_REF] Nayak | SUMO-specific proteases/isopeptidases: SENPs and beyond[END_REF][START_REF] Shin | DeSUMOylating isopeptidase: a second class of SUMO protease[END_REF].

One of the major roles of SUMOylation cycles is the control of transcription processes through the individual modification of trans-acting factors and chromatin remodelling [START_REF] Verger | Modification with SUMO. A role in transcriptional regulation[END_REF][START_REF] Heun | SUMOrganization of the nucleus[END_REF][START_REF] Neyret-Kahn | Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation[END_REF]. The properties of the transcription factors WT1, Nurr1 and SF-1 that are essential for development and function of adrenal cortex, are also regulated by SUMOylation [START_REF] Smolen | SUMO-1 modification of the Wilms' tumor suppressor WT1[END_REF][START_REF] Galleguillos | PIASgamma represses the transcriptional activation induced by the nuclear receptor Nurr1[END_REF][START_REF] Komatsu | Small ubiquitin-like modifier 1 (SUMO-1) modification of the synergy control motif of Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) regulates synergistic transcription between Ad4BP/SF-1 and Sox9[END_REF][START_REF] Chen | SUMO modification of repression domains modulates function of nuclear receptor 5A1 (steroidogenic factor-1)[END_REF][START_REF] Lee | The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification[END_REF]. However, physiological evidence of the impact of SUMOylation on adrenal cortex function remains scarce but was elegantly brought into light by knock-in experiment from H.

Ingraham's group [START_REF] Lee | Eliminating SF-1 (NR5A1) sumoylation in vivo results in ectopic hedgehog signaling and disruption of endocrine development[END_REF]. Indeed, the complete lack of SUMO-conjugated SF-1 in mice expressing a unSUMOylatable Sf1 2KR allele was shown to affect adrenal zonation (delayed X-zone regression), expanded the progenitor compartment, disturbed cell identities (ectopic gonadal traits) and resulted in mild endocrine hypofunction. This highlighted the importance of SF-1 SUMOylation in fine-tuning the genetic programs of endocrine differentiation but our knowledge of overall SUMOylation patterns in the adrenal gland and hormonal regulation of this process is still lacking. Interestingly, increased cAMP levels was shown to influence the expression of members of the SUMO pathway in ovarian granulosa cells [START_REF] Yang | Liver receptor homolog-1 localization in the nuclear body is regulated by sumoylation and cAMP signaling in rat granulosa cells[END_REF].

Here, we hypothesized that SUMOylation, cortex architecture and hormonal regulation of adrenocortical function should be coordinated. To test this hypothesis, we analysed the influence of ACTH/PKA signalling on the SUMOylation process in cell culture, wild-type mice, genetic mouse models of adrenal diseases and in human PPNAD tissues. This enabled us to propose that cell signallings critical for adrenal cortex homeostasis and stress response tighly coordinate the SUMOylation pathway which in turn is found altered in cortical diseases.

MATERIALS AND METHODS

Adrenocortical cell cultures. Adrenocortical Tumor Cells 1 were established from an adrenal tumor derived from a mouse expressing the SV40 T antigen under the control of the Akr1b7 promoter specific to the adrenal cortex (AdTAg mouse) [START_REF] Ragazzon | Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoidproducing adrenocortical cell lines derived from targeted tumorigenesis[END_REF][START_REF] Batisse-Lignier | P53/Rb inhibition induces metastatic adrenocortical carcinomas in a preclinical transgenic model[END_REF]. The primary cultures were obtained from adrenals of female mice aged of 3 weeks following the protocol described above (45). The cells were cultured on poly-D-lysine-coated dishes (Sigma-Aldrich) in a DMEM-F12 medium (Life Technologies SAS.) At 37 ° C. in the presence of 5% CO2, insulin (10 µg/mL), transferrin (5.5 µg/mL), selenium (6.7 ng/mL) (ITS, Life Technologies SAS), L-glutamine (2mM), penicillin 0.1U/ml ), streptomycin (0.1 µg/mL), 2.5% horse serum and 2.5% fetal calf serum [START_REF] Ragazzon | Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoidproducing adrenocortical cell lines derived from targeted tumorigenesis[END_REF]. For PKA induction experiments, cells were seeded in 6-well plates at a density of 2.10 5 cells per well, cultured to subconfluence and then weaned 12h (serum-free medium) prior to the addition of forskolin, ACTH, cycloheximide, actinomycin, at the times and concentrations indicated in the legends of the Fig. s.

Animals and treatments. Two-month-old SWISS CD-1 mice received an i.p. injection of immediate ACTH (0.05 mg/30 g, Synacthene® 0.25 mg/mL, Novartis Pharma S.A.) 2h or 4h before adrenal sampling. Others received i.m. injection of long-acting ACTH (12 µg/30 g, Synacthene Retard 1 mg/mL, Novartis Pharma S.A) and the adrenals were dissected 6h or 24h later. For the 24h point, the mice received a second injection 12h before sampling. Immediate ACTH, with a short half-life, allows to observe the early effects of stimulation. The delayed ACTH is stabilized because complexed with zinc salts, the diffusion is slowed which allows to observe effects in the longer term. Alternatively, 6-month-old females were injected s.c. with dexamethasone 21-acetate for 5 days in order to deplete endogenous ACTH production (75 µg twice daily in corn oil) and were injected with immediate ACTH for 2h or 6h. The female Sf1-Cre::Prkar1a fl/fl and ∆Cat (Akr1b7-Cre::Catnb lox(ex3) ) mice used were 2.5-and 18-month-old, respectively [START_REF] Berthon | Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development[END_REF][START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF]. The mice were sacrificed by decapitation and the adrenals immediately frozen in liquid nitrogen.

Protein extraction and western blot. Cell and tissue samples were lysed in RIPA (Radio Immunoprecipitation Assays) buffer (50 mM Tris-HCl, pH7.4, 1% NP40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented extemporaneously with phosphatase inhibitors (1 mM Na3VO4, 0.5 mM NaF), protease inhibitors (Roche Diagnostics) and SUMO inhibitors proteases N-ethylmaleimide (NEM, Sigma-Aldrich) (3.13 mg/mL).

The cell debris was removed by centrifugation at 13,000 rpm for 15 minutes at 4 °C. The protein concentration was determined by the Bradford method (Bio-Rad) and 50 µg of proteins were denatured for 5 minutes at 95 ° C in Laemmli (50 mM Tris, 100 mM DTT, 2% SDS, 0.1% Bromophenol blue, 12% glycerol). The proteins were then separated on a 7% SDS-PAGE (Fig. 2D) or precast gels 4-15% (BioRad) (Supplemental Fig. S3D) and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Biosciences) overnight at 4 °C at 50V followed by one hour at 120V. Non-specific protein binding sites were saturated for 1h at room temperature by incubation in TBS-T (50 mM Tris-HCl (pH 8), 150 mM NaCl and 0.1% Tween 20) containing 5% Regilait®.

Incubation with the appropriate primary antibody (Supplemental Table S1A) was carried out in the same buffer overnight at 4 °C with gentle stirring. After rinsing in TBS-T, the membranes were incubated for 1h at room temperature with the appropriate secondary antibody conjugated with peroxidase. The specific complexes were revealed by chemiluminescence (ECL) system (Enhancer chemoluminescent, Amersham). The fluorescence signals were quantified using the MultiGauge V 3.2 software.

RT-qPCR analyses. The cell samples were lysed in Trizol (Invitrogen) and the total RNAs were extracted according to the manufacturer's recommendations. Tissue samples were ground in RNA II lysis buffer (Macherey Nagel) and total RNAs were extracted using the NucleoSpin RNA II kit as recommended by the manufacturer. Five hundred nanograms of mRNA were reverse-transcribed for 1h at 37 ° C with 5 pmoles (reverse transcriptase), 2.5 mM dNTP and 20 units of RNAse inhibitors (recombinant RNAsin, N2615, Promega) in a final volume of 25 µL. The cDNAs obtained were used (at 1/4 dilution) as template for RT-qPCR analysis using the qPCR SYBR (TAKRR820W, Takara) premix under standard conditions (40 cycles at 95 °C for 15 sec, 60 °C for 15 sec and 72°C for 20 sec). The relative accumulation of mRNAs was determined from the average of duplicates by the ΔΔCt method (46), using the Actin household gene. The primers are listed in Supplemental Table S1B.

Immunohistochemistry. After collection, the adrenals were fixed in 4% PFA for 24 hours, dehydrated in an increasing gradient of ethanol and then placed in Histoclear (HS200, National Diagnostics, Atlanta, USA) for 2 h before being included in paraffin. 5 µm thick sections were made using a microtome (HM 340E, ThermoFisher Scientific, Massachusetts, USA). The tissue sections were then dewaxed by passing through the Histoclear and progressively rehydrated in an increasing gradient of ethanol. Immunohistological analyses were performed according to the conditions described in Supplemental Table S1C. The tissues were photographed using an Axioplan2 Imaging microscope (Zeiss, Jena, Germany). In order to evaluate the presence of non-SUMOylated nuclei, a counter-staining with haematoxylin was performed on the same sections previously used for SUMO1 immunohistochemistry. Quantification of SUMO signals were performed using Zen Software (Zeiss).

Patients

RESULTS

SUMO conjugation is repressed by ACTH/PKA signalling in adrenocortical cells in vitro.

To evaluate the role of PKA signalling on adrenocortical proteins SUMOylation, we analysed the effects of forskolin, an adenylate cyclase activator, on the formation of SUMO conjugates in lysates of ATC1 mouse adrenocortical cells [START_REF] Ragazzon | Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoidproducing adrenocortical cell lines derived from targeted tumorigenesis[END_REF]. Immunoblotting with either anti-SUMO1 (Fig. 1A) or anti-SUMO2/3 (Fig. 1B) showed that sumoylated proteins in cell extracts were represented by a smear of bands of high molecular mass whose intensity decreased with forskolin, reaching a nadir after 6h-12h treatment. Quantification of the westernblots showed that steady-state levels of Sumo1-and Sumo2/3-conjugated proteins were reduced by 54% and 40%, respectively, after 6h of forskolin treatment.

To explore the mechanism leading to PKA-mediated inhibition of protein SUMOylation, the hormonal responsiveness of genes encoding SUMO modules and enzymes of the SUMO conjugation/deconjugation pathway (Fig. 1C) was assessed by RTqPCR, in ATC1 cell line and primary adrenocortical cells (Fig. 1D). In both cell types, strong induction of Akr1b7 mRNA levels, a well characterised ACTH/PKA-responsive gene [START_REF] Ragazzon | Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoidproducing adrenocortical cell lines derived from targeted tumorigenesis[END_REF], validated treatments at all time-points, including the expected more robust effect of forskolin compared to ACTH, the physiological inducer (Supplemental Fig. S1A). In both cell culture systems, PKA stimulation downregulated mRNA accumulation of Sumo1, Sae1, Pias3 and, to a lesser extent, Ube2i, with a maximal effect occurring between 6h and 12h (Fig. 1D). Conversely, these treatments induced a two-fold increase in Senp2 mRNA expression that culminated between 2-6h. This positive PKA responsiveness was also observed at protein levels (Fig. 1E) suggesting that Senp2 gene regulation by ACTH/PKA was mainly transcriptional as confirmed by the sensitivity to actinomycin D and resistance to cycloheximide (Supplemental Fig. S1B).

Mechanisms of PKA-dependent repression of genes involved in SUMO conjugation (Sumo1, Ube2i, Sae1 and Pias3) were less clear, but presumably required de novo expression of a labile repressor as suggested by Pias3 mRNA sensitivity to both actinomycin D and cycloheximide (Supplemental Fig. S1B). Interestingly, although Ube2i transcripts levels were still elevated 6 hours post-induction in ATC1 cells, protein levels declined between 2 and 6h (Fig. 1E). Therefore, this further suggested that PKA-dependent repression might also be achieved by post-translational mechanisms. The responsiveness of the other effectors of SUMOylation appeared either unaffected by PKA stimulation or not reproduced under ACTH treatment (Supplemental Fig. S1C).

Taken together these results indicate that PKA-induced overall decrease of SUMO-conjugated protein substrates in adrenocortical cells could be the consequence of two coordinated and convergent mechanisms: increased gene transcription of deSUMOylating enzyme (here Senp2) and repression of genes encoding SUMOylating proteins including SUMO peptides.

Acute and chronic effects of ACTH/PKA repress SUMOylation in mouse adrenal glands.

To evaluate in vivo, the hormonal responsiveness of genes of the SUMO-conjugation or -deconjugation pathways, WT young adult mice were injected with rapid-or long-acting forms of ACTH and sacrificed after 2-4h or 6-24h, respectively. RT-qPCR analyses in the adrenal cortex showed that activation of PKA signalling repressed mRNA levels of Sumo1, Sumo2 and Sumo3 by 50%, 25% and 30%, after 4h of treatment, respectively (Fig. 2A,B). Extended ACTH treatments showed that this decline was transient and could be maintained over 6h. However, expression re-increased at 24h (Supplemental Fig. S2A). A similar repression pattern was observed for Pias3 mRNA levels that reached a nadir after 2-4h of treatment (75% drop) and re-increased at 24h (Fig. 2B and supplemental Fig. S2A). In contrast, transcripts levels for the SUMO protease Senp2 were increased by two-fold after 2h, returning to pre-treatment values after 4h (Fig. 2B). Remarkably, mRNA levels for SUMO protease Desi1 remained stable for the first 6h of treatment and were increased more than 1.5-fold after 24h (Fig. 2B and supplemental Fig. S2A). Regardless of the treatment duration, the expression of Ube2i transcripts was unaltered (Fig. 2B and supplemental Fig. S2A). We next investigated the effect of chronic activation of PKA signalling on genes ensuring SUMO conjugation/deconjugation dynamics. For this, we used Sf1-Cre::Prkar1a fl/fl mice with adrenal-specific deletion of PKA regulatory R1a subunit [START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF]. RT-qPCR analysis revealed a 38% reduction of Pias3 expression compared to wild-type levels while Senp2 and Desi1 expression was induced by 1.5-and 2-fold, respectively. Senp3 and Senp5 mRNA levels also showed a modest but significant increase (Fig. 2C). Interestingly, under these chronic stimulation conditions, there was no downregulation of Sumo genes and expression levels of all other conjugating/deconjugating genes were unaltered or only slightly increased (Supplemental Fig. S2B). In agreement with the repression of Pias3 and simultaneous stimulation of genes for deSUMOylating isopeptidases, western-blot analysis of adrenal lysates showed that Sumo2/3 conjugation levels in mutant mice were approximately 30% of wild-type (Fig. 2D).

Taken together, these data suggest that ACTH stimulation in vivo, limits the SUMOylation process in the adrenal gland through both acute (≤4h) and chronic (≥24h) transcriptional effects. These lead to the simultaneous and opposite regulation of a subset of PKA-responsive genes of the SUMOylation pathway, with repression of actors of the conjugation process (Sumo1-3 and Pias3), and stimulation of deconjugation enzymes (Senp2, Senp3, Senp5 and Desi1).

Regionalisation of SUMOylation in the adrenal cortex is dependent on ACTH/PKA signalling.

In the adrenal cortex, ACTH/PKA signalling is required for differentiation, maintenance and function of the zF [START_REF] Chida | Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis[END_REF][START_REF] Dumontet | PKA signaling drives reticularis differentiation and sexually dimorphic adrenal cortex renewal[END_REF]. Active SUMOylation processes can be assessed in tissue sections by monitoring changes in the intensity of nuclear SUMO signal [START_REF] Yang | Transient focal cerebral ischemia induces a dramatic activation of small ubiquitin-like modifier conjugation[END_REF][START_REF] Tirard | In vivo localization and identification of SUMOylated proteins in the brain of His6-HA-SUMO1 knock-in mice[END_REF]. Immunohistochemical staining of mouse adrenal samples revealed a cortical gradient of Sumo1 and Sumo2/3 staining in steroidogenic cells, with high nuclear signal in the peripheral subcapsular/glomerulosa region, intermediate signal at the glomerulosa-fasciculata junction and low labelling in fasciculata cells (Fig. 3A,B & supplemental Fig. S3A). Sumo1 and Sumo2/3 staining pattern was similar in both sexes although the decrease SUMOylation levels between zG to zF appeared more pronounced in females (Supplemental Fig. S3B). Interestingly, SUMO labelling was not restricted to steroidogenic cells. Indeed, capsular as well as endothelial cell nuclei were strongly positive for Sumo2/3 whereas Sumo1 staining was much less pronounced in these cell populations (Fig. 3A,B & supplemental Fig. S3B).

In order to evaluate the consequences of changes in the hypothalamic-pituitary adrenal (HPA) axis on SUMO staining profile, mice were either treated for 5 days with dexamethasone to block endogenous ACTH secretion, or supplemented with (rapid-acting) ACTH injection on the last day (Fig. 3C). Compared to basal conditions, HPA axis blockade had no significant effect on either Sumo1 or Sumo2/3 nuclear staining patterns. In contrast, ACTH supplementation resulted in a transient decrease in SUMO nuclear labelling that affected all cortical zones after 2h and almost returned to pre-treatment labelling after 6h. Interestingly, the negative effect of ACTH was restricted to steroidogenic cells since nuclear Sumo2/3 staining intensity was unaltered in capsular or endothelial cells. In agreement with histological data, western blotting analysis of the corresponding adrenal extracts showed a parallel change of Sumo2/3 and Sumo1 conjugate levels (Supplemental Fig. S3D). General staining pattern for SUMO was also assessed in adrenal sections from Sf1-Cre::Prkar1a fl/fl mice. This showed that chronic stimulation of PKA signalling in the cortex resulted in the relative loss of centripetal gradient of nuclear Sumo1 labelling typical of WT cortex (Supplemental Fig. S3C). Altogether, these results indicate that there is a centripetally decreasing gradient of SUMOylation throughout adrenal cortical zones that relies on the activity of ACTH/PKA signalling, which maintains relative hypoSUMOylation context in steroidogenic cells of the zF.

Differential SUMOylation pattern of adrenal cortex is conserved in human adrenal and is altered in primary pigmented nodular adrenal hyperplasia (PPNAD) from Carney complex patients.

To assess whether our observations were transferable to human, we analysed by immunohistochemistry, the SUMOylation pattern of adrenal samples from either healthy patients or from hypercortisolic patients suffering from pathological activation of PKA due to Carney complex disease (CNC). The SUMO1 and SUMO2/3 immunolabelling performed on normal human adrenal sections revealed a centripetally decreasing gradient of steroidogenic cells nuclear staining intensity similar to that found in mice (Fig. 4A & supplemental Fig. S4A).

Accordingly, nuclear staining intensity was maximal in cells of the outer cortex (zG and zG/zF transition zone) and weaker in the zF and zR. Interestingly, SUMO1 immunolabelling of adrenal samples from CNC patients (n=3+3), revealed obvious hypoSUMOylation of cells forming the hyperplastic nodules (as evidenced by hematoxylin counterstaining). This contrasted with the intense SUMO1 nuclear staining found in adjacent internodular tissue (Fig. 4B & supplemental Fig. S4B). Reciprocally, the signal for SUMO protease SENP1 was more pronounced within nodules (Fig. 4C & supplemental Fig. S4C). Altogether, these data suggested that the SUMOylation potential was inhibited in a context of constitutive PKA activity due to R1a loss within PPNAD nodules (PRKAR1A LOH) and was maintained or even increased in a context of blunted PKA activity, as found in the atrophic internodular fasciculata tissue (due to repressed ACTH levels [START_REF] Tirosh | Genetics of micronodular adrenal hyperplasia and Carney complex[END_REF]). We conclude that as suggested by mice studies, SUMOylation follows a centripetal decreasing gradient in human adrenal cortex that seems to negatively correlate with activation of PKA signalling in normal gland (zF) and in adrenal nodules from patients suffering from CNC.

WNT/b-catenin signalling favours SUMOylation in the adrenal cortex.

We have previously established that WNT and PKA signalling pathways exert complementary and antagonistic actions to maintain adrenal cortex zonation: WNT signalling is essential for zG identity, whereas PKA signalling drives identity of the inner cortex (zF and zR) [START_REF] Dumontet | PKA signaling drives reticularis differentiation and sexually dimorphic adrenal cortex renewal[END_REF][START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF]. Therefore, we hypothesized that relative hyperSUMOylation of the outer cortex could rely on WNT/b-catenin signalling. To evaluate this hypothesis, we analysed changes in SUMO and b-catenin immunofluorescent staining in adrenal sections of ∆Cat mice that present stochastic constitutive activation of b-catenin due to Ctnnb1 exon 3 excision (Akr1b7-Cre::Catnb fl(ex3) ) (11)(Fig. 5A). As previously described, constitutive activation of WNT signalling resulted in expansion of the nucleo-cytoplasmic staining of b-catenin beyond zG and throughout the cortex. This resulted in formation of large hyperplastic cells clusters (Fig. 5B) in which b-catenin staining intensity was massively increased compared to the adjacent non-recombined tissue (Supplemental Fig. S5A). Co-immunofluorescent detection of either Sumo1 or Sumo2/3, revealed that hyperplastic clusters (b-catenin positive) and areas with highest Sumo nuclear labelling overlap. This was confirmed by quantifying Sumo nuclear signals in hyperplastic area (bcatenin high) and intact adjacent cortex (b-catenin low). This showed that increased SUMOylation correlated with b-catenin activation (Fig. 5C &5D). We assessed possible concurrent changes in SUMO pathway gene expression in ∆Cat adrenal glands using RT-qPCR. As illustrated in Fig. 5E, mRNA levels for genes involved in SUMO conjugation (Sumo2, Sumo3, Pias1 and Pias2) were induced, while mRNA expression of SUMO protease Desi1 was reduced by 50% in adrenals from ∆Cat mice. With the exception of genes for SUMO protease Senp1 and Senp6 which were upregulated, expression of all other tested genes was unaltered in ∆Cat adrenals (Supplemental Fig. S5B). We conclude that WNT/b-catenin signalling pathway activation favours SUMOylation in vivo and can participate in establishing the centripetally decreasing gradient of SUMOylation in the adrenal cortex by maintaining high SUMOylation potential in the outer cortex.

DISCUSSION

The results presented here show that there is a negative correlation between the activation of PKA signalling (genetically or hormonally induced) and the SUMOylation of adrenocortical proteins in cell culture and in vivo.

This PKA-dependent hypoSUMOylation relies on the coordinated transcriptional stimulation of deSUMOylating isopeptidase genes (Senp2, Desi1, SENP1) and repression of genes encoding SUMO peptides and SUMO (Pias3) ligase. In agreement with this inhibitory effect, we show that in both human and mouse adrenal cortices, the SUMOylation pattern appears as a centripetal decreasing gradient. This is inversely correlated with PKA sensitivity of the cells, as emphasised by strong nuclear staining in zG and weaker staining in zF/zR. Activation of PKA signalling by ACTH injections leads to a transient decrease in SUMOylation in zF, which suggests a possible active role of this post-translational modification in zonation and/or stress response.

Conversely, this gradient seems less sensitive to ACTH suppression as dexamethasone treatment leads to a slight though nonsignificant increased SUMOylation (Supplemental Fig. S3D). Moreover, the hypoSUMOylating effect of PKA signalling could participate in the aetiology of PPNAD. Indeed, we show that global SUMOylation is reduced in the nodules of patients with PPNAD as well as in the adrenals of a mouse model of PPNAD (Sf1-Cre::Prkar1a fl/fl ).

In adrenocortical cells, kinetics of SUMO pathway genes expression in response to PKA stimulation are similar to that of steroidogenic genes [START_REF] Ragazzon | Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoidproducing adrenocortical cell lines derived from targeted tumorigenesis[END_REF]. This observation suggests that the SUMOylation process may be subordinated to the same regulatory mechanisms as steroidogenesis. Although PKA-mediated transcriptional activation is well documented, data on mechanisms relaying its repressive effects are scarce, even though they are quite prominent. Indeed, our transcriptome analyses of mice with adrenal Prkar1a gene inactivation with various Cre drivers [START_REF] Dumontet | PKA signaling drives reticularis differentiation and sexually dimorphic adrenal cortex renewal[END_REF][START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF] showed similar numbers of down-and upregulated genes, following constitutive PKA activation. The only negative molecular mechanism identified to date in the adrenal gland, mobilizes the oscillating SIK-CRTC (Salt inducible kinase-CREB regulated transcription coactivator) cascade in which SIK represses the activity of CREB in a transient PKA-dependent manner [START_REF] Lin | SIK (Salt-inducible kinase): regulation of ACTH-mediated steroidogenic gene expression and nuclear/cytosol redistribution[END_REF][START_REF] Takemori | Regulation of CREB-mediated gene expression by salt inducible kinase[END_REF][START_REF] Jagannath | The CRTC1-SIK1 pathway regulates entrainment of the circadian clock[END_REF]. The participation of SIK-CRTC in the transcriptional repression of Sumo1-3 and Pias3 genes will require further investigation.

In contrast to SUMO conjugating genes, and in agreement with the overall hypoSUMOylation induced by PKA, expression of deSUMOylases Senp2 and Desi1 is stimulated by activation of PKA signalling, in the same way as steroidogenic genes. The induction of Senp2 mRNA expression by ACTH/PKA might rely on the recruitment of CREB to a functional cAMP responsive element (CRE) located in the proximal promoter region that was described to mediate PKA responsiveness in 3T3L1 preadipocytes [START_REF] Chung | Control of adipogenesis by the SUMO-specific protease SENP2[END_REF]. No functional data are available for the Desi1 regulatory regions although several putative CREs are predicted by bioinformatics (not shown).

However, the kinetics of ACTH responsiveness in vivo, with fast up-regulation of Senp2 transcripts (2h) and slow up-regulation of Desi1 (24h), suggests that mechanisms contributing to their regulation by PKA may differ. In addition, Senp2 and Desi1 proteins are supposed to diverge in their subcellular localization. Indeed, whereas Senp2 is mainly nuclear (54) Desi1 is cytoplasmic [START_REF] Shin | DeSUMOylating isopeptidase: a second class of SUMO protease[END_REF]. This suggests that their target substrates may differ.

The centripetal decreasing gradient of SUMOylation within the adrenal cortex (zG>zF/zR) together with its sensitivity to the HPA axis, are suggestive of a role for SUMOylation in the response to stress. Accordingly, requirement of SUMO pathway for adaptative response to stress seems to have been conserved in evolution [START_REF] Tempé | SUMO under stress[END_REF]. Indeed, proteomic analyses have demonstrated the coordinated SUMOylation of protein groups involved in the same biochemical pathway after genotoxic stress in yeast [START_REF] Psakhye | Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair[END_REF]. SENP2-dependent deSUMOylation is required to adapt cells to etoposide-induced genotoxic stress while SENP1-mediated deSUMOylation is required to counteract colon tumour growth, induced by genotoxic E. coli strains [START_REF] Lee | NF-κB induction of the SUMO protease SENP2: A negative feedback loop to attenuate cell survival response to genotoxic stress[END_REF][START_REF] Cougnoux | Bacterial genotoxin colibactin promotes colon tumour growth by inducing a senescence-associated secretory phenotype[END_REF]. In agreement with this concept, the repressor effect of PKA signalling on the SUMOylation process could participate in the transcriptional response of adrenocortical cells following stress. The constitutive hypoSUMOylation in zF could exert a permissive action on the response to stress, by lifting the "tonic" repression exerted by SUMOylation on transcription factors such as SF-1 and Nurr1 [START_REF] Galleguillos | PIASgamma represses the transcriptional activation induced by the nuclear receptor Nurr1[END_REF][START_REF] Campbell | Decreased recognition of SUMO-sensitive target genes following modification of SF-1 (NR5A1)[END_REF]. Under stress conditions, ACTH-induced transient hypoSUMOylation would further facilitate steroidogenic response. Furthermore, activation of some PDEs following their SUMOylation [START_REF] Li | Selective SUMO modification of cAMP-specific phosphodiesterase-4D5 (PDE4D5) regulates the functional consequences of phosphorylation by PKA and ERK[END_REF], suggests that such an intracellular feedback mechanism would allow the adrenal cortex to sustain elevated intracellular levels of cAMP and promote activation of PKA. The hypoSUMOylation observed in PPNAD nodules suggests the involvement of this modification in the aetiology of the disease. These results are consistent with the increasing number of studies involving SUMOylation in tumour mechanisms [START_REF] Seeler | SUMO and the robustness of cancer[END_REF].

Finally, the decreasing gradient of SUMOylation in the adrenal cortex also suggests its possible involvement in the establishment and/or maintenance of cortical zonation. Increased SUMOylation consecutive to depletion of SENP1/2/7 proteins was reported in various models, to activate the WNT/b-catenin pathway by increasing bcatenin stability [START_REF] Karami | Novel SUMO-Protease SENP7S Regulates β-catenin Signaling and Mammary Epithelial Cell Transformation[END_REF][START_REF] Huang | β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells[END_REF] and by promoting its nuclear translocation [START_REF] Choi | Reversible SUMOylation of TBL1-TBLR1 regulates β-catenin-mediated Wnt signaling[END_REF][START_REF] Tan | SENP2 regulates MMP13 expression in a bladder cancer cell line through SUMOylation of TBL1/TBLR1[END_REF]. The ability of SUMOylation to activate the WNT pathway is likely to explain the intense staining observed in zG. Reciprocally, we report here that constitutive b-catenin activation in mouse adrenals, not only induces zG tumour formation [START_REF] Berthon | Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development[END_REF][START_REF] Berthon | WNT/beta-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production[END_REF] but also expands the hyperSUMOylation domain. This suggests that a positive feedback loop between SUMOinduced WNT pathway and b-catenin-dependent SUMOylation increase could allow maintaining higher SUMO staining in the zG. Conversely, the hypoSUMOylating effects of PKA signalling activation could participate (e.g. through Senp2/SENP1 upregulated expression) in the antagonism between WNT/b-catenin and ACTH/PKA pathways, which is needed to establish proper functional zonation and to counteract b-catenininduced tumour formation [START_REF] Dumontet | PKA signaling drives reticularis differentiation and sexually dimorphic adrenal cortex renewal[END_REF][START_REF] Drelon | PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development[END_REF].

In conclusion, our findings reveal that protein SUMOylation follows a conserved centripetal decreasing gradient within the adrenal cortex. This relies on antagonistic actions of ACTH/PKA and WNT/b-catenin signalling pathways: ACTH/PKA signalling acutely reduces SUMOylation in zF by coordinating the expression of both SUMO pathway inducers and repressors, whereas WNT/b-catenin induces hypersumoylation. Our data suggest that programming of the SUMOylation pattern could play a role in endocrine function and stress response, maintenance of zonation and pathology (hypersecretion syndromes and cancer).

Ubc9

Relative mRNA level

Ube2i

Relative mRNA level

Fig. 1 .

 1 Fig. 1. Activation of PKA signalling decreases global SUMOylation in adrenocortical cell cultures. A-B) Time course effects of PKA activation on the accumulation of adrenocortical proteins conjugated to Sumo1 (A) and Sumo2/3 (B). Murine adrenocortical ATC1 cell line was treated with forskolin (10 -5 M) for the indicated time (h) or with vehicle (DMSO) for the longest treatment time. The accumulation of Sumo1 and Sumo2/3 conjugates was analysed by western blot (representative Fig.) and quantified (histograms). Tubulin signal was used to normalise the quantification of SUMO signals. C) Schematic representation of the SUMOylation cycle. Sumo1, Sumo2 and Sumo3 peptides are conjugated to their substrate proteins by an enzymatic process involving E1, E2 and E3 enzymes. The dynamism of the SUMOylation is ensured by the proteases Senp and Desi1 which allow the removal of the SUMO peptide from the targeted protein. D) Time course effects of PKA activation on the mRNA expression of SUMOylation pathway genes. Primary cultured cells from mouse adrenal cortex and ATC1 cell line were treated with forskolin (10 -5 M) for the indicated time or with DMSO (Vhl) for the longest treatment time. Primary cultures were also treated with ACTH (10 -9 M). RT-qPCR analyses were normalised with respect to Actin gene expression. E) PKA responsiveness of Senp2 and Ube2i protein levels. ATC1 cells were treated with forskolin (10 -5 M) for the indicated time (h) or with DMSO (Vhl) for the longest treatment time. Protein accumulation of Senp2 and Ube2i was analysed by western blot. Tubulin signal serves as loading control. Quantification histograms represent mean ± SEM from 3-4 experiments. Statistical analyses were conducted by 1-way ANOVA followed by Dunett's test. *p<0.05; **p<0.01; ***p<0.005.

Fig. 2 .

 2 Fig. 2. Acute hormonal induction or constitutive activation of PKA signalling affect protein SUMOylation and SUMO pathway gene expression in mouse adrenals. A-B) Wild-type mice were injected with immediate ACTH and adrenals were removed 2h or 4h later, control adrenals were dissected from untreated mice (0h) and subjected to RNA extraction. A) Experimental setup. B) RT-qPCR analyses of SUMOylation pathway gene expression normalised with respect to Actin gene expression. Statistical analyses were conducted by 1-way ANOVA followed by Dunett's test. C-D) Carney complex mouse model with constitutive activation of PKA following adrenal inactivation of Prkar1a (Sf1-Cre::Prkar1a fl/fl mice). C) Experimental setup. Adrenals from Sf1-Cre::Prkar1a +/+ (WT) and Sf1-Cre::Prkar1a fl/fl (KO) mice were removed and subjected to RNA and protein extraction. RT-qPCR analyses of SUMOylation pathway gene expression normalised with respect to Actin gene expression and, D) western blot analyses of Sumo2/3 conjugates in adrenal protein extracts and quantification (histogram). Tubulin signal was used to normalise the quantification of SUMO signals. Statistical analyses were conducted by Student t-test. Quantification histograms represent mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Fig. 3 .

 3 Fig. 3. SUMOylation is regionalised in mouse adrenal cortex and modulated by ACTH/PKA signalling. A-B) SUMOylation profile forms a decreasing gradient of intensity from the outer to the inner cortex. The accumulation of conjugated proteins to Sumo1 (A) and Sumo2/3 (B) was analysed by immunohistochemistry in adrenals of 6-month-old female mice. The quantification graphs represent mean ± SEM of SUMO nuclear staining intensity in each histological zone and inter-zone all along the cortex and in medulla, for 3 adrenals from 3 different animals and 50-60 nuclei per adrenal. Statistical analyses were conducted by Kruskal-Wallis followed by Dunn's test. Ca: capsule; zG: zona glomerosa; zF ext : external zona fasciculata; zF int : internal zona fasciculata; M: medulla. C) Stimulation of the corticotropic axis changes the cortical SUMOylation profile. Sixmonth-old female mice were treated with dexamethasone (Dex.) for 5 days, injected or not the last morning with ACTH and sacrificed 2h or 6h later. Basal conditions correspond to untreated mice. The accumulation of conjugated nuclear proteins to Sumo1 and Sumo2/3 was analysed by immunohistochemistry. The quantification graphs represent mean ± SEM of SUMO nuclear staining intensity in zF cells for 6 adrenals from 6 different animals and 10 nuclei per adrenal. Black and white arrows: nuclear Sumo staining in capsular and endothelial cells, respectively. Statistical analyses were conducted by 1-way ANOVA followed by Tukey post hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars = 50 µm.

Fig. 4 .

 4 Fig. 4. SUMOylation is regionalized in human adrenal cortex and altered in nodular hyperplasia from Carney complex patients. A) SUMOylation profile forms a decreasing gradient of intensity from the outer to the inner cortex. The accumulation of conjugated proteins to SUMO1 was analysed by immunohistochemistry in a control human adrenal. The quantification graph represents mean ± SEM of SUMO1 nuclear staining intensity in steroidogenic cells in each histological zone and inter-zone all along the cortex. Statistical analyses were conducted by 1-way ANOVA followed by Dunett's test. ***p<0.001. zG: zona glomerosa, zF: zona fasciculata; zR: zona reticularis. B-C) HypoSUMOylation of PPNAD nodules. PPNAD sections from patients with PRKAR1A germinal mutation were immunostained for SUMO1 (B) or for SENP1 (C) and counterstained with hematoxylin (H&E). HypoSUMOylated nuclei (black arrow). N: nodule. Blue scale bars = 2 cm, black scale bars = 100 µm.

Fig. 5 .Fig. 1 .

 51 Fig. 5. Adrenal cortex SUMOylation is enhanced by constitutive activation of WNT/β-catenin signalling. A) Experimental setup. β-catenin stabilization in adrenal cortex was allowed by deletion of exon 3 of Ctnnb1 (Catnb lox(ex3) allele) using Akr1b7-Cre line (referred to as ∆Cat mice). B) Coimmunofluorescent labeling of Sumo1 (upper) or Sumo2/3 (bottom) (green), β-catenin (red) and merged signals, in adrenal sections from ∆Cat female mice of 18 months. Inset, magnification view showing nuclei from cells with low (white arrowheads) or high (white asterisk) β-catenin staining. Scale bars = 20 µm. C) The quantification graphs represent mean ± SEM of SUMO nuclear Sumo1 and Sumo2/3 staining intensity in cells that express low or high levels of β-

Fig. 2 .

 2 Fig. 2. Acute hormonal induction or constitutive activation of PKA signalling affect protein SUMOylation and SUMO pathway gene expression in mouse adrenals. A-B) Wild-type mice were injected with immediate ACTH and adrenals were removed 2h or 4h later, control adrenals were dissected from untreated mice (0h) and subjected to RNA extraction. A) Experimental setup. B) RT-qPCR analyses of SUMOylation pathway gene expression normalised with respect to Actin gene expression. Statistical analyses were conducted by 1-way ANOVA followed by Dunett's test. C-D) Carney complex mouse model with constitutive activation of PKA following adrenal inactivation of Prkar1a (Sf1-Cre::Prkar1a fl/fl mice). C) Experimental setup. Adrenals from Sf1-Cre::Prkar1a +/+ (WT) and Sf1-Cre::Prkar1a fl/fl (KO) mice were removed and subjected to RNA and protein extraction. RT-qPCR analyses of SUMOylation pathway gene expression normalised with respect to Actin gene expression and, D) western blot analyses of Sumo2/3 conjugates in adrenal protein extracts and quantification (histogram). Tubulin signal was used to normalise the quantification of SUMO signals. Statistical analyses were conducted by Student t-test. Quantification histograms represent mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Senp1

Fig. 3 .Fig. 4 .

 34 Fig.3. SUMOylation is regionalised in mouse adrenal cortex and modulated by ACTH/PKA signalling. A-B) SUMOylation profile forms a decreasing gradient of intensity from the outer to the inner cortex. The accumulation of conjugated proteins to Sumo1 (A) and Sumo2/3 (B) was analysed by immunohistochemistry in adrenals of 6-month-old female mice. The quantification graphs represent mean ± SEM of SUMO nuclear staining intensity in each histological zone and inter-zone all along the cortex and in medulla, for 3 adrenals from 3 different animals and 50-60 nuclei per adrenal. Statistical analyses were conducted by Kruskal-Wallis followed by Dunn's test. Ca: capsule; zG: zona glomerosa; zF ext : external zona fasciculata; zF int : internal zona fasciculata; M: medulla. C) Stimulation of the corticotropic axis changes the cortical SUMOylation profile. Six-month-old female mice were treated with dexamethasone (Dex.) for 5 days, injected or not the last morning with ACTH and sacrificed 2h or 6h later. Basal conditions correspond to untreated mice. The accumulation of conjugated nuclear proteins to Sumo1 and Sumo2/3 was analysed by immunohistochemistry. The quantification graphs represent mean ± SEM of SUMO nuclear staining intensity in zF cells for 6 adrenals from 6 different animals and 10 nuclei per adrenal. Black and white arrows: nuclear Sumo staining in capsular and endothelial cells, respectively. Statistical analyses were conducted by 1-way ANOVA followed by Tukey post hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bars = 50 µm.

5 BFig. 5 .Dumontet

 55 Fig. 5. Adrenal cortex SUMOylation is enhanced by constitutive activation of WNT/β-catenin signalling. A) Experimental setup. β-catenin stabilization in adrenal cortex was allowed by deletion of exon 3 of Ctnnb1 (Catnb lox(ex3) allele) using Akr1b7-Cre line (referred to as ∆Cat mice). B) Coimmunofluorescent labeling of Sumo1 (upper) or Sumo2/3 (bottom) (green), β-catenin (red) and merged signals, in adrenal sections from ∆Cat female mice of 18 months. Inset, magnification view showing nuclei from cells with low (white arrowheads) or high (white asterisk) β-catenin staining. Scale bars = 20 µm. C) The quantification graphs represent mean ± SEM of SUMO nuclear Sumo1 and Sumo2/3 staining intensity in cells that express low or high levels of β-catenin (3 adrenals from 3 different animals and 10-15 nuclei per adrenal). D) Sumo1 or Sumo2/3 nuclear staining is correlated with β-catenin activation. Spearman correlation between staining intensity values of Sumo1 or Sumo2/3 nuclear signal and staining intensity of β-catenin nucleo-cytoplasmic signal. r: Spearman correlation coefficient. E) Adrenals from Akr1b7-Cre::Catnb +/+ (WT) and Akr1b7-Cre::Catnb lox(ex3)/+ (∆Cat) mice were removed and subjected to RNA extraction. RT-qPCR analyses of SUMOylation pathway gene expression normalised with respect to Actin gene expression. Statistical analyses were conducted by Student t-test. Quantification histograms represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001

.

  Adrenal tissue analysis and genetic diagnosis was obtained from patients after signed consent. The study was approved by an institutional review committee (Advisory Committee for the Protection of Persons in Biomedical Research, Cochin Hospital, Paris). PPNAD paraffin sections were performed from adrenal samples of PPNAD patients who underwent bilateral adrenalectomy for ACTH-independent Cushing's syndrome. Most patients were carriers of PRKAR1A inactivating germinal mutations.

	Statistical analysis. Statistical analyses were performed using a Student t-test or Mann-Witney test depending
	on Gaussian values distribution to compare two groups. For multiple comparisons, one way ANOVA (Gaussian
	values distribution) test or Kruskal-Wallis (non-Gaussian values distribution) test followed by Dunett's, Dunn's
	or Tukey post hoc tests were performed. A p-value of less than or equal to 0.05 was considered statistically
	significant. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001).
	Study approval. All animal work was conducted according to French and European directives for the use and
	care of animals for research purposes and was approved by the local ethics committee, C2E2A (Comité
	d'Ethique pour l'Expérimentation Animale en Auvergne).

  Wild-type mice were injected with retard ACTH and adrenals were removed after 6h or 24h, control adrenals were dissected from untreated mice (0h) and subjected to RNA extraction. Experimental setup. RT-qPCR analyses of SUMOylation pathway gene expression normalised with respect to Actin gene expression. Statistical analyses were conducted by 1-way ANOVA followed by Dunett's test.

	Supplemental Fig. S2. A)
	Relative mRNA level	2.0 1.5

B) Adrenals from Sf1-Cre::Prkar1a +/+ (WT) and Sf1-Cre::Prkar1a fl/fl (KO) mice were removed and subjected to RNA extraction. RT-qPCR analyses of SUMOylation pathway gene expression normalised with respect to Actin gene expression. Statistical analyses were conducted by Student t-test. Quantification histograms represent mean ± SEM. *p<0.05; **p<0.01; ***p<0.005.

ACKNOWLEDGMENTS

We also wish to thank Sandrine Plantade, Philippe Mazuel and Khirredine Ouchen for care of the transgenic mice. We thank Kaitlin J. Basham (University of Michigan) for critical reading of the manuscript. This work was funded through institutional support from Centre national de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Clermont-Auvergne, the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25) and supported by grants from Région Auvergne-Rhône-Alpes to T.D., from Fondation Association pour la Recherche sur le Cancer to T.D., from Agence Nationale pour la Recherche (ANR-14-CE12-000) to A.M.

AUTHOR CONTRIBUTION

TD, ISB, DD, AMLM, PV, and AM conceived and designed the experiments; TD, ISB, DD, AMLM, AB, NM, CD, JCP and FRB performed the experiments; TD, ISB, DD, AMLM and AM analysed the data; BR, MBL, IT and JB provided human samples; AM supervised the project, and TD and AM wrote the paper; PV and all authors edited the paper. Supplemental Fig. S3. SUMOylation is regionalised in mouse adrenal cortex from both female and male mice. The accumulation of conjugated proteins to Sumo1 and Sumo2/3 was analysed by immunohistochemistry in adrenals of 6month-old mice. A) SUMOylation profile forms a decreasing gradient of intensity from the outer to the inner cortex.

Pictures of Sumo1 and Sumo2/3 immunostaining illustrating the delimitation of zones and inter-zones chosen for quantification of nuclear staining intensity in Fig. 3. Ten nuclei (red dots) were analysed per area. B) Sumo1 and Sumo2/3 immunostaining patterns were compared in adrenals from 4 male and 5 female mice. The quantification graphs represent mean ± SEM of SUMO nuclear staining intensity in zG and zF from male (white lozenge) and female (grey lozenge) mice (20 nuclei per adrenal zone). Percentage decrease of SUMO staining between zG and zF was indicated for both sexes. Ca: capsule; zG: zona glomerosa; zF ext : external zona fasciculata; zF int : internal zona fasciculata; M: medulla. Black and white arrows: nuclear Sumo staining in capsular and endothelial cells, respectively.