INHIBITION OF CASPASE-3 BY S-NITROSATION AND OXIDATION CAUSED BY NITRIC-OXIDE

Apoptotic signaling cascades converge in the activation of caspases (i nterleukin-1 beta converting enzyme like proteases). Treatment of the human promyelocytic leukaemia cell line U937 with actinomycin D result ed in the activation of caspase-3 also known as CPP32. Protease activi ty was measured in cytosolic extracts by fluorometric analysis of the time-dependent cleavage of acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC), a caspase-3 substrate. Caspase activity was inhibited by t hiol modifying agents such as N-ethylmaleimide or iodoacetamide and NO donors such as S-nitrosoglutathione (GSNO), BF4NO, and spermine-ETO. NO-mediated enzyme inhibition was fully reversible upon the addition o f DTT (dithiothreitol). NO. itself was not primarily responsible for d ownregulation of caspase-3, as we found no correlation between rates o f NO. release and the magnitude of enzyme inhibition. It is likely tha t S-nitrosation accounts for enzyme inhibition by various NO donors. S IN-1 and peroxynitrite were inhibitory as well. In this case, however, enzyme activity was not restored upon DTT addition, suggesting oxidat ion as an additional thiol modification mechanism. Our studies provide evidence that caspases are targeted by NO via S-nitrosation and oxida tion of critical thiol groups. (C) 1997 Academic Press.