A Proteolytic Site‐Directed Affinity Label to Inhibit the Human ATP‐Dependent Protease Caseinolytic Complex XP. (2nd April 2020)

• Record Type: Journal Article
• Authors: Sha, Zhou; Chilakala, Sujatha; Crabill, George; Cheng, Iteen; Xu, Yan; Fishovitz, Jennifer; Lee, Irene
• Is Part Of: Chembiochem. Volume 21:Number 14(2020)
• Page Start: 2049
• Page End: 2059
• Publication Date: 2020-04-02
• Subjects: Enzymes -- peptides -- AAA+ protease -- inhibitors -- chloromethyl ketone; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202000031
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Human caseinolytic protease component X and P (hClpXP) is a heterooligomeric ATP‐dependent protease. The hClpX subunit catalyzes ATP hydrolysis whereas the hClpP subunit catalyzes peptide bond cleavage. In this study, we generated a peptidyl chloromethyl ketone (dansyl‐FAPAL‐CMK) that inhibited the hClpP subunit through alkylation of the catalytic His122, which was detected by LC‐MS. This inhibitor is composed of a peptide sequence derived from a hydrolyzed peptide product of a substrate cleaved by hClpXP. Binding of FAPAL positions the electrophilic chloromethyl ketone moiety near His122 where alkylation occurs. Dansyl FAPAL‐CMK exhibits selectivity for hClpXP over other ATP‐dependent proteases such as hLon and the 26S proteasome and abolishes hClpXP activity in HeLa cell lysate. Using the fluorogenic peptide substrate FR‐Cleptide as reporter, we detected biphasic inhibition time courses; this supports a slow‐binding, time‐dependent, covalent inhibition mechanism that is often found in active‐site directed affinity labels. Because this inhibitor reacts only with hClpXP but not hLon or the proteasome, it has the potential to serve as a chemical tool to help validate endogenous protein substrates of hClpXP in cell lysate, thereby benefiting investigation of the physiological functions of hClpXP in different cell types or tissue samples. Abstract : Enzyme‐specific inhibitors of the ATP‐dependent Lon and ClpXP proteases are necessary to distinguish the activity of these two similar enzymes in a protein mixture. We present here the design, synthesis, and characterization of a ClpXP‐specific inhibitor that allows mitochondrial Lon to be monitored in HeLa cell lysate.