An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.

Proteasome substrate capture and gate opening by the accessory factor PafE from Mycobacterium tuberculosis

“…Although HspR is the only PafE-mediated proteasome substrate established so far, we previously identified several other proteins that accumulate in an Mtb pafE null mutant or in Mtb treated with a proteasome inhibitor (11). Furthermore, we also identified numerous proteins that specifically co-purify with a proteasome trap (11).…”

“…These results indicated that we achieved our goal of acquiring a dodecameric PafE ring with only six GQYL-containing termini. We next examined if this variant PafE could activate Mtb 20S proteolysis by using HspR (heat shock protein repressor), a known PafE-proteasome substrate in Mtb (11). In contrast to the robust degradation of HspR using WT PafE and 20S CPs, PafE-PafE could not degrade HspR (Fig.…”

“…In addition to ATP, the Mpa-proteasome requires proteins to be posttranslationally modified with the small protein Pup (prokaryotic ubiquitin-like protein) to target them for degradation (9,10). In contrast, the PafEproteasome neither requires ATP nor interaction with a post-translational modification on a doomed protein for degradation (8,11,12). Instead, it appears that PafE can facilitate the degradation of unfolded proteins and at least one native protein, HspR, without a post-translational modification (11)(12)(13).…”

“…In contrast, the PafEproteasome neither requires ATP nor interaction with a post-translational modification on a doomed protein for degradation (8,11,12). Instead, it appears that PafE can facilitate the degradation of unfolded proteins and at least one native protein, HspR, without a post-translational modification (11)(12)(13). A failure to degrade HspR by the PafEproteasome results in slowed growth in vitro (12) and likely contributes to the attenuated growth phenotype in vivo (11).…”

“…However, there are several caveats to the interpretation of this structure. The study used a mutated 20S CP to which PafE would more strongly bind: the amino (N)-terminal eight residues of the α-subunits (PrcA) of the 20S CP were deleted, creating an "open-gate" mutant 20S CP (6); and, the catalytic threonine (Thr) of each 20S CP b-subunit (PrcB) was changed to alanine ("T1A" mutant), which increases binding with PafE (and Mpa and substrates) (11). Thus, the 20S CP was used in a "pre-activated" form, preventing any conclusions from being drawn regarding the impact of PafE binding on proteasome activation.…”