Article

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: 10.75). 06/2010; 29(12):2037-47. DOI: 10.1038/emboj.2010.95
Source: PubMed

ABSTRACT 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.
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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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    PLoS ONE 09/2013; 8(9):e74576. DOI:10.1371/journal.pone.0074576 · 3.23 Impact Factor
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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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