A Proteolytic Site-Directed Affinity Label to Inhibit the Human ATP-Dependent Protease Caseinolytic Complex XP.
AAA+ protease
Enzymes
chloromethyl ketone
inhibitors
peptides
Journal
Chembiochem : a European journal of chemical biology
ISSN: 1439-7633
Titre abrégé: Chembiochem
Pays: Germany
ID NLM: 100937360
Informations de publication
Date de publication:
16 07 2020
16 07 2020
Historique:
received:
20
01
2020
revised:
01
03
2020
pubmed:
18
3
2020
medline:
23
6
2021
entrez:
18
3
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32180302
doi: 10.1002/cbic.202000031
doi:
Substances chimiques
Protease Inhibitors
0
Adenosine Triphosphate
8L70Q75FXE
ClpP protein, human
EC 3.4.21.92
Endopeptidase Clp
EC 3.4.21.92
CLPX protein, human
EC 3.6.1.3
Types de publication
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
2049-2059Informations de copyright
© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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