Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

Department of Biology, Brookhaven National Laboratory, Upton, NY, USA.
The EMBO Journal (Impact Factor: 10.43). 06/2010; 29(12):2037-47. DOI: 10.1038/emboj.2010.95
Source: PubMed


Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the beta-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven alpha-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

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    • "Upon apposition of two half-proteasomes, the propeptide of β-subunits is processed to yield a fully assembled and proteolytically active holo-proteasome complex (Zuhl et al., 1997). While the β-propeptide usually promotes the proteasome assembly in both prokaryotic and eukaryotic 20S proteasomes, the βpropeptide of the M. tuberculosis 20S appears to serve as a thermodynamic hurdle for 20S assembly (Zuhl et al., 1997; Kwon et al., 2004; Li et al., 2010). "
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    ABSTRACT: Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.
    Full-text · Article · Sep 2014 · The Journal of Microbiology
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    • "The Mtb proteasome complex presents a tractable in-cell system for studying the interactions between Pup and the proteasomal ATPase, Mpa. Crystal structures of the Mtb proteasome CP [14,20], the Pup-Mpa coiled coil domain complex [21], as well as in vitro NMR solution studies of Pup-Mpa interactions [19,22,23] are available. E. coli is a relevant prokaryotic host that provides a proper milieu for studying the Pup-Mpa interaction without interfering factors. "
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    ABSTRACT: The Mycobacterium tuberculosis proteasome is required for maximum virulence and to resist killing by the host immune system. The prokaryotic ubiquitin-like protein, Pup-GGE, targets proteins for proteasome-mediated degradation. We demonstrate that Pup-GGQ, a precursor of Pup-GGE, is not a substrate for proteasomal degradation. Using STINT-NMR, an in-cell NMR technique, we studied the interactions between Pup-GGQ, mycobacterial proteasomal ATPase, Mpa, and Mtb proteasome core particle (CP) inside a living cell at amino acid residue resolution. We showed that under in-cell conditions, in the absence of the proteasome CP, Pup-GGQ interacts with Mpa only weakly, primarily through its C-terminal region. When Mpa and non-stoichiometric amounts of proteasome CP are present, both the N-terminal and C-terminal regions of Pup-GGQ bind strongly to Mpa. This suggests a mechanism by which transient binding of Mpa to the proteasome CP controls the fate of Pup.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "The 20S core particle appears to be more ancient than the ubiquitin system, as it operates in both prokaryotic and archaeal ancestors. Crystal structures of the 20S proteasomes from Actinomycetes eubacteria Rhodococcus [56] or M. tuberculosis [57], from the archaeon T. acidophilum [46], from yeast S. cerevisiae [47] and from mammals [48] [58] revealed cylindrical particles with active sites within a large central cavity. The minimal prokaryotic prototype is a homo-dodecamer made of two hexameric rings stacked head to head. "
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    ABSTRACT: The 26S proteasome is a chambered protease in which the majority of selective cellular protein degradation takes place. Throughout evolution, access of protein substrates to chambered proteases is restricted and depends on AAA-ATPases. Mechanical force generated through cycles of ATP binding and hydrolysis is used to unfold substrates, open the gated proteolytic chamber and translocate the substrate into the active proteases within the cavity. Six distinct AAA-ATPases (Rpt1-6) at the ring base of the 19S regulatory particle of the proteasome are responsible for these three functions while interacting with the 20S catalytic chamber. Although high resolution structures of the eukaryotic 26S proteasome are not yet available, exciting recent studies shed light on the assembly of the hetero-hexameric Rpt ring and its consequent spatial arrangement, on the role of Rpt C-termini in opening the 20S 'gate', and on the contribution of each individual Rpt subunit to various cellular processes. These studies are illuminated by paradigms generated through studying PAN, the simpler homo-hexameric AAA-ATPase of the archaeal proteasome. The similarities between PAN and Rpts highlight the evolutionary conserved role of AAA-ATPase in protein degradation, whereas unique properties of divergent Rpts reflect the increased complexity and tighter regulation attributed to the eukaryotic proteasome.
    Full-text · Article · Jul 2011 · Biochimica et Biophysica Acta
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