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ANR funded project

Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale (Blanc SVSE 8) 2012
Projet ARCHELYSE

Novel protein degradation pathways in Archaea

Protein destruction within cells regulates many cellular functions and rid the cell of abnormal proteins. Intracellular proteases are therefore involved in stress response and aging, and deregulation of proteolysis is responsible for many human degenerative diseases and cancers. The ubiquitin-proteasome machinery is the main protein destruction system. However, despite a decade of research one still don’t understand how the proteasome activity is regulated and the existence of other degradation pathways is suspected. The ARCHELYSE project aims to address these important questions by studding the primitive proteasome regulatory system from Archaea, a separate domain of Life with many aspects of their biology closer to eukaryotes, including proteolysis.
Recent findings showed that a ubiquitin-like proteasome targeting system exists in Archaea. However, the machinery responsible for the processing of the conjugated proteins and their targeting toward the proteasome is still unknown. In Eukarya, this function is fulfilled by the 19S regulatory particle. The first objective of the project is to characterize the 19S prototype in Archaea. The structural study of a less complex and hyperstable homologue from Pyrococcus, a deep sea hyperthermophile, will allow to understand the eukaryotic 19S function. It will also provide a tractable system to give insights into the evolutionary mechanisms underlying the emergence of the eukaryotic elaborated ubiquitination and 26S proteasome machineries.
In Archaea, the PAN complex is a homologue of the eukaryotic proteins forming the base of the 19S complex. PAN unfolds proteins and stimulates the proteasome activity. In preparation to this project, we succeeded in obtaining the first structural data of the intact, assembled PAN complex. The objective is to continue this effort and to determine the structure of PAN alone and in association with HydX, a putative deubiquitinase representing the core of the archaeal 19S complex. The peptidase activity of HydX-PAN will be determined. The other missing components of the archaeal 19S complex will be sought by using a specific pull down proteomics approach developed by partner 2. The PAN and HydX interactants will thus be identified. The interactions will be verified in vitro. Then, the activities and structures of the most relevant proteins will be studied individually. Finally one will reconstruct the primitive proteasome regulatory particle. A combination of small angle scattering, crystallography and electron microscopy techniques will be used to determine its structure.
In vivo studies indicated that PAN interacts with uncharacterized peptidase complexes different from the 20S proteasome. We have identified and purified 4 unassigned peptidases that form giant complexes. The second objective of the project is to determine the structure and the activity of novel types of large proteolytical machines, possibly conserved in eukaryotes. In eukaryotes as in Archaea, compensatory peptidase activities were detected in proteasome defective cells. Thus the project may reveal the identity of the peptidases responsible for non-proteasomal protein destruction pathways. These studies are associated with medical issues since the proteasome system represent a important pharmaceutical target. Moreover, the new enzymes activities might be patented
The ARCHELYSE project builds on solids preliminary results and on a strong expertise in the biochemistry and integrated structural biology of large peptidases complexes. The partnership with the extremophile microbiology laboratory represents a great opportunity in the field. Beside the biological interest of the research theme, the coordinator’s group was created at IBS to develop small angle scattering, anomalous phasing and high-pressure crystallography methods to improve the structure determination of large complexes. These methods will greatly increase our chance to succeed in the most challenging part of the project.

Partners

CNRS-CEA-UJF. UMR5075 Institut de Biologie Structurale

UBO-CNRS-IFREMER. UMR 6197 Laboratoire de Microbiologie des environnements extremes

ANR grant: 499 723 euros
Beginning and duration: janvier 2013 - 48 mois

 

ANR Programme: Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale (Blanc SVSE 8) 2012

Project ID: ANR-12-BSV8-0019

Project coordinator:
Monsieur Bruno FRANZETTI (Institut de Biologie Structurale)
franzetti@nullibs.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.