Études fonctionnelles de deux nouvelles protéines centrosomales, NPHP5 et Cep76, et leurs implications dans les maladies humaines

Centrosomes are small organelles that regulate diverse cellular processes such as polarity or mitosis in mammalian cells. They are composed of two centrioles surrounded by a pericentriolar matrix. These centrosomes are the major microtubule organizing centers. Moreover, they promote the formation of cilia, protrusions on the surface of quiescent cells that are critical for signal transduction. A wide variety of human diseases such as cancers or ciliopathies are linked to a malfunction of centrosomes and cilia. Therefore the aim of my research is to understand the mechanisms necessary for the biogenesis and function of centrosomes and cilia. First, I have characterized a novel centrosomal protein called nephrocystin - 5 (NPHP5). This protein is localized, in interphase cells, in the distal region of centrioles. Its depletion inhibits the migration of centrosomes to the cell surface during the early stage of cilia formation. NPHP5 interacts with CEP290 via its C-terminal region that is essential for ciliogenesis. It also interacts with calmodulin, which prevents its self-aggregation. I have demonstrated that the Cep290- and CaM-binding domains as well as the centrosomal localization domain of NPHP5 are separable. Moreover, I have shown that NPHP5 proteins with pathogenic mutations can no longer interact with CEP290 and are not localized to centrosomes, rendering these proteins non-functional. Finally, using a pharmacological approach to modulate the downstream events in the ciliogenic pathway, I showed that cilia formation can be restored even without NPHP5. On the other hand, I studied the role of NPHP5 in the assembly and trafficking of the BBSome into the cilium. The BBSome consists of eight different subunits that assemble into a functional complex of which little is known about the spatiotemporal regulation of its assembly process. I have previously shown that NPHP5 favored the formation of cilia and its dysfunction contributes to the development of nephronophthisis (NPHP). Although the NPHP and BBS syndrome (BBS) are ciliopathies that share common clinical features, molecular basis of these phenotypic similarities is not understood. I found that NPHP5, located at the base of the cilium, contains two separate binding sites for BBSome. Furthermore, I demonstrated that NPHP5 and his partner CEP290 interact dynamically with the BBSome during the transition from quiescence to proliferation. Depletion NPHP5 or CEP290 leads to the dissociation of at least two subunits of BBSome forming a sub-complex that can still traffic into the cilium. I have shown that the transport of cargo to the ciliary compartment through this sub-complex is only partially altered. Finally, I have also focused my research on another centrosomal protein poorly characterized. The centrosomal protein of 76 kDa (Cep76) was previously involved in the maintenance of a single duplication of centrioles per cell cycle, and interacts with the cyclindependent kinase 2 (CDK2). Cep76 is preferentially phosphorylated by cyclin A/CDK2 on the single site S83. This event is essential to suppress centrioles amplification in S phase. I have demonstrated that Cep76 inhibits amplification by blocking the phosphorylation of Plk1 at the centrosome. Moreover, Cep76 can be acetylated at the K279 site in G2 phase, which negatively regulates its activity and phosphorylation on the site S83. These studies will improve our understanding of the biology of centrosomes and cilia and could lead to development of new diagnostic and therapeutic applications.

Keyphrases: Cil, NPHP5, Cep76, Cep290, BBSome, Cell cycle, Cilia, Centrosome