A Selective Fluorogenic Peptide Substrate for the Human Mitochondrial ATP‐Dependent Protease Complex ClpXP. (2nd April 2020)

• Record Type: Journal Article
• Authors: Sha, Zhou; Fishovitz, Jennifer; Wang, Susan; Chilakala, Sujatha; Xu, Yan; Lee, Irene
• Is Part Of: Chembiochem. Volume 21:Number 14(2020)
• Page Start: 2037
• Page End: 2048
• Publication Date: 2020-04-02
• Subjects: Enzymes -- hydrolases -- peptides -- AAA+ protease -- enzyme kinetics; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202000030
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: The goal of this work is to identify differences in the substrate determinants of two human mitochondrial matrix ATP‐dependent proteases, human ClpXP (hClpXP) and human Lon (hLon). This information allows the generation of protease‐specific peptide substrates that can be used as chemical biology tools to investigate the physiological functions of hClpXP. These enzymes play a role in protein quality control, but currently the physiological functions of human ClpXP are not well defined. In this study, the degradation profile of casein, an alanine positional scanning decapeptide library, and a specific peptide sequence found in an endogenous substrate of bacterial ClpXP by hClpXP as well as hLon were examined. Based on our findings, we generated a specific fluorogenic peptide substrate, FR‐Cleptide, for hClpXP with a k cat of 2.44±0.15 s −1 and K m =262±43 μM, respectively. The FR‐Cleptide substrate was successfully used to identify a leucine methyl ketone as a potent lead inhibitor, and to detect endogenous hClpXP activity in HeLa cell lysate. We propose that the fluorogenic peptide substrate is a valuable tool for quantitatively monitoring the activity of hClpXP in cell lysate, as well as mechanistic characterization of hClpXP. The peptide‐based chemical tools developed in this study will complement the substrates developed for human Lon in aiding the investigation of the physiological functions of the respective protease. Abstract : Lon and ClpXP are ATP‐dependent proteases that help maintain human mitochondrial integrity. The two have overlapping substrate specificity, making it difficult to distinguish their activities. In this study, a fluorogenic peptide substrate was designed and shown to be selective in monitoring ClpXP peptidase activity in isolated HeLa cell lysate.