Abstract
Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation.
Highlights
Most individuals who are infected with Mycobacterium tuberculosis (Mtb) do not develop tuberculosis
We used a tandem affinity purification (TAP) approach to isolate proteins covalently associated with Prokaryotic ubiquitin-like protein (Pup) in Mtb under routine culture conditions
Two proteins had been identified as pupylation targets in the non-pathogenic saprophyte M. smegmatis (Ino1, SodA); the same Lys in Ino1 was modified in both mycobacterial species [7] (Table 1)
Summary
Most individuals who are infected with Mtb do not develop tuberculosis. Numerous aspects of the immune system are responsible for slowing the growth of Mtb, the antimicrobial molecule nitric oxide (NO) appears to be essential for this process (reviewed in [2]). In a previous effort to identify new targets for tuberculosis therapy, Mtb mutants were screened for hyper-susceptibility to NO [3]. This screen identified two previously uncharacterized proteins, Mycobacterium proteasomal ATPase (Mpa) and proteasome accessory factor A (PafA). Proteasome protease activity encoded by prcBA appears to be essential for optimal in vitro growth of Mtb [4,5,6] but not the non-pathogenic relative M. smegmatis [7,8]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
