Structural, dynamical and functional study of the proteasome activating complex (PAN) ; Etude de la structure et du mécanisme d’action du complexe unfoldase PAN, un activateur du protéasome 20S

Intracellular protein degradation is a fundamental process occurring in all organisms, from bacteria to human. The continual degradation of proteins is necessary for regulating the intracellular levels of enzymes that control all metabolic reactions, as well as the general content of all other proteins, in response to physiological changes. A state of a dynamic equilibrium is created where the intracellular concentration of a protein can be modulated by changes in the synthesis rate as well as the degradation rate. The work presented in this thesis deals with the proteasome activating complex from archaeal cells (PAN). PAN is an energy dependent hexameric complex discovered in archaea and involved in the unfolding of protein substrates to facilitate their degradation by the 20S proteasome. All the previous structural studies on the assembled PAN complex have failed in revealing the structure of the whole complex because of the difficulties encountered during sample preparation and stabilization. In the first part of this work we determined a Cryo-electron microscopy structure and a pseudo-atomic model of the hexameric PAN complex from Pyrococcus horikoshii. In addition, the study of the different conformational states of the PAN complex induced by nucleotide binding helped in gaining several information about the AAA+ unfoldases mechanism and to propose a mode of action of the PAN complex. The second part of the study led to elucidate the question about the dynamic and the conformational changes of the AAA+ unfoldases in general and the PAN complex in particular. The method of contrast variation in Small Angle Neutron Scattering (SANS) coupled with online fluorescence spectroscopy applied to study, in real-time, the substrate unfolding process by the PAN complex from Methanococcus jannaschii allowed to reveal, for the first time, a contraction movements of PAN during substrate unfolding induced by ATP hydrolysis and followed by a relaxation of the molecule at the end of the process. The unfolding mechanism ...