Novel proteasome regulation systems in Archaea

Group members associated: Federico Musso, Cécile Cadoux, Eric Girard, Bruno Franzetti

In all cell types the 20S proteasome is responsible for the efficient destruction of damaged cellular components in stress conditions. Understanding how the proteasomes select disabled molecular edifices represent a major challenge in fundamental and biomedical research. Archaea possess a primitive version of the 20S eukaryotic proteasome. The regulation of the archaeal proteasome remains poorly explored and little is known about the its physiological role and importance in extremophilic adaptation. We seek to discover novel proteasome targeting machines that guarantee proteome fitness in hyper-thermophilic Archaea. We characterize the P. abyssi cellular interactomes of PAN-20S proteasome and of novel interactants using in vitro pull-downs/Co-IP at IBS and in vivo pull-downs (collaboration with D. Flament/ M. Jebbar team in Brest for genetics and Y. Couté from Edyp-Lab for proteomics). This work, carried out with several his- and strep-tagged bait proteins, reveals a strong connection with specific cellular functions. It also identified a dozen unknown proteasome partners some of them able to activate proteasome degradation of model proteins in vitro. We study the structures, mode of action of these large complexes using high-resolution cryo-EM and time resolved studies (collaboration with G. Schoehn- EM group IBS). We use native interactomics, peptidomics-MS and biochemistry to identify their respective natural substrates in order to determine their cellular functions. The structural study of these machineries in various archaeal species, including methanogens should make it possible to determine whether these systems are associated with certain types of extremophilic adaptation.

Funding

Collaborations

• C. Brochier : Bioinformatics and molecular evolution (LBBE, Lyon 1 University)
• Y Couté : Proteomics (Edyp Laboratory-Grenoble)
• D. Flament and M. Jebbar. Laboratoire BEEP (UBO-Ifremer), Brest

Selected Publications

• Observing protein degradation by the PAN-20S proteasome by time-resolved neutron scattering

• Emilie Mahieu, Jacques Covès, Georg Krüger, Anne Martel, Martine Moulin, Nico Carl, Michael Härtlein, Teresa Carlomagno, Bruno Franzetti, Frank Gabel
  Biophysical Journal, 2020, 119 (2), pp.375-388. ⟨10.1016/j.bpj.2020.06.015⟩

• An NMR study of a 300-kDa AAA+ unfoldase
  Georg Krüger, John Kirkpatrick, Emilie Mahieu, Bruno Franzetti, Frank Gabel, Teresa Carlomagno. Journal of Molecular Biology, 2023, 435 (11), pp.167997. ⟨10.1016/j.jmb.2023.167997⟩

• Characterization of a small tRNA ‐binding protein that interacts with the archaeal proteasome complex.
  Gaëlle Hogrel, Laura Marino-Puertas, Sébastien Laurent, Ziad Ibrahim, Jacques Covès, Eric Girard, Frank Gabel, Daphna Fenel, Marie‐claire Daugeron, Béatrice Clouet-d’Orval, Tamara Basta, Didier Flament, Bruno Franzetti. Molecular Microbiology, 2022, Bacterial macromolecular machineries, 118 (1-2), pp.16-29. ⟨10.1111/mmi.14948⟩

• Structural Insight into Ubiquitin-Like Protein Recognition and Oligomeric States of JAMM/MPN(+) Proteases.
  Shiyun Cao, Sylvain Engilberge, Eric Girard, Frank Gabel, Bruno Franzetti, Julie A Maupin-Furlow. Structure (London, England : 1993), 2017, ⟨10.1016/j.str.2017.04.002⟩

• Chamieh H, Marty V, Guetta D, Perollier A and Franzetti B
  Stress regulation of the PAN-proteasome system in the extreme halophilic archaeon Halobacterium. Extremophiles (2012) 16(2): 215-225

• Chamieh H, Guetta D and Franzetti B
  The two PAN ATPases from Halobacterium display N-terminal heterogeneity and form labile complexes with the 20S proteasome.
  Biochemical Journal (2008) 411(2): 387-397