Bacterial Proteasome Activator Bpa (Rv3780) Is a Novel Ring-Shaped Interactor of the Mycobacterial Proteasome

Abstract

The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins.

Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E ( pafE ) Mutant

“…PafE forms rings, caps 20S CPs, and enhances the degradation of peptides and a model unfolded protein in vitro. Another group reported similar in vitro degradation phenotypes and refer to PafE as Bpa (bacterial proteasome activator) (11). We also found that PafE forms a dodecameric structure, which is unique in proteasome biology (9,12).…”

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

“…While this manuscript was under review, Delley et al reported their independent discovery of PafE as an activator of proteasomal degradation (77). However, Delley et al concluded that PafE forms hexamers whereas we concluded it forms dodecamers.…”

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

“…proteasomal ATPase (Mpa) and an ATPindependent pathway mediated by PafE (also known as Bpa for bacterial proteasome activator) (3,8). To stimulate degradation, Mpa or PafE cap either one or both ends of a 20S CP using GQYL motifs at the carboxyl (C)-termini of each monomer of an activator complex.…”

“…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).…”