Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone.
Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome.
Dick
L R
LR
ProScript, Inc., Cambridge, Massachusetts 02139, USA.
Cruikshank
A A
AA
Grenier
L
L
Melandri
F D
FD
Nunes
S L
SL
Stein
R L
RL
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007783:Lactones / Q000737:chemistry / Q000494:pharmacology*; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D013302:Streptomyces
D015853:Cysteine Proteinase Inhibitors; D007783:Lactones; D009097:Multienzyme Complexes; C067713:lactacystin; C098869:clasto-lactacystin beta-lactone; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine