Molecular prospecting for novel bioactivities and understanding cellular proteolysis: Studies on proteases and unfoldases from hyperthermophilic Archaea

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TET proteases, found recently in Archaea, are extremophilic enzymes that form large hollow molecular edifices (12 to 24 subunits) and process large substrates. However their function and activities remain poorly explored. Therefore, the project aims to characterise the enzymatic activity and the structure of these complexes with two objectives: I) specify their role into the cell, and II) explore their possible biotechnological use.

The efforts of the project will be focused first in the enzymatic characterisation (conditions for activity, substrate specificity) of four recombinant TET-like proteases from the hyperthermophilic archaeal genus Pyrococcus. Then, a complete study about the structure of these enzymes will be carry out by using analytical ultra centrifugation, electron cryo-microscopy, crystallography and neutron scattering. These investigations will lead to understand the enzymatic mechanisms of the TET proteases and the molecular basis of their substrate specificity. Moreover, since the study is centred on enzymes that are stable and active at temperatures around 100°C, the results obtained could give place to new biotechnological applications. The second part of the project will focus on the cellular role of these proteases.

Because of their complex quaternary structures and their co-purification with molecular chaperone complexes, the TET proteases could participate into the controlled elimination of proteins into the cytoplasm as partners of the 20S proteasome and/or by interacting with protein unfoldase complexes such as PAN. In order to specify this, protease assays will be performed in the presence of recombinant PAN complexes and/or purified 20S proteasome. Since the study of the archaeal systems can be a resource for solving the more intricate problems that are found within the eukaryotic biology, and since these cellular proteolytic processes remain bad understood, the outcomes of the PROTARC project will be of high physiological value.

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CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE

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3, rue Michel-Ange
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