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Updated: Jun 10, 2026
Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis
Published on: September 26, 2025
Proteome-wide identification of mycobacterial pupylation targets
Christian Poulsen1, Yusuf Akhter, Amy Hye-Won Jeon
1EMBL-Hamburg Unit, European Molecular Biology Laboratory, Hamburg, Germany.
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.
Insights
Mycobacteria modify proteins using prokaryotic ubiquitin-like protein (PUP) conjugation. This study identified 243 pupylation targets in Mycobacterium smegmatis, revealing dynamic substrate targeting and functional gene clustering.
Area of Science:
- Microbiology
- Proteomics
- Molecular Biology
Background:
- Mycobacteria utilize a unique protein modification system involving prokaryotic ubiquitin-like protein (PUP) conjugation.
- Pupylation is a post-translational modification analogous to ubiquitination in other organisms.
Purpose of the Study:
- To conduct a comprehensive, proteome-wide analysis of endogenous pupylation targets in Mycobacterium smegmatis.
- To identify specific lysine residues targeted for pupylation and explore the functional roles of pupylated proteins.
Main Methods:
- Affinity capture techniques were employed to isolate and enrich pupylated proteins from Mycobacterium smegmatis.
- Two complementary proteomics approaches, including collision-induced dissociation (CID) mass spectrometry, were used for identification and site determination.
- Comparative analysis of pupylomes under different culture conditions was performed.
Main Results:
- A total of 243 candidate pupylation targets were identified.
- Direct evidence for 48 lysine-mediated pupylation sites on 41 proteins was obtained.
- Pupylation targets are significantly represented in intermediary metabolism, respiration, lipid metabolism, and virulence pathways.
- Functional linkages were suggested by the location of approximately one-third of target genes within seven gene clusters.
- Pupylation substrate targeting was observed to be dynamic under varying cell culture conditions.
Conclusions:
- This study provides the first large-scale characterization of the mycobacterial pupylome.
- Pupylation plays a significant role in diverse cellular processes in Mycobacterium species, including metabolism and virulence.
- The dynamic nature of pupylation suggests its involvement in adaptive responses to environmental changes.
