Physical and functional plasticity of proteasomes. – PLASTIZOME

To answer the questions stated above, we plan to rely on the yeast Saccharomyces cerevisiae as a model system. This project combines genome-wide and specific approaches, genetic and biochemical strategies and is based on a solid knowledge of the proteasome system and a solid expertise in various technical approaches. It should improve the global understanding of proteasome functions and regulations but also provide detailed molecular mechanisms of some of these functions and regulations. We will then transpose our findings to mammalian systems.

Recently, we have been interested in proteasome assembly, which participates in positive regulation of proteasome activity. Some specific proteins called ‘assembly chaperones’ are in charge of promoting an harmonious and efficient formation of the proteasome machinery. We have been interested in the molecular requirements for the specific interaction between assembly chaperones and proteasome subunits. We have reconstituted in vitro an assembly complex containing three base subunits in association with the Hsm3 chaperone. We have established which protein is in direct interaction with another and have delineated the domain of each partner involved in the interaction. Moreover, , we have recently obtained a structure of the complex by X-ray crystallography . Our study sheds light on the molecular requirements for the specificity of interaction surfaces within such complex machineries.

An elevated proteasome activity contributes to tumorigenesis and proteasome inhibitors are presently used in anti-cancer treatment. Further understanding on how the proteasome dependent degradation pathway is regulated may provide molecular basis for developing new strategies in preventing formation of intracellular protein aggregates in aging and neurodegenerative disorders and improving some anti-cancer therapies based on proteasome inhibition. Assembly chaperones could provide additional interesting targets to enhance both efficiency and specificity of anti-proteasome treatments.

This study underscores the multi-modal and amazing properties of assembly chaperones in promoting the biogenesis of large marcromolecular machines. It provides rational basis to design specific inhibitors of proteasome assembly.
Barrault MB, Richet N, Godard C, Murciano B, Le Tallec B, Rousseau E, Legrand P, Charbonnier JB, Le Du MH, Guérois R, Ochsenbein F, Peyroche A . (2012) Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly. Proc Natl Acad Sci U S A. Apr 24;109(17):E1001-10. Epub 2012 Mar 29.

The 26S proteasome is a large multi-subunit protease complex located in the cytoplasm and the nucleus of cells. This proteolytic system is conserved in all eukaryotes. This proteasome is the central catalytic unit of the Ubiquitin-Proteasome system. The ubiquitin/proteasome system has become increasingly recognized as a controller of numerous physiological processes, including signal transduction, DNA repair, chromosome maintenance, transcriptional activation, cell cycle progression, cell survival, and certain immune cell functions. This is in addition to its more established roles in the removal of misfolded or damaged proteins. Hence, deciphering new specific functions for proteasomes is still a major challenge.
The 26S proteasome is responsible for regulated proteolysis of most intracellular proteins yet the focus of intense regulatory action itself. Proteasome abundance is responsive to cell needs or stress conditions, and dynamically localized to concentrations of substrates. Proteasomes are continually assembled and disassembled, and their subunits subject to a variety of posttranslational modifications. A growing body of evidence indicates that a number of proteins transiently associate with the proteasome complexes to perform specific activity. Hence, proteasomes are larger and more diverse in composition than previously assumed. Many regulators are certainly modulating the proteasome activity upon specific physiological conditions and are yet to be discovered. Proteasomes appear as highly dynamic structures, whose specific multi-task functions and regulations need to be further investigated and determined. We propose to contribute to decipher proteasome complexes in the current project combining multiple approaches.

Combining genetic and deep biochemical approach, we previously unraveled five proteasome-dedicated chaperones, which assist the assembly of distinct proteasome subcomplexes (Le Tallec et al., 2007; Le Tallec et al., 2009). Our study highlights a striking link between proteasome function and DNA damage response, which is of particular interest since nuclear functions of proteasome remain elusive. Besides, it allowed us to uncover a new specific phenotype in response to the carcinogenic bulky alkylating agent 4-Nitroquinoline 1-oxide (4NQO). This phenotype is a new sensitive tool to detect any impairment in proteasome functioning.
An elevated proteasome activity contributes to tumorigenesis and proteasome inhibitors are presently used in anti-cancer treatment. It thus seems important to identify factors that modulate the level of expression and activity of the proteasome. We propose to study such factors using S. cerevisiae as a cellular model and then examine the functional conservation of the candidates in mammal cells. The aim of the project is to identify and characterize functional and physical partners of the proteasome and to study proteasome modulation in response to DNA damage or to entry into quiescence. The project is based on the development of different genetics and chimiogenomics screens and quantitative proteomics and 2-hybrid approaches.