Biology subjects

Schmitz, K. R.

Publications and source records attributed to Schmitz, K. R..

3 recordsLinked to original sources

bioRxiv2023

Bioinformatic identification of ClpI, a distinct class of Clp unfoldases in Actinomycetota

All clades of bacteria possess Hsp100/Clp family unfoldase enzymes that contribute to aspects of protein quality control. In Actinomycetota, these include ClpB, which functions as an independent chaperone and disaggregase, and ClpC, which cooperates with the ClpP1P2 peptidase to carry out regulated proteolysis of client proteins. We initially sought to algorithmically catalog Clp unfoldase orthologs from Actinomycetota into ClpB and ClpC categories. In the process, we uncovered a phylogenetically distinct third group of double-ringed Clp enzymes, which we term ClpI. ClpI enzymes are architecturally similar to ClpB and ClpC, with intact ATPase modules and motifs associated with substrate unfolding and translation. While ClpI possess an M-domain similar in length to that of ClpC, their N-terminal domain is more variable than the strongly conserved N-terminal domain of ClpC. Surprisingly, we identified separate sets of ClpI sequences that possess or lack the LGF-motifs required for stable assembly with ClpP1P2. In species where they occur, we suggest that ClpI enzymes provides additional pathways and points of regulatory control over protein quality control programs, supplementing the conserved roles of ClpB and ClpC.

bioinformatics↗

bioRxiv2022

Interactome analysis identifies MSMEI_3879 as a substrate of Mycolicibacterium smegmatis ClpC1

The prevalence of drug resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new mycobacterial drug targets. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize ClpC1-targeting compounds are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a co-immunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a relative of M. tuberculosis. We identify a diverse panel of interaction partners, many of which make co-immunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis identifies MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires an exposed N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics, to help address the challenge of M. tuberculosis drug resistance.

biochemistry↗

bioRxiv2021

Identification of diverse targets of arginine phosphorylation in Mycolicibacterium smegmatis by shotgun proteomics

Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate of Mtb. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on several functionally diverse targets within the Msm proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species.

microbiology↗