Proteome‐wide identification of mycobacterial pupylation targets

Abstract

Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of a small protein, referred to as prokaryotic ubiquitin-like protein (PUP). In this study, we report a proteome-wide analysis of endogenous pupylation targets in the model organism Mycobacterium smegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by two complementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of 48 lysine-mediated pupylation acceptor sites was obtained by collision-induced dissociation spectra. For the majority of these pupylation targets (38 of 41), orthologous genes are found in the M. tuberculosis genome. Interestingly, approximately half of these proteins are involved in intermediary metabolism and respiration pathways. A considerable fraction of the remaining targets are involved in lipid metabolism, information pathways, and virulence, detoxification and adaptation. Approximately one-third of the genes encoding these targets are located in seven gene clusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of the pupylome under different cell culture conditions indicates that substrate targeting for pupylation is rather dynamic.

Genetic and Proteomic Analyses of Pupylation in Streptomyces coelicolor

“…With respect to subcellular localization, we estimate that 8 of the 110 proteins are likely to be localized to the cell wall. Collectively, these statistics are largely consistent with those reported for mycobacteria (16)(17)(18)(19).…”

“…In analogy to published approaches used to characterize the pupylomes of M. smegmatis and M. tuberculosis (17)(18)(19)22), we engineered a gene encoding a variant of S. coelicolor Pup with an N-terminal hexahistidine tag that when expressed in S. coelicolor strains of interest would enable pupylated proteins to be captured from lysates via Ni affinity chromatography for easy identification via proteomic methods. Further, we designed our experiments to limit artifacts that could complicate the interpretation of proteomic data.…”

“…The latter observations have been invoked to explain the capacity of M. tuberculosis to evade the innate immune response and persist in the host (13,14). The genetic analyses of the Pup-proteasome system in mycobacteria have been complemented by biochemical analyses wherein pupylated proteins (i.e., the pupylome) have been identified in M. tuberculosis and Mycobacterium smegmatis (16)(17)(18)(19). In these mycobacterial species, as many as 55 pupylated proteins have been identified in cell lysates.…”

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.…”

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].…”