Dataset Information

Interactome analysis by shotgun proteomics identifies a novel substrate for Mycolicibacterium smegmatis unfoldase ClpC1

ABSTRACT: Tuberculosis remains a leading cause of worldwide infectious mortality and one of the top ten leading causes of death overall. While advances in public health have contributed to a reduction in tuberculosis cases, the prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) (MDR-TB) infections has created an urgent need to exploit novel drug targets. One such target is the ClpC1P1P2 protease, which degrades folded cytosolic proteins through the cooperation of the ATP-dependent unfoldase ClpC1 and the ClpP1P2 peptidase. Both protease components are strictly essential for Mtb viability and are validated therapeutic targets. However, efforts to develop anti-Mtb compounds are constrained by a limited understanding of Clp protease function and essentiality. Thus, it is crucial to identify physiological substrates and pathways regulated by this protease. In this study, we identify cellular proteins that interact with the ClpC1 unfoldase in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate for Mtb. Using a FLAG-tagged ClpC1 variant with mutations within the Walker B motifs, candidate ClpC1 interaction partners were captured by co-immunoprecipitation and identified by mass spectrometry-based proteomics (LC-MS/MS). Notably, our work reveals a novel proteolytic substrate, 5-oxoprolinase, which is recognized by ClpC1P1P2 via an N-terminal degradation sequence.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mycobacterium Smegmatis (strain Atcc 700084 / Mc(2)155)

SUBMITTER: Emmanuel Ogbonna

LAB HEAD: Karl Robert Schmitz

PROVIDER: PXD030385 | Pride | 2022-12-12

REPOSITORIES: Pride

ACCESS DATA

Publications

Interactome Analysis Identifies MSMEI_3879 as a Substrate of Mycolicibacterium smegmatis ClpC1.

Ogbonna Emmanuel C EC Anderson Henry R HR Beardslee Patrick C PC Bheemreddy Priyanka P Schmitz Karl R KR

Microbiology spectrum 20230621 4

The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of C ...[more]

PMID: 37341639

Similar Datasets

Project description:Background: Conflicting results have been reported about the role of the two-component sensor and transcriptional regulator DosS/DosR, controlling the expression of the dormancy DosR regulon, for in vivo virulence of M. tuberculosis. Here, we have used a new approach to further analyze the relevance of the dosRS system, by driving DosR (Rv3133c) expression under the control of a constitutive promoter (phsp60). Methodology/Principal Findings: M. tuberculosis H37Rv constitutively expressing the transcriptional regulator DosR (Mtb::DosR) was compared to wild type M. tuberculosis (Mtb+/+) for in vitro growth kinetics and expression of the target genes of the DosR dormancy regulon, for in vivo virulence and for immunogenicity in mice. Under aerobic conditions, hsp60-driven DosR induced the expression of 28 out of 39 tested DosR regulon genes. In vitro growth characteristics were comparable for both strains, but Mtb::DosR showed an attenuated in vivo phenotype in immunocompetent mice, as indicated by reduced bacterial replication, reduced pulmonary immunopathology, reduced cachexia and significantly prolonged survival time as compared Mtb+/+. In immunodeficient SCID mice, Mtb::DosR was fully virulent. RT-qPCR analysis revealed a strong and comparable pulmonary TNF-?? and IL-23 expression following intratracheal infection, whereas IL-12 and IL-17 expression was slightly higher with wild type Mtb+/+. Finally, mice persistently infected with Mtb::DosR for 8 months showed five to tenfold higher lung IFN-?? responses against ten of the 48 DosR regulon encoded antigens (Rv1733c, Rv1734, Rv1738, Rv1996, Rv1997, Rv2029c, Rv2623, Rv2627c, Rv2628 and Rv3127) than mice actively infected with Mtb+/+. In spleen however, DosR regulon encoded antigen specific IFN-?? responses were similar in both groups. Conclusions/Significance. Collectively, these results suggest that increased DosR regulon encoded antigen specific pulmonary T cell responses are responsible for the attenuated phenotype of Mtb::DosR and that infection with Mtb::DosR could be used as a new animal model for studying key aspects of latent tuberculosis. Set of arrays that are part of repeated experiments

Dataset Information

Interactome analysis by shotgun proteomics identifies a novel substrate for Mycolicibacterium smegmatis unfoldase ClpC1

Publications

Interactome Analysis Identifies MSMEI_3879 as a Substrate of Mycolicibacterium smegmatis ClpC1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure