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Étude du mécanisme d’action du protéasome PAN-20S par diffusion de neutrons aux petits angles résolue en temps

Abstract : Proteasomes are large ubiquitous macromolecular assemblies composed of a 20S catalytic complex and a regulatory particle containing an AAA-ATPase module. This cellular machine is responsible for selectively degradation of intracellular proteins in order to allow proteome renewal, elimination of defective proteins and control of many biological functions. The objective of this thesis was to reveal the mechanisms by which the AAA-ATPase complexes selectively unfold substrate proteins and translocate them into the 20S particle, where they are destroyed. To this end, an innovative approach was used by combining time-resolved small-angle neutron scattering (TR-SANS) combined with fluorescence spectroscopy to monitor biochemical activity. The proteasome of the hyperthermophilic archaea Methanocaldococcus jannaschii was used as a model system. It is composed of the 20S protease of the regulatory particle AAA-ATPase PAN. A variant of the fluorescent protein GFP was used as a substrate protein.The data obtained show that PAN unfolding activity generates denatured species of GFP forming aggregates. The association with the 20S particle prevents the formation of these species and indicates that once the unfolding of a substrate by PAN is engaged, translocation into the 20S complex and degradation processes are closely coupled. Analysis of the neutron scattering spectra of the GFP substrate reveal that the native GFP population is rapidly disappearing in favor of peptides generated by the 20S protease, as confirmed by mass spectrometric analysis. This demonstrates the highly processive nature of the proteasome. Finally, two modes of action of PAN have been identified depending on the amount of proteins to be degraded compared to the PAN-20S proteasome. This work allows to experimentally validate one of the proteasome function models previously proposed and emphases the importance of controlling the association of proteasomes in vivo. This study also highlights the interest of TR-SANS technique to study the functional dynamics of large cellular machines.
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Submitted on : Friday, September 4, 2020 - 2:15:10 PM
Last modification on : Tuesday, October 6, 2020 - 4:12:09 PM


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  • HAL Id : tel-02930469, version 1



Emilie Mahieu. Étude du mécanisme d’action du protéasome PAN-20S par diffusion de neutrons aux petits angles résolue en temps. Biophysique. Université Grenoble Alpes, 2019. Français. ⟨NNT : 2019GREAY073⟩. ⟨tel-02930469⟩



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