MECHANISTIC STUDIES ON THE INACTIVATION OF THE PROTEASOME BY LACTACYSTIN IN CULTURED-CELLS

Citation
Lr. Dick et al., MECHANISTIC STUDIES ON THE INACTIVATION OF THE PROTEASOME BY LACTACYSTIN IN CULTURED-CELLS, The Journal of biological chemistry, 272(1), 1997, pp. 182-188
Citations number
26
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
272
Issue
1
Year of publication
1997
Pages
182 - 188
Database
ISI
SICI code
0021-9258(1997)272:1<182:MSOTIO>2.0.ZU;2-H
Abstract
The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential compon ent of the ubiquitin-proteasome pathway for intracellular protein degr adation, In vitro, lactacystin does not react with the proteasome; rat her, it undergoes a spontaneous conversion (lactonization) to the acti ve proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of g lutathione to form a thioester adduct that is both structurally and fu nctionally analogous to lactacystin, We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can u ndergo lactonization to yield back the active beta-lactonee, We have s tudied the kinetics of this reaction under appropriate in vitro condit ions as well as the kinetics of lactathione accumulation and proteasom e inhibition in cells treated with lactacystin or beta-lactone, The re sults indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentr ate the inhibitor inside cells, providing a reservoir for prolonged re lease of the active beta-lactone.