Abstract
HSP90 is a chaperone that stabilizes many proteins involved in cell proliferation mechanisms. As a result, it plays an important role in tumorigenesis and is the target of many cancer treatments. However, HSP90 has many client proteins, and its inhibition can lead to unwanted pleiotropic effects. HSP90 works with co-chaperones that are specific to certain client protein classes and could be targeted in treatments. It is therefore important to understand how these co-chaperones work and what their substrates are. The R2TP, also called PAQosome (Particle for Arrangment of Quaternary structure), is a co-chaperon of HSP90 which has the unique property of being involved in the assembly of macromolecular complexes. It contains the AAA+ ATPase RUVBL1/2, which have chaperone activity themselves, and the RPAP3 and PIH1D1 proteins which serve as adapters and regulators. Remarkably, most of the known substrates of R2TP are involved in cell proliferation mechanisms. This includes PIKKs regulators, including mTOR, as well as basal machinery such as RNA polymerases or snoRNPs/snRNPs.The N-terminal domain of PIH1D1 (PIH1D1_NTER) is known to recognize certain client proteins with a DSDD/E domain in which serine is phosphorylated. In order to better characterize the HSP90/R2TP system, I searched for new partners of PIH1D1_NTER by proteomic studies after affinity purification. In addition to the known substrates of R2TP, I have identified the subunits of the TSC complex (Tuberous Sclerosis complex): TSC1, TSC2 and TBC1D7. This is very interesting because this complex is a tumor suppressor. Indeed, mutations in TSC1 or TSC2 lead to a genetic disease, tuberous sclerosis, where patients develop benign tumors in many organs. TSC acts as a negative regulator of the mTOR pathway, specifically the mTORC1 complex that controls cell growth in response to nutrients, growth factors and energy levels.My thesis project has two objectives: 1) to characterize the physical and functional links between HSP90/R2TP system and TSC complex; 2) to understand the role of this system in the regulation of mTORC1 pathway.To answer to the first objective, I have first characterized the interaction between HSP90/R2TP and TSC subunits at the molecular level. For this, I have carried out proteomics, phospho-proteomics experiments, systematic interaction tests by double-hybrid or IP LUMIER. The results obtained allow to identify several interactions between component of R2TP and TSC subunits and especially a direct interaction between PIH1D1 and TSC1. Then, I have performed functional studies to show that HSP90/R2TP system favors TSC complex assembly.To answer the second objective, I wanted to assess the impact of inactivation of PIH1D1 on mTORC1 pathway. For this, I have studied substrates of this pathway after activation through insulin treatment in human cells knock out for PIH1D1 compared to parental cells. In a collaborative work, I have studied mTORC1 and its substrates in intestine cells from a murin model with inducible knock out of RPAP3.In the medium term, a structural study of the interaction between PIH1D1 and TSC1 will be done, which will allow to better understand how R2TP recognizes its substrates. In the long term, understanding the role of HSP90/R2TP in regulating the mTORC1 pathway would bring new perspectives to design more specific anti-cancer treatments with fewer side effects.