Identification and characterization of novel Clp protease interaction partners in Mycolicibacterium smegmatis
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
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 dissertation, we characterized the essential ClpC1P1P2 protease, specifically a novel protease substrate and regulator, whilst also providing evidence of posttranslational modification previously shown to be a potential degradation signal. ☐ In Chapter 2, we identify cellular proteins that interact with the ClpC1 unfoldase in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate for Mtb, as captured by co-immunoprecipitation and identified by mass spectrometry-based proteomics (LC-MS/MS). Notably, this work revealed a novel proteolytic substrate, 5-oxoprolinase (Msmei_3879), which is recognized and degraded by ClpC1P1P2 with the help of its N-terminal sequence. A construct based on this substrate (5-oxoprolinase fused to GFP at its C-terminus) can be employed as a valid reporter in high throughput library screening for novel ClpC1P1P2-targeting antibiotics. Hence, this study lays the groundwork for future efforts to target Clp protease in mycobacteria. ☐ There is currently only one known adaptor or regulator of ClpC1, which is the adaptor ClpS. There is a crucial need to identify and characterize more regulators to understand the intricacies of modulation of the ClpC1P1P2 protease. Another protein identified to interact with Msm ClpC1 in Chapter 2 was N-methylhydantoinase (Msmei_3878/Msmeg_3973). In Chapter 3, we characterized N-methylhydantoinase as a novel negative regulator of the unfoldase ClpC1. In fact, our data showed that the presence of Msm N-methylhydantoinase affects the ATPase and unfoldase activities of ClpC1. It also impacts its ability to stimulate the peptidase activity of ClpP1P2, thus inhibiting the proteolytic activity of the ClpC1P1P2 protease. This lays the groundwork for future efforts to expound on the roles potentially played by negative Clp protease regulators in mycobacteria. Future work will be required to delineate the exact mechanism of regulation, and how this activity contributes to the physiology of mycobacteria and other actinobacterial species. ☐ An expanded understanding of the physiology of the ClpC1P1P2 protease – especially in terms of specific substrate recognition – would bolster efforts to develop novel antimicrobial compounds. Recent studies in Bacillus subtilis revealed that the orthologous ClpCP protease recognizes proteolytic substrates through post-translational arginine phosphorylation (pArg). Several lines of evidence suggest that ClpC1P1P2 similarly recognizes pArg-bearing proteins. However, the existence of pArg modifications in mycobacteria has remained in question. In Chapter 4, we confirm the existence of post-translational phosphoarginine modifications in Msm. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on specific targets within the Msm proteome. Our findings provide new evidence supporting the existence of phosphoarginine modifications in mycobacteria and other actinobacterial species. Future work will be required to decipher how arginine phosphorylation is regulated (including identifying the arginine kinase and phosphatase), and its role in mycobacterial physiology.
Description
Keywords
Biochemical assay, Interactome, Mass spectrometry, Mycolicibacterium smegmatis, Proteomics, Tuberculosis