Kristin E. Burns

Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein.

The striking identification of an apparent proteasome core in Mycobacteria and allied actinomycetes suggested that additional elements of this otherwise strictly eukaryotic system for regulated protein degradation might be conserved. The genes encoding this prokaryotic proteasome are clustered in an operon with a short open reading frame that encodes a small protein of 64 amino acids resembling ubiquitin with a carboxyl-terminal di-glycine-glutamine motif (herein called Pup for prokaryotic ubiquitin-like protein). Expression of a polyhistidine-tagged Pup followed by pulldown revealed that a broad spectrum of proteins were post-translationally modified by Pup. Two-dimensional gel electrophoresis allowed us to conclusively identify two targets of this modification as myoinositol-1-phosphate synthase and superoxide dismutase. Deletion of the penultimate di-glycine motif or the terminal glutamine completely abrogated modification of cellular proteins with Pup. Further mass spectral analysis demonstrated that Pup was attached to a lysine residue on its target protein via the carboxyl-terminal glutamine with deamidation of this residue. Finally, we showed that cell lysates of wild type (but not a proteasome mutant) efficiently degraded Pup-modified proteins. These data therefore establish that, despite differences in both sequence and target linkage, Pup plays an analogous role to ubiquitin in targeting proteins to the proteasome for degradation.

Characterization of Two Kinases Involved in Thiamine Pyrophosphate and Pyridoxal Phosphate Biosynthesis in Bacillus subtilis: 4-Amino-5-Hydroxymethyl-2-Methylpyrimidine Kinase and Pyridoxal Kinase

Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized. YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B. subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.

Reconstitution of the Mycobacterium tuberculosis pupylation pathway in Escherichia coli

Prokaryotic ubiquitin‐like protein (Pup) is a post‐translational modifier that attaches to more than 50 proteins in Mycobacteria. Proteasome accessory factor A (PafA) is responsible for Pup conjugation to substrates, but the manner in which proteins are selected for pupylation is unknown. To address this issue, we reconstituted the pupylation of model Mycobacterium proteasome substrates in Escherichia coli, which does not encode Pup or PafA. Surprisingly, Pup and PafA were sufficient to pupylate at least 51 E. coli proteins in addition to the mycobacterial proteins. These data suggest that pupylation signals are intrinsic to targeted proteins and might not require Mycobacterium‐specific cofactors for substrate recognition by PafA in vivo.

Pupylation versus ubiquitylation: tagging for proteasome-dependent degradation.

Prokaryotic ubiquitin‐like protein (Pup) is the first identified prokaryotic protein that is functionally analogous to ubiquitin. Despite using the proteasome as the end‐point for proteolysis, Pup differs from ubiquitin both biochemically and structurally. We will discuss these differences that have been highlighted by several recent studies. Finally, we will speculate on the possible interactions between the two analogous pathways in pathogen and host.

Crystal Structure of a Sulfur Carrier Protein Complex Found in the Cysteine Biosynthetic Pathway of Mycobacterium tuberculosis

The structure of the protein complex CysM−CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 Å resolution. CysM (Rv1336) is a PLP-containing β-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 Å resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM−CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF−ThiS and MoeB−MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.

Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

Background: Dop is critical for the full virulence of Mycobacterium tuberculosis; however, its mechanism is not understood. Results: Asp-95 was identified as a catalytically significant residue. Conclusion: This work suggests that Asp-95 functions either as a direct nucleophile forming a unique anhydride intermediate or is part of a catalytic center that includes polarized water as the nucleophile. Significance: Understanding the mechanism of Dop can help guide the design and selection of inhibitors. Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.

Synthesis and Evaluation of a Selective Fluorogenic Pup Derived Assay Reagent for Dop, a Potential Drug Target in Mycobacterium tuberculosis

A litter of pups: The synthesis and in vitro evaluation of new Pup-based fluorogenic substrates for Dop, the mycobacterial depupylase, are described. A full-length Pup-amidomethylcoumarin conjugate as well as an amino-terminus-truncated analogue exhibited high sensitivity and specificity towards hydrolysis by Dop. The substrates developed here might find application as high-throughput screening assay reagents for the identification of Dop inhibitors.