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.
Highlights
Large protease complexes that isolate their active sites on the inner walls of a cylindrical compartment exist in all organisms [1, 2]
A functionally analogous substrate recruitment and degradation system has been described in mycobacteria [21, 22], where the homohexameric proteasomal ATPase ring Mpa recognizes protein substrates modified with the prokaryotic ubiquitin-like protein Pup, unfolds them and transfers them into the 20S proteasome for degradation [23, 24]
It has been shown that the mycobacterial proteasomal ATPase Mpa employs a Cterminal hydrophobic-tyrosine-X motif (HbYX motif) in the interaction with the 20S proteasome [23, 24]
Summary
Large protease complexes that isolate their active sites on the inner walls of a cylindrical compartment exist in all organisms [1, 2]. Two types of interaction partners have been described for the proteasome core cylinder (referred to as 20S proteasome), the ATPase-active 19S regulatory complex and the non-ATPase 11S complex ( PA28 or REG) and PA200 [11,12,13,14,15] Both interactors dock to the 20S proteasome via a C-terminal hydrophobic-tyrosine-X motif (HbYX motif) that inserts into hydrophobic pockets on the a-rings of the proteasome [16]. It leaves open the possibility that a proteasomal assembly chaperone is needed for promoting a conformational change in the 20S particle supporting gate-opening in vivo Such assembly factors have been described for both eukaryotic as well as archaeal proteasomes and were shown to function in a HbYX motif-dependent fashion [26]
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