Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

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.

RhodococcusProkaryotic Ubiquitin-Like Protein (Pup) Is Degraded by Deaminase of Pup (Dop)

“…Several proteins have been identified as pupylated target proteins in M. tuberculosis and M. smegmatis. 13,24,25) Consistent with previous reports, all pupylated sequences were detected as GGE-K linkages, and no Pup$Pup conjugation (poly-Pup) was detected. 13,20,21) We selected PPase as a model substrate and prepared pupylated PPase.…”

“…13,24,25) Consistent with previous reports, all pupylated sequences were detected as GGE-K linkages, and no Pup$Pup conjugation (poly-Pup) was detected. 13,20,21) We selected PPase as a model substrate and prepared pupylated PPase. Strep-Pup and His 6 -PPase were coexpressed in R. erythropolis DPA698 and the pupylated PPase (Strep-Pup$His 6 -PPase) was isolated by affinity chromatography.…”

“…Moreover, no all pupylated proteins lead to degradation. 13) Dop may have a crucial role in the degradation and functional regulation of pupylated proteins using other ATPdependent proteases.…”

Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E ( pafE ) Mutant

“…Three biological replicate cultures of the indicated M. tuberculosis strains were grown in 7H9-ADN to an OD 580 of ϳ0.7, and RNA was purified and analyzed as described previously (9,34). Briefly, M. tuberculosis cultures were mixed at a 1:1 (vol/vol) ratio with GTC buffer (5 M guanidine thiocyanate, 25 mM trisodium citrate, 0.5% Sarkosyl, 0.8% ␤-mercaptoethanol) and , and 400 l of the upper layer was removed and added to 500 l of ethanol.…”

Proteomic progress in studying tuberculosis from 2010 to 2011

“…Pupylation is one of important post-translations which regulates protein's degradation. Usually, pupylated proteins were affinity purified and identified by mass spectrometry [15][16][17]. Through expression of His7 tagged PUP and exploitation of the characteristic +243 Da mass shift attributed to tryp- sinized PUPylated peptides, a global pull-down of protein targets for PUPylation in Mycobacterium smegmatis (Mycobacterium tuberculosis (Mtb)'s avirulent strain) revealed 103 candidate PUPylation targets and 52 confirmed targets [17].…”