New insights into the basic processes of targeted intracellular protein destruction.
The targeted destruction of proteins by the proteasome system regulates directly many biological functions, allows the cells to get rid off non-functional proteins and participitate to extreme environmental adaptation. Proteasome malfunctions are implicated in ageing, pathogenicity, cancer and degenerative diseases. Despite important efforts, many caveats remain in the understanding of this complex process. The archelyse projects aims to propose new paradigms in this research field by studying the «primitive«, but highly efficient, archaeal 19S Prototype wich represent a simplified eukaryotic-like systems. The PAN complex is a highly dynamic molecular edifice. For this reason, there is still no structure of the full-length PAN complex. The first objective is to determine, by using a combination of advanced structural biology techniques, the structure and mode of action of the archaeal 19S Prototype and to study how these regulators perform mechanical work to unfold the proteins prior to their destruction. Archaea also possess a specific ubiquitine like protein modification system. However nothing is known about how these modified proteins are brought to the PAN complex. The second objective is to identify using in vivo and in vitro interactomic studies proteins interacting with PAN which may represent the primitive ubiquitine modifying enzymes or adpatators that regulate the recognition of protein to be destroyed. Finally, we shall study novel types of large unassigned peptidases complexes that represent partners of the 20S proteasome core particle. The work should demonstrate the first direct evidence for a simplified 19S core complex in Archaea. It will allow a better understanding of the evolution and basic mode of action of the proteasome system in the same time providing insights into the adaptation of archaea to deep-sea chemio-synthetic environments.
The PAN complex is a highly dynamic molecular edifice, highly refractory to conventional x-ray crystallography. Different types of PAN complexes and putative interacting proteins such as deubiquitinase-like enzymes arizing from different deep-sea archaeal strains will be expressed in E.coli and purified. Protocols will be developed for each complex to obtain monodisperse and stable preparations. Unfoldases assays and Biacore experiments will be developed to study the structural dynamics of the system. Protein fragments will also be purified for X-ray crystallography. Integrated structural biology approach combining Small Angle X-ray Scattering (SAXS), cryo-electron microscopy and crystallography will allow to obtain a pseudo-atomic models of the PAN structure. The conformational changes associated with ATP hydrolysis or substrate binding and unfolding will be also determined in proteasome-free and proteasome-bound states. This study will represent proof of concept for the development of innovative small angle neutron scattering (SANS) studies that may permit real time studies on the unfoldase process. In this project, we will also use the intact assembled PAN complex as bait to pull down and co-immunoprecipitate the components of the archaeal 19S-like complex from cell extracts. The proteins will be purified and their functional and physical interaction with the PAN complex will be tested in vitro. Their functions will be determined and their structures will be solved by X-ray crystallography as for the unassigned large peptidases complexes that we identified in the archaeal proteomes. Among them, potential proteasome associates aminopeptidases (PAAP) were already identified. Their substrate specificities and functional regulation will be studied in order to specify their role in protein degradation.
Appolaire A, Dura MA, Ferruit M, Andrieu JP, Godfroy A, Gribaldo S and Franzetti B. The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties. Mol. Microbiol. (2014) doi : 10.1111/mmi.12775.
Appolaire A, Girard E, Colombo M, Durá MA, Moulin M, Härtlein M, Franzetti B and Gabel F. Small angle neutron scattering reveals the assembling mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase. Acta Crystallogr D Biol Crystallogr. D70 (2014) doi:10.1107/S1399004714018446.
Appolaire A, Rosenbaum E, Dura MA, Colombo M, Marty V, Noirclerc Savoye M, Godfroy A, Schoehn G, Girard E, Gabel F and Franzetti B. Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation. Journal of Biological Chemistry (2013) 288(31) : 22542-22554
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.
Monsieur Bruno FRANZETTI (Institut de Biologie Structurale) – firstname.lastname@example.org
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.
CNRS-CEA-UJF. UMR5075 Institut de Biologie Structurale
UBO-CNRS-IFREMER. UMR 6197 Laboratoire de Microbiologie des environnements extremes
Help of the ANR 499,723 euros
Beginning and duration of the scientific project: December 2012 - 48 Months