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• W4295698652 abstract "Mycobacteria use a proteasome system that is similar to a eukaryotic proteasome but do not use ubiquitin to target proteins for degradation. Instead, mycobacteria encode a prokaryotic ubiquitin-like protein (Pup) that posttranslationally modifies proteins to mark them for proteolysis. Pupylation occurs on lysines of targeted proteins and is catalyzed by the ligase PafA. Like ubiquitylation, pupylation can be reversed by the depupylase Dop, which shares high structural similarity with PafA. Unique to Dop near its active site is a disordered loop of approximately 40 amino acids that is highly conserved among diverse dop-containing bacterial genera. To understand the function of this domain, we deleted discrete sequences from the Dop loop and assessed pupylation in mutant strains of Mycobacterium tuberculosis. We determined that various Dop loop mutations resulted in altered pupylome profiles, in particular when mutant dop alleles were overexpressed. Taken together, our data suggest these conserved amino acids play a role in substrate selectivity for Dop. Mycobacteria use a proteasome system that is similar to a eukaryotic proteasome but do not use ubiquitin to target proteins for degradation. Instead, mycobacteria encode a prokaryotic ubiquitin-like protein (Pup) that posttranslationally modifies proteins to mark them for proteolysis. Pupylation occurs on lysines of targeted proteins and is catalyzed by the ligase PafA. Like ubiquitylation, pupylation can be reversed by the depupylase Dop, which shares high structural similarity with PafA. Unique to Dop near its active site is a disordered loop of approximately 40 amino acids that is highly conserved among diverse dop-containing bacterial genera. To understand the function of this domain, we deleted discrete sequences from the Dop loop and assessed pupylation in mutant strains of Mycobacterium tuberculosis. We determined that various Dop loop mutations resulted in altered pupylome profiles, in particular when mutant dop alleles were overexpressed. Taken together, our data suggest these conserved amino acids play a role in substrate selectivity for Dop. Mycobacterium tuberculosis is a human exclusive pathogen that is transmitted by aerosols and causes the disease tuberculosis (TB). Although TB can be effectively treated with several antibiotics, treatment is prolonged, which often results in poor compliance and the emergence of drug-resistant strains. In an effort to find new targets for TB treatment, a screen for mutants sensitive to the host effector nitric oxide (NO) identified mutations in components of the bacterial proteasome system (1Darwin K.H. Ehrt S. Gutierrez-Ramos J.C. Weich N. Nathan C.F. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide.Science. 2003; 302: 1963-1966Crossref PubMed Scopus (429) Google Scholar). In eukaryotes, proteins targeted for proteasomal degradation are posttranslationally modified by the small protein ubiquitin (reviewed in (2Komander D. Rape M. The ubiquitin code.Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (2386) Google Scholar)), whereas bacteria have a different modification called Pup. In M. tuberculosis, Pup is translated as a 64 amino acid protein ending in glutamine (Gln) that must be deamidated to glutamate (Glu) by deamidase of Pup (Dop) prior to attachment by the only known Pup ligase, proteasome accessory factor A (PafA), to substrate lysines (3Pearce M.J. Mintseris J. Ferreyra J. Gygi S.P. Darwin K.H. Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis.Science. 2008; 322: 1104-1107Crossref PubMed Scopus (318) Google Scholar, 4Guth E. Thommen M. Weber-Ban E. Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate.J. Biol. Chem. 2011; 286: 4412-4419Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 5Striebel F. Imkamp F. Sutter M. Steiner M. Mamedov A. Weber-Ban E. Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.Nat. Struct. Mol. Biol. 2009; 16: 647-651Crossref PubMed Scopus (166) Google Scholar). The pupylation status of any protein is likely dynamic given that Dop can also remove Pup from substrates (depupylation), rescuing them from degradation (6Burns K.E. Cerda-Maira F.A. Wang T. Li H. Bishai W.R. Darwin K.H. Depupylation of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates.Mol. Cell. 2010; 39: 821-827Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Imkamp F. Striebel F. Sutter M. Ozcelik D. Zimmermann N. Sander P. et al.Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway.EMBO Rep. 2010; 11: 791-797Crossref PubMed Scopus (84) Google Scholar), and PafA can potentially move Pup from one substrate to another (8Zhang S. Burns-Huang K.E. Janssen G.V. Li H. Ovaa H. Hedstrom L. et al.Mycobacterium tuberculosis proteasome accessory factor A (PafA) can transfer prokaryotic ubiquitin-like protein (pup) between substrates.MBio. 2017; 8e00122-17Crossref Scopus (18) Google Scholar). Given that over 60 proteins are targets of pupylation that comprise the “pupylome” (9Festa R.A. McAllister F. Pearce M.J. Mintseris J. Burns K.E. Gygi S.P. et al.Prokaryotic ubiquitin-like protein (Pup) proteome of Mycobacterium tuberculosis [corrected].PLoS One. 2010; 5e8589Crossref PubMed Google Scholar, 10Watrous J. Burns K. Liu W.T. Patel A. Hook V. Bafna V. et al.Expansion of the mycobacterial PUPylome.Mol. Biosyst. 2010; 6: 376-385Crossref PubMed Scopus (77) Google Scholar, 11Schubert O.T. Mouritsen J. Ludwig C. Rost H.L. Rosenberger G. Arthur P.K. et al.The Mtb proteome library: a resource of assays to quantify the complete proteome of Mycobacterium tuberculosis.Cell Host Microbe. 2013; 13: 602-612Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), it is perhaps unsurprising that components of the Pup-proteasome system (PPS) are essential for the robust virulence of M. tuberculosis in animal models (1Darwin K.H. Ehrt S. Gutierrez-Ramos J.C. Weich N. Nathan C.F. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide.Science. 2003; 302: 1963-1966Crossref PubMed Scopus (429) Google Scholar, 12Darwin K.H. Lin G. Chen Z. Li H. Nathan C.F. Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue.Mol. Microbiol. 2005; 55: 561-571Crossref PubMed Scopus (112) Google Scholar, 13Cerda-Maira F.A. Pearce M.J. Fuortes M. Bishai W.R. Hubbard S.R. Darwin K.H. Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.Mol. Microbiol. 2010; 77: 1123-1135Crossref PubMed Scopus (84) Google Scholar, 14Gandotra S. Schnappinger D. Monteleone M. Hillen W. Ehrt S. In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice.Nat. Med. 2007; 13: 1515-1520Crossref PubMed Scopus (208) Google Scholar). In fact, the accumulation of a single proteasome substrate, Log, results in a buildup of aldehydes that synergize with NO to kill bacteria and attenuate growth in mice, demonstrating the essential robustness of the PPS for resistance to host defenses and potentially other stressors (15Samanovic M.I. Tu S. Novak O. Iyer L.M. McAllister F.E. Aravind L. et al.Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.Mol. Cell. 2015; 57: 984-994Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). A major gap in understanding the PPS is how proteins are selected for pupylation and depupylation. The expression of M. tuberculosis dop, pup, and pafA in Escherichia coli, which lacks a PPS, results in the pupylation of numerous proteins (16Cerda-Maira F.A. McAllister F. Bode N.J. Burns K.E. Gygi S.P. Darwin K.H. Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli.EMBO Rep. 2011; 12: 863-870Crossref PubMed Scopus (44) Google Scholar), suggesting that there is no mycobacteria-specific sequence motif that PafA must recognize to pupylate a protein. PafA and Dop are members of the glutamine synthetase superfamily and share numerous conserved residues in their active sites (13Cerda-Maira F.A. Pearce M.J. Fuortes M. Bishai W.R. Hubbard S.R. Darwin K.H. Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.Mol. Microbiol. 2010; 77: 1123-1135Crossref PubMed Scopus (84) Google Scholar, 17Ozcelik D. Barandun J. Schmitz N. Sutter M. Guth E. Damberger F.F. et al.Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.Nat. Commun. 2012; 3: 1014Crossref PubMed Scopus (57) Google Scholar, 18Iyer L.M. Burroughs A.M. Aravind L. Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination.Biol. Direct. 2008; 3: 45Crossref PubMed Scopus (90) Google Scholar). While PafA catalyzes a reaction similar to glutamine synthetases, Dop does not. Dop has an amidase activity that appears unique to it and its close homologs (17Ozcelik D. Barandun J. Schmitz N. Sutter M. Guth E. Damberger F.F. et al.Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.Nat. Commun. 2012; 3: 1014Crossref PubMed Scopus (57) Google Scholar, 19Burns K.E. McAllister F.E. Schwerdtfeger C. Mintseris J. Cerda-Maira F. Noens E.E. et al.Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase.J. Biol. Chem. 2012; 287: 37522-37529Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 20Cui H. Muller A.U. Leibundgut M. Tian J. Ban N. Weber-Ban E. Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation.Nat. Commun. 2021; 12: 6635Crossref PubMed Scopus (4) Google Scholar). Furthermore, Dop has a disordered loop sequence that is absent in PafA and is a highly conserved region among Dops from diverse actinobacterial species (17Ozcelik D. Barandun J. Schmitz N. Sutter M. Guth E. Damberger F.F. et al.Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.Nat. Commun. 2012; 3: 1014Crossref PubMed Scopus (57) Google Scholar). Deletion of the Dop loop does not diminish its activity nor does it convert Dop into a ligase. However, deletion of the loop and addition of an alpha helix from PafA confers ligase activity to Mycobacterium smegmatis Dop (21Hecht N. Monteil C.L. Perriere G. Vishkautzan M. Gur E. Exploring protein space: from hydrolase to ligase by substitution.Mol. Biol. Evol. 2021; 38: 761-776Crossref PubMed Scopus (6) Google Scholar). In a study by the Gur lab, in vitro analysis found that deletions in the M. smegmatis Dop loop result in enzymes that more rapidly depupylate model substrates. Steady state pupylomes in M. smegmatis expressing mutant dop are reduced compared to the pupylome from a strain expressing wild-type (wt) dop, suggesting these Dop loop mutant alleles also hyperdepupylate in vivo (22Hecht N. Becher M. Korman M. Vishkautzan M. Gur E. Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.FEBS J. 2020; 287: 4389-4400Crossref PubMed Scopus (7) Google Scholar). The authors of this work also showed that Dop binding to one substrate, Pup∼IdeR, is unaffected by the Dop loop deletion, concluding the Dop loop regulates catalysis and not substrate binding. In contrast, the Weber-Ban lab found that replacement of loop residues with different amino acids made Corynebacterium glutamicum Dop more slowly depupylate a model substrate. Moreover, the authors proposed that the Dop loop promotes the dephosphorylation of an active site nucleotide (ATP), releasing a phosphate needed for amidase activity (20Cui H. Muller A.U. Leibundgut M. Tian J. Ban N. Weber-Ban E. Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation.Nat. Commun. 2021; 12: 6635Crossref PubMed Scopus (4) Google Scholar). It is possible that differences in Dop loop function described in these studies were in part due to the use of Dop from different species (M. smegmatis Dop is 50% identical/75% similar to C. glutamicum Dop). We sought to understand how this highly conserved and unstructured region of Dop affects the proteome of M. tuberculosis. We complemented a dop transposon mutation with either integrative or overexpression plasmids encoding various dop alleles, including a large deletion encompassing most of the conserved amino acids or several smaller deletions within the loop, and assessed the pupylomes of these strains. Deletion of the Dop loop resulted in an overall reduced pupylome and the accumulation of several established proteasome substrates, supporting observations in M. smegmatis (22Hecht N. Becher M. Korman M. Vishkautzan M. Gur E. Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.FEBS J. 2020; 287: 4389-4400Crossref PubMed Scopus (7) Google Scholar). Smaller deletions of the Dop loop had variable effects, affecting only a handful of established PPS substrates. Most interestingly, the overexpression of mutant dop loop alleles resulted in dramatically different pupylomes. In particular, the expression of a specific dop loop deletion allele resulted in the accumulation of a single pupylated protein, suggesting the deleted amino acids are important for depupylating this substrate. Collectively, we propose that residues in the Dop loop help regulate depupylation, possibly by affecting access to substrates. In M. smegmatis, Dop lacking the loop depupylates faster than wt Dop in vitro and in vivo, suggesting that the Dop loop inhibits depupylation (22Hecht N. Becher M. Korman M. Vishkautzan M. Gur E. Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.FEBS J. 2020; 287: 4389-4400Crossref PubMed Scopus (7) Google Scholar). To test if deletion of the loop would have a similar effect in M. tuberculosis, we complemented an M. tuberculosis dop transposon mutation with an integrative plasmid encoding various deletions from the dop loop sequence; dop alleles were expressed from the native dop promoter (see Table 1). We deleted the coding sequence for the 24 most conserved amino acids (“Δloop”) as well as made shorter deletions within the loop (Fig. 1A) and assessed pupylome levels at steady state by immunoblotting (Fig. 1B). As previously reported in M. tuberculosis, complementation of this dop mutant with WT dop restores a robust pupylome (Fig. 1B, lanes 1 versus 2) (13Cerda-Maira F.A. Pearce M.J. Fuortes M. Bishai W.R. Hubbard S.R. Darwin K.H. Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.Mol. Microbiol. 2010; 77: 1123-1135Crossref PubMed Scopus (84) Google Scholar). The strain complemented with Δloop had a reduced pupylome (Fig. 1B, lane 3), similar to what was previously observed in M. smegmatis producing Dop lacking either 14 or 37 residues from its loop (22Hecht N. Becher M. Korman M. Vishkautzan M. Gur E. Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.FEBS J. 2020; 287: 4389-4400Crossref PubMed Scopus (7) Google Scholar).Table 1Bacterial strains, plasmids, and primers used in this workE. coli:Relevant genotype:Source or reference:DH5αF-, θ80ΔlacZM15 Δ(lacZYA-argF)U169 deoR recA1 endA1hsdR17 (rk-mk+) phoA supE44 λ- thi-1 gyrA96 relA1Gibco, BRL.M. tuberculosis:CDC1551wild typeW. Bishai collectionMHD58 (MT2172)CDC1551 dop::MycoMarT7; Kanr(13Cerda-Maira F.A. Pearce M.J. Fuortes M. Bishai W.R. Hubbard S.R. Darwin K.H. Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.Mol. Microbiol. 2010; 77: 1123-1135Crossref PubMed Scopus (84) Google Scholar)MHD375MHD58 pMV306; Hygr, Kanr(13Cerda-Maira F.A. Pearce M.J. Fuortes M. Bishai W.R. Hubbard S.R. Darwin K.H. Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.Mol. Microbiol. 2010; 77: 1123-1135Crossref PubMed Scopus (84) Google Scholar)MHD376MHD58 pMV-dop; Hygr, Kanr(13Cerda-Maira F.A. Pearce M.J. Fuortes M. Bishai W.R. Hubbard S.R. Darwin K.H. Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.Mol. Microbiol. 2010; 77: 1123-1135Crossref PubMed Scopus (84) Google Scholar)MHD1628MHD58 pMV-dopΔloop; Hygr, KanrThis work.MHD1631MHD58 pMV-dopΔWDYEV; Hygr, KanrThis work.MHD1632MHD58 pMV-dopΔESPLR; Hygr, KanrThis work.MHD1630MHD58 pMV-dopΔRGF; Hygr, KanrThis work.MHD1633MHD58 pMV-dopΔDLS; Hygr, KanrThis work.MHD1629MHD58 pMV-dopΔRSAGPP.; Hygr, KanrThis work.MHD671MHD58 pOLYG; HygrThis work.MHD1097MHD 58 pOLYG-dop; TAP-tagged; Hygr, KanrThis work.MHD1663MHD 58 pOLYG-dopΔloop; TAP-tagged; Hygr, KanrThis work.MHD1664MHD 58 pOLYG-dopΔWDYEV; TAP-tagged; Hygr, KanrThis work.MHD1681MHD 58 pOLYG-dopΔESPLR; TAP-tagged; Hygr, KanrThis work.MHD1682MHD 58 pOLYG-dopΔRGF; TAP-tagged; Hygr, KanrThis work.MHD1683MHD 58 pOLYG-dopΔDLS; TAP-tagged; Hygr, KanrThis work.MHD1684MHD 58 pOLYG-dopΔRSAGPP; TAP-tagged; Hygr, KanrThis work.ΔnuoANCDC1551 with a deletion of nuoA through nuoN(23Vilcheze C. Weinrick B. Leung L.W. Jacobs Jr., W.R. Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 1599-1604Crossref PubMed Scopus (38) Google Scholar)MHD1701ΔnuoAN pOLYG; HygrThis work.MHD1702ΔnuoAN pOLYG-dop; TAP-tagged; HygrThis work.MHD1703ΔnuoAN pOLYG-dopΔWDYEV; TAP-tagged; HygrThis work.PlasmidsDescriptionReferencepOLYGHygr; shuttle plasmid for gene overexpression in mycobacteria(36Garbe T.R. Barathi J. Barnini S. Zhang Y. Abou-Zeid C. Tang D. et al.Transformation of mycobacterial species using hygromycin resistance as selectable marker.Microbiology. 1994; 140: 133-138Crossref PubMed Scopus (118) Google Scholar)pMV306Hygr; mycobacterial plasmid that integrates at attB site on mycobacterial chromosomes(37Stover C.K. de la Cruz V.F. Fuerst T.R. Burlein J.E. Benson L.A. Bennett L.T. et al.New use of BCG for recombinant vaccines.Nature. 1991; 351: 456-460Crossref PubMed Scopus (1218) Google Scholar)Primers (sequences are 5′ to 3′):PrimersSequence (5′ to 3′)pOLYGforCATGACCAACTTCGATAACGpOLYGrevGCACGACAGGTTTCCCGACTGdopTAP_loop-WDYEV_RGCGCAGCGGCGATTCACGGGTGCGTTTGGCdopTAP_loop-WDYEV_FGCCAAACGCACCCGTGAATCGCCGCTGCGCdopTAP_loop-ESPLR_RGAAGCCCCGGGCGTCCACCTCGTAGTCCCAdopTAP_loop-ESPLR_FTGGGACTACGAGGTGGACGCCCGGGGCTTCdopTAP_loop-DA_RCAAATCGAAGCCCCGGCGCAGCGGCGATTCdopTAP_loop-DA_FGAATCGCCGCTGCGCCGGGGCTTCGATTTGdopTAP_loop-RGF_RCGAGCGACTCAAATCGGCGTCGCGCAGCGGCGATTCCACCdopTAP_loop-RGF_FGGTGGAATCGCCGCTGCGCGACGCCGATTTGAGTCGCTCGdopTAP_loop-DLS_RCGGCCCGGCCGAGCGGAAGCCCCGGGCGTCGCGCAGCGGCdopTAP_loop-DLS_FGCCGCTGCGCGACGCCCGGGGCTTCCGCTCGGCCGGGCCGdopTAP_loop-RSAGPP_RGGCGTCGACCACCGGACTCAAATCGAAGCCdopTAP_loop-RSAGPP_FGGCTTCGATTTGAGTCCGGTGGTCGACGCCDop_24cleandel_FAGCGTGCCAAACGCACCCGTCCGGTGGTCGACGCCGACGADop_24cleandel_RTCGTCGGCGTCGACCACCGGACGGGTGCGTTTGGCACGCTpMV306forCGGTTCCTGGCCTTTTGCTGGCCpMV306seqRCCTGTCGTTCACGGCTCTA Open table in a new tab The smaller amino acid deletions in the Dop loop also resulted in decreased pupylome abundance. Deletions nearer to the amino terminus had greater decreases in pupylome levels; the strain producing Dop lacking the amino acids tryptophan, aspartate, tyrosine, glutamate, and valine (“ΔWDYEV”) had the most similar pupylome to the Δloop strain (Fig. 1B, lanes 3 versus 4). This decrease in pupylome abundance was specifically due to the deleted residues and not just the shortening of the Dop loop, given that deletion of six residues (arginine, serine, alanine, glycine, proline, proline; ΔRSAGPP) at the carboxyl terminus of the loop resulted in a pupylome like the wt-complemented strain (Fig. 1B, lanes 1 versus 8). Deletion of the loop from M. smegmatis Dop does not affect deamidation activity (22Hecht N. Becher M. Korman M. Vishkautzan M. Gur E. Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.FEBS J. 2020; 287: 4389-4400Crossref PubMed Scopus (7) Google Scholar). Thus, it seemed unlikely that the decreases in pupylome levels seen in Figure 1 were due to the reduced conversion of newly translated PupGln to PupGlu. Instead, we hypothesized that the reduced pupylome levels were due to either slower or faster depupylation by the various Dop alleles. Hypodepupylation would result in more protein getting targeted to the proteasome, thus reducing the abundance of known proteasome substrates. In contrast, hyperdepupylation could rescue these substrates from degradation, thereby increasing the amount of a substrate relative to its abundance in wt bacteria. To determine which of these scenarios was more likely, we quantified and compared the proteome of the dop-null mutant to the proteomes of strains producing wt, Δloop, and ΔWDYEV Dop using tandem-mass tag mass spectrometry (TMT-MS). As expected, the dop-null mutant had the highest accumulation of several established proteasome substrates given that there is no pupylation in this strain (Tables 2, and S1). In the strains producing Δloop or ΔWDYEV alleles, several proteasome substrates accumulated but to a lesser degree than what were observed in the dop-null strain (Tables 2 and S1). Nonetheless, this result suggested these mutant loop Dop alleles hyperdepupylated several known proteasome substrates, rescuing them from proteasomal degradation.Table 2Mutations in the Dop loop resulted in increased levels of a subset of pupylated substrates. + indicates the protein was statistically significantly more abundant in the respective strain compared to a strain producing wt DopSubstrate:MW (kD):Dop nullΔloopΔWDYEVFabD31+++KasA43+++Icl47+++Log20++PanB29++Ino140++FusA77++Bcp17+LeuD22+MtrA25+NuoE27+Rv2859c32+Rv007336+FadA42+MurA44+PhoH247+PafA50+GlmU52+SahH54+Mpa67+RecA85+See Table S1 for full list of quantified proteins.Abbreviation: MW, molecular weight. Open table in a new tab See Table S1 for full list of quantified proteins. Abbreviation: MW, molecular weight. Defective protein degradation by proteasomes is associated with an increased susceptibility of M. tuberculosis to NO due to the failed degradation of the proteasome substrate Log (15Samanovic M.I. Tu S. Novak O. Iyer L.M. McAllister F.E. Aravind L. et al.Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.Mol. Cell. 2015; 57: 984-994Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Log did not accumulate in any of the tested loop mutants (Table S1), but we nonetheless tested whether or not the small loop deletions affected NO susceptibility. Consistent with our observation that Log did not accumulate in any of the tested loop mutant strains, none of these strains was hypersensitive to NO (Fig. 1C). The decrease in pupylome levels was unlikely due to changes in the abundance of the proteasome subunits (PrcA and PrcB) and mycobacterial proteasome activator Mpa because they were present at similar levels in the analyzed strains (Table S1). In contrast, there was less Pup in the mutant strains relative to the strain making wt Dop (Table S1). Pup is highly unstable when not conjugated to another protein in M. tuberculosis (13Cerda-Maira F.A. Pearce M.J. Fuortes M. Bishai W.R. Hubbard S.R. Darwin K.H. Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.Mol. Microbiol. 2010; 77: 1123-1135Crossref PubMed Scopus (84) Google Scholar). Thus, the reduced Pup levels in the Dop loop mutants, along with the accumulation of known proteasome substrates, is consistent with a model in which hyperdepupylation occurs in these bacteria. However, we could not rule out an alternative explanation in which Dop loop mutations negatively influenced the ability of Dop to depupylate certain substrates, an activity that could also affect the overall Pup pool. While the relative amounts of pupylated protein varied, the banding pattern of the pupylomes in our immunoblots did not appear different among the strains expressing the various loop alleles (Fig. 1B). However, an accumulated species of about 100 kD was apparent in the strain producing the ΔWDYEV allele (Fig. 1B, lane 4, arrowhead). Based on this observation, we hypothesized that specific residues in the Dop loop contributed to the depupylation of certain proteins. To begin to test this hypothesis, we overexpressed wt dop and mutant loop alleles in the dop-null M. tuberculosis strain, with the expectation that overexpression might magnify differences among the Dop alleles. We performed immunoblot analysis on total cell lysates of these strains and observed that several of the mutant dop allele-expressing strains had distinct pupylomes, with multiple pupylated proteins that were more prominent in several strains compared to each other or the WT dop-expressing strain (Fig. 2, lane 2 versus lanes 3–8). In most of the loop mutants, an approximately 100 kD species, herein called “protein X,” was present at greater levels than in the wt dop-expressing strain and most dramatically accumulated in the ΔWDYEV strain (Fig. 2, lane 4); it was likely that protein X was the same species seen in Figure 1B, lane 4. We hypothesized that the identity of protein X could give some insight into the significance of the WDYEV sequence in the Dop loop. To identify protein X, we performed immunoprecipitations using mAbs to Pup. After separating immunoprecipitated proteins by SDS-PAGE, we excised the region around 100 kD for MS analysis. After tryptic digestion and MS analysis, the top proteins with more than five peptide spectral matches included Pup and NuoG (Fig. 3A). NuoG is an 85 kD protein and part of the 14-subunit type 1 NADH dehydrogenase complex that is encoded by the nuoA operon (23Vilcheze C. Weinrick B. Leung L.W. Jacobs Jr., W.R. Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 1599-1604Crossref PubMed Scopus (38) Google Scholar). To further test if NuoG was indeed protein X, we tested for protein X accumulation in a ΔnuoAN mutant lacking the entire operon and overexpressing wt or ΔWDYEV dop alleles. Robust pupylomes were seen in both the parental and ΔnuoAN strains when transformed with empty vector (Fig. 3B, lanes 1 and 4), whereas the overexpression of wt dop resulted in dramatically reduced pupylomes (Fig. 3B, lanes 2 and 5), most likely due to hyperdepupylation. Nonetheless, the overproduction of the ΔWDYEV mutant resulted in the appearance of protein X in the parental strain as seen in Figure 2 but not in the ΔnuoAN strain. Because none of the other proteins encoded in the nuoA operon was identified by our proteomics analysis and all of the Nuo proteins except NuoG are 66 kD or smaller, we concluded that protein X is Pup∼NuoG. NuoG is a part of the peripheral arm of the type 1 NADH dehydrogenase complex (24Schimpf J. Oppermann S. Gerasimova T. Santos Seica A.F. Hellwig P. Grishkovskaya I. et al.Structure of the peripheral arm of a minimalistic respiratory complex I.Structure. 2022; 30: 80-94.e84Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) and has never been identified as a proteasome substrate in M. tuberculosis. Under routine culture conditions used in this work, we did not observe an accumulation of NuoG in the dop mutant, which we would expect if NuoG were a proteasome substrate (Table S1). In contrast, NuoE, which is also a part of this complex, is a confirmed pupylated substrate that accumulated in the dop-null mutant (Table 2) (9Festa R.A. McAllister F. Pearce M.J. Mintseris J. Burns K.E. Gygi S.P. et al.Prokaryotic ubiquitin-like protein (Pup) proteome of Mycobacterium tuberculosis [corrected].PLoS One. 2010; 5e8589Crossref PubMed Google Scholar). Although we do not know which lysine in NuoG is pupylated, it is possible that access to this residue is affected by its location within the NADH dehydrogenase complex (Fig. 4). In this study, we sought to understand the in vivo function of a highly conserved loop sequence in the M. tuberculosis amidase Dop. We showed that the effect of the loop deletions depended on which residues were deleted, and deletion of as few as three amino acids from the Dop loop had global effects on pupylome levels. The overexpression of a specific dop allele, ΔWDYEV, resulted in the dramatic accumulation of Pup∼NuoG, suggesting this substrate could not be efficiently depupylated by this mutant Dop. Thus, our data suggest highly conserved amino acids in the Dop loop regulate the ability of Dop to depupylate certain substrates in M. tuberculosis. Previous work by two other groups worked to understand the function of the Dop loop. Both studies concluded that the loop affected the rate of catalysis by Dop but in contradictory ways (20Cui H. Muller A.U. Leibundgut M. Tian J. Ban N. Weber-Ban E. Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation.Nat. Commun. 2021; 12: 6635Crossref PubMed Scopus (4) Google Scholar, 22Hecht N. Becher M. Korman M. Vishkautzan M. Gur E. Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.FEBS J. 2020; 287: 4389-4400Crossref PubMed Scopus (7) Google Scholar). In one study, deletion of the entire loop sequence or replacement of seven highly cons" @default.
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• W4295698652 title "A conserved loop sequence of the proteasome system depupylase Dop regulates substrate selectivity in Mycobacterium tuberculosis" @default.
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