A Proteolytic Site‐Directed Affinity Label to Inhibit the Human ATP‐Dependent Protease Caseinolytic Complex XP
In: ChemBioChem, Jg. 21 (2020-04-02), S. 2049-2059
Online
unknown
Zugriff:
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.
Titel: |
A Proteolytic Site‐Directed Affinity Label to Inhibit the Human ATP‐Dependent Protease Caseinolytic Complex XP
|
---|---|
Autor/in / Beteiligte Person: | Crabill, George ; Chilakala, Sujatha ; Lee, Irene ; Fishovitz, Jennifer ; Cheng, Iteen ; Sha, Zhou ; Xu, Yan |
Link: | |
Zeitschrift: | ChemBioChem, Jg. 21 (2020-04-02), S. 2049-2059 |
Veröffentlichung: | Wiley, 2020 |
Medientyp: | unknown |
ISSN: | 1439-7633 (print) ; 1439-4227 (print) |
DOI: | 10.1002/cbic.202000031 |
Schlagwort: |
|
Sonstiges: |
|