Bpa acts as a proteasomal activator.

<p>A, Peptidase activity measured by fluorescence detection of AMC molecules (λ_ex: 360 nm, λ_em: 460 nm) released from the fluorogenic peptide L-Suc-LLVY-AMC (150 µM). PrcAB exhibits a turnover number (TON) of 0.057±0.004 s⁻¹, which is more than three times less active than Δ7PrcAB with a TON of 0.185±0.014 s⁻¹. Addition of an excess of Bpa to the reaction increases peptidase activity of PrcAB about 1.8 fold to a TON of 0.105±0.008 s⁻¹. B, Degradation of β-casein (27 µM) at 37°C by Δ7PrcAB or PrcAB (0.2 µM) in presence or absence of Bpa or BpaΔHbYX (6 µM protomer) is sampled at different time points and analyzed by Coomassie-stained SDS-PAGE.</p

Bpa interacts with the 20S proteasome through its C-terminal HbYX motif.

<p>A, Vector constructs for the bacterial two-hybrid screen. PrcA and Δ7PrcA were fused to the C-terminus of the catalytic T18 domain, Bpa and BpaΔHbYX to the C-terminus of the catalytic T25 domain of adenylate cyclase. B, MacConkey agar matrix of all pairwise combinations of the pUT18C and pKT25 constructs in triplicates. Successful interaction between T18 and T25 switches the colony from lac− to lac+ and the resulting acidification of the agar is visualized by the pH indicator turning red. C, A quantitative β-galactosidase assay of the same hybrid experiments as shown in B. The assay was performed on chloroform-treated <i>E. coli</i> cells grown overnight in liquid LB medium containing 0.5 mM IPTG. The background activity is indicated by the negative control (no insert, corresponding to pKT25 and pUT18C carrying only the adenylate cyclase domains without fusion). Bars represent averages ± SEM of at least three replicates. D, Mpa-mediated proteasomal PanB-Pup degradation is inhibited in presence of association-competent Bpa but not in presence of BpaΔHbYX. Concentrations: Mpa (0.2 µM), 20S proteasome (0.1 µM), Bpa or BpaΔHbYX (14 µM protomer).</p

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

<div><p>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 <i>Mycobacterium tuberculosis</i> (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 <i>Mycobacterium tuberculosis</i> 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 <i>in vitro</i> 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.</p></div

Bpa is conserved in actinobacteria encoding the proteasomal core particle genes.

<p>A, Multiple sequence alignment of Bpa orthologs (Rv3780 in Mtb) from different actinobacteria. The predominant residues in positions with an identity score above 0.5 are shaded in blue where increasing similarity is indicated by a gradient from light to dark blue. The completely conserved penultimate tyrosine of the HbYX motif is colored in red. B, Occurrence and location of the Pup-proteasome gene locus and the bpa gene. Each line represents the location of all open reading frames (orfs) of a bacterium in relation to the Pup proteasome gene locus. The position and orientation (if a corresponding homolog exists) of the proteasomal genes <i>prcB</i> and <i>prcA</i> are indicated by turquoise and <i>bpa</i> by a red arrow, respectively. The pupylation genes are given in shades of grey. Organisms are abbreviated as follows: <i>M. tuberculosis</i> (Mtb), <i>M. leprae</i> (Mlep), <i>M. smegmatis</i> (Msm), <i>N. farcinica</i> (Nfar), <i>R. erythropolis</i> (Re<i>), S. coelicolor</i> (Scoe), <i>T. fusca</i> (Tfus), <i>K. radiotolerans</i> (Krad), <i>Janibacter sp.</i> (Jani), <i>A. cellulolyticus</i> (Acel), <i>S. erythraea</i> (Ser), <i>Nocardioides sp.</i> (Noc), <i>S. tropica</i> (Strop), <i>Frankia sp.</i> (Frankia), <i>A. aurescens</i> (Aaur), <i>R. salmoninarum</i> (Rsal), <i>A. ferrooxidans</i> (Afer), <i>Brevibacterium sp.</i> (Brevi), <i>B. mcbrellneri</i> (Bmcb), <i>F. alni</i> (Fal), <i>L. ferrooxidans</i> (Lfer), <i>P. acnes</i> (Pac), <i>A. odontolyticus</i> (Aodo), <i>B. adolescentis</i> (Bado), <i>C. diphtheria</i> (Cdip), <i>C. glutamicum</i> (Cglu), <i>K. rhizophila</i> (Krhi), <i>M. luteus</i> (Mlut).</p

Bpa can be retained by immobilized Δ7PrcAB or half-proteasomes.

<p>A, Elution fractions of the pull-downs of Bpa or BpaΔHbYX with different Strep-immobilized proteasome particles. Strep-tagged proteasome particles were eluted from Strep-Tactin Sepharose with 2.5 mM desthiobiotin and the elution fractions were visualized on Coomassie stained SDS-PAGE. Bpa binds to Δ7PrcAB, while BpaΔHbYX does not. PrcAB interaction with Bpa is not detectable, while half-proteasomes (proPrcAB) retain a small amount of Bpa. B, Maturation of half-proteasomes at 37°C for 24 hours followed by SDS-PAGE. No difference in processing speed can be observed in the reactions supplemented with Bpa compared to PrcAB alone within this time frame.</p