Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity

Abstract

SummaryGenes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 ∞ C allowed formation of an a 7 b 7 b 7 a 7 , 750 kDa cylindrical stack of four rings in which all 14 b -subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete , Rhodococcus erythropolis , Mtb's b -chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg 2 + or Ca 2 + . Electron microscopy revealed what appeared to be obstructed a -rings in the Mtb 750 kDa particle. Deletion of the Nterminal octapeptide from Mtb's a -chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes , the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.

Role of the β1 Subunit in the Function and Stability of the 20S Proteasome in the Hyperthermophilic ArchaeonPyrococcus furiosus

“…K m values measured were all between 20 and 70 M, which are similar to values for CPs from Rhodococcus and Mycobacterium species (25). V max values obtained were also consistent with previously reported values for other prokaryotic CPs (25,42) and consistent with the native P. furiosus CP (2). Based on the information in Fig.…”

“…Table 2 shows kinetic parameters determined at 95°C for recombinant versions of the P. furiosus CP assembled at 90°C and 105°C on three different peptide substrates; note that a standard Michaelis-Menten model was used for LLVY and AAF, while substrate inhibition was included in the model for VKM (39). K m values measured were all between 20 and 70 M, which are similar to values for CPs from Rhodococcus and Mycobacterium species (25). V max values obtained were also consistent with previously reported values for other prokaryotic CPs (25,42) and consistent with the native P. furiosus CP (2).…”

“…CP composition may, in some cases, also be variable in prokaryotes, although the possibilities are much more limited. In bacteria, 20S proteasomes have been identified in Rhodococcus erythropolis (8) as well as in Mycobacterium tuberculosis (25), both of which appear to be based on single ␣ and ␤ proteins. In Haloferax volcanii, the proteasome CP is found in at least two different isoforms, based on combinations of one ␤ protein and two ␣ proteins (7,21).…”

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

“…Recently it has become clear that bacteria encoding a proteasome [9][10][11], like for example Mycobacterium tuberculosis (Mtb), use a similar tagging system termed pupylation [12][13][14]. In this process, the prokaryotic ubiquitin-like protein (Pup) is conjugated to substrate-lysines via its deamidated C-terminal GGQ-motif [12,14].…”

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

“…The Mtb 20S proteasome core consists of 14 α subunits encoded by prcA and 14 β subunits encoded by prcB, which assemble in an α 7 β 7 β 7 α 7 complex 12,13 . Bacterial proteasomes, which are unique to actinomycetes 14,15 , are not essential for in vitro growth of Streptomyces coelicolor, S. lividans, or M. smegmatis [16][17][18] .…”