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Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes., Author summary Spermatogenesis defects are a common cause of male sterility. Spermatogenesis occurs in the seminiferous tubules of the testes and involves adhesive interactions between developing germ cells and Sertoli cells. Knock-out mouse models identified several adhesion molecules that are critically involved in spermatogenesis. We previously demonstrated that the Junctional Adhesion Molecule-C (JAM-C) plays a crucial role in establishing spermatids polarity. The latter involves rearrangements of the Golgi apparatus in spermatids which contribute to acrosome formation. The present study demonstrated that the C-terminal cytosolic region of JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55) encoded by Gorasp2 and that spermatogenesis was impaired in Gorasp2-deficient mice. We developed an inhibitor of GRASP55 interaction with JAM-C and demonstrated that treatment of wild-type mice with the inhibitory compound induced germ cell loss. Therefore, the male infertility-associated pathway identified in this study is important not only from a genetic point of view, but also as a potential target for male contraception.

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core., PLoS ONE, 9 (8), ISSN:1932-6203

International audience; Second cause of death due to cancer worldwide, colorectal cancer (CRC) is a major public health issue. The discovery of new therapeutic targets is thus essential. The pseudokinase PTK7 intervenes in the regulation of the Wnt/βcatenin pathway signaling, in part, through a kinase-domain dependent interaction with the β-catenin protein. PTK7 is overexpressed in CRC; an event associated with metastatic development and reduced survival of non-metastatic patient. In addition, numerous alterations have been identified in CRC inducing constitutive activation of Wnt/β-catenin pathway signaling through β-catenin accumulation. Thus,targeting PTK7/β-catenin interaction could be of interest for future drug development. We have developed a NanoBRET TM screening assay recapitulating the interaction between PTK7 and βcatenin to identify compounds able to disrupt this protein-protein interaction. A high-throughput screening allowed us to identify small molecule inhibitors targeting the Wnt pathway signaling and inducing anti-proliferative and anti-tumor effect in vitro in CRC cells harboring β-catenin or APC mutations. Thus, inhibition of the PTK7/β-catenin interaction could represent a new therapeutic strategy to inhibit cell growth dependent on Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

Differentially screening the Fr-PPIChem chemical library on the BET BRD4-BDII versus -BDI bromodomains led to the discovery of a BDII selective tetrahydropyridothienopyrimidinone (THPTP)-based compound. Structure-activity relationship (SAR) and hit-to-lead approaches allowed us to develop CRCM5484, a potent inhibitor of BET proteins with a preferential and 475-fold selectivity for the second bromodomain of the BRD3 protein (BRD3-BDII) over its first bromodomain (BRD3-BDI). Its very low activity was demonstrated in various cell-based assays, corresponding with recent data describing other selective BDII compounds. However, screening on a drug sensitivity and resistance-profiling platform revealed its ability to modulate the anti-leukemic activity in combination with various FDA-approved and/or in-development drugs in a cell- and context-dependent differential manner. Altogether, the results confirm the originality of the THPTP molecular mode of action in the BD cavity and its potential as starting scaffold for the development of potent and selective bromodomain inhibitors.