Premium
A Proteolytic Site‐Directed Affinity Label to Inhibit the Human ATP‐Dependent Protease Caseinolytic Complex XP
Author(s) -
Sha Zhou,
Chilakala Sujatha,
Crabill George,
Cheng Iteen,
Xu Yan,
Fishovitz Jennifer,
Lee Irene
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000031
Subject(s) - peptide , biochemistry , chemistry , proteases , protease , protein subunit , proteasome , hela , affinity label , cleavage (geology) , enzyme , stereochemistry , biology , cell , paleontology , fracture (geology) , gene
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.