Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

The Hsp90-R2TP machinery and the assembly of macromolecular complexes: role in RNP biogenesis. – Hsp90assembly.com

Architecture of the Hsp90-R2TP chaperone system: a therapeutic target in oncology.

We wish to understand the mechanism of action of a new therapeutic target, the Hsp90-R2TP chaperone system, in the biogenesis of cellular machineries that control important phenomena in cancer such as cell growth or DNA repair.

Understand the spatial organization of the Hsp90-R2TP system to illuminate its function.

The Hsp90 molecular chaperone and its R2TP cochaperone form an assembly and maturation system for complex molecular machines that are essential to cell life.<br />To shed light on the mode of action of the Hsp90-R2TP system, we will analyze its architecture and the interactions it forms during the assembly of cellular machineries such as snoRNP. Our main objectives are: the mapping of protein interactions within the system and in particular the characterization of ATP-dependent interactions made by Rvb1 and Rvb2 proteins; the resolution of the three-dimensional structures of Hsp90-Tah1-Pih1 chaperone and snoRNP precursors complexes. These results should allow us to propose a comprehensive structural model of the system and explains its chaperone function during assembly of the snoRNP.<br />This project should enable a better understanding of the assembly mechanisms of cellular machinery by the Hsp90-R2TP system. Given the importance of the system in many human diseases including cancer, this information will help the development of new therapeutic approaches.

The mapping of protein-protein interactions is obtained by a random mutagenesis analysis coupled with molecular genetics in yeast.
The three-dimensional structures of chaperone complexes and precursors of cellular machineries are obtained by various approaches combining biochemistry, biophysics, mass spectrometry, X-ray crystallography and nuclear magnetic resonance.
The structural and functional comprehensive model of the chaperone system during assembly of snoRNP cellular machineries will be obtained by modeling using all available constraints previously established (interaction map, structures by crystallography or nuclear magnetic resonance and mass spectrometry).

We recently solved the high-resolution three-dimensional structures of the Tah1 protein free and bound to the Hsp90 chaperone by nuclear magnetic resonance. These structures, made in partnership with the ETH Zürich (Switzerland), allow the first detailed description of the recruitment mechanism of R2TP complex on the Hsp90 chaperone.

The three-dimensional structure of the Hsp90-Tah1 complex gives us valuable information on the way towards a comprehensive model of the Hsp90-R2TP chaperone system. Theses detailed structural information will also help the rational design of inhibitors for therapeutic purposes. This new type of inhibitors should block the formation of the Hsp90-R2TP chaperone system, essential for the assembly of cellular machinery, and could thus have potential applications in oncology.

publication submitted

The Hsp90 molecular chaperone is responsible for the folding and conformational activation of essential proteins involved in cell signaling and regulation. Many of these so-called “client” proteins control essential mechanisms of malignant transformation such as cell proliferation, immortalization, angiogenesis or apoptosis. Because specific Hsp90 inhibition results in the proteasomal degradation of theses oncogenic clients, the chaperone has attracted enormous interest as an anti-cancer target capable of acting on all the hallmarks of cancer.
Since then, proteomic approaches have demonstrated a much larger cellular role for Hsp90 with possibly hundreds of dependent processes, and major roles in the dynamics of many macromolecular assemblies. However, in contrast to other chaperones, the molecular function of Hsp90 remains elusive. Indeed, Hsp90 recognizes a quasi-native state in the latest stage of protein folding, thus the great functional and structural diversity of Hsp90 clientele opens numerous questions about the mechanism of client protein recognition. A first level of analysis arises from the concomitant discovery of new cochaperone protein that may assist Hsp90 in this process. Indeed, cochaperones seem to play an important role in the selectivity of Hsp90 machinery towards its clients. Specific cochaperones could thus deliver a specific family of clients to Hsp90, however, little is known about the molecular mechanism behind this specificity.
Hsp90 and the R2TP cochaperone have recently emerged as a central player in the assembly of large cellular machines. We have shown that they are essential for the assembly of both snoRNP and RNA polymerases, while others showed their involvement in the biogenesis of PIKKs such as mTOR, ATM or DNA-PKcs. The R2TP complex is composed of the co-chaperones Tah1, Pih1 and the AAA+ ATPases Rvb1 and Rvb2. We have shown that the Tah1-Pih1 cochaperone complex blocks the Hsp90 ATPase cycle in order to allow the loading of client proteins. These and other preliminary data allowed us to propose a mechanism for the HSP90/R2TP system, where the transfer of client proteins to target complexes depends on dynamic interactions regulated by coordinated ATPase cycles of Hsp90 and Rvb1/2. To understand how the Hsp90/R2TP system works, we will use a structural approach coupled to functional studies, and we will focus our attention on the assembly of snoRNP complexes.

Our main objectives are the following:
1-High resolution mapping of all the protein-protein interactions involved, using a transposition approach coupled to two-hybrid screens. Use yeast molecular genetics to assess their relevance in vivo.
2-Solve the structure of the Hsp90-Tah1-Pih1 complex by crystallography, NMR and biochemical or biophysical approaches.
3-Solve the structure of the early pre-snoRNP complex Rsa1-Snu13-Hit1, with or without Nop58, and its link with Pih1. Use NMR to solve the structure of sub-complexes and to constrain the modeling of the full complex.
4-Characterize the ATP driven dynamics of Rvb1/Rvb2 interaction with snoRNP core proteins and assembly factors, using mutagenesis of their nucleotide binding sites and binding assays to precisely map the interaction sites.
5-Build a structural model of the chaperone-pre-snoRNP complex by in vitro or in vivo assembly coupled to structural modeling, using available constraints from protein-protein interactions mapping, crystallography, SAXS and native mass spectrometry.
6-Obtain a mechanism for the HSP90-R2TP chaperone system, using our models and the known structure and interaction data of Rvb1/2 proteins.

This project should provide major novel insights in the mechanism of assembly of Hsp90-R2TP-dependent macromolecular complexes. Given the importance of theses molecular machines in several diseases including cancer, the structures that we propose to solve should provide a great potential for new therapeutic targets with improved selectivity.

Project coordination

Philippe MEYER (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B) – meyer@lebs.cnrs-gif.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IGMM/CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE LANGUEDOC-ROUSSILLON
UMR 7214 CNRS-UHP UNIVERSITE DE NANCY I [HENRY POINCARE]
IPHC - CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
BMCE - IBPC/CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B

Help of the ANR 550,000 euros
Beginning and duration of the scientific project: November 2011 - 48 Months

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