Inhibition of the interleukin-1 beta converting enzyme family rescues neurons from apoptotic death.
The interleukin-1 beta converting enzyme (ICE) family of cysteine proteases has been implicated in apoptosis. This study tested the effects of a novel pan-ICE family inhibitor, bocaspartyl(OMe)-fluoromethylketone (boc-Asp-CH2F), against low potassium-induced apoptosis of cultured rat cerebellar granule neurons (CGN). A single application of this cell-permeant compound (20 microM) inhibited apoptotic cell death up to 48 h. Classical apoptotic changes were monitored by fluorescence microscopy, DNA fragmentation and scanning electron microscopy (SEM). A control peptidic fluoromethylketone (boc-Thr-CH2F), and inhibitors to calpain (Ac-Leu-Leu-norleucinal), cathepsin B (Z-Phe-Ala-CH2F), and CPP32-like proteases (Z-DEVD-CH2F), failed to prevent apoptotic death. An 35S-methionine incorporation assay verified that, unlike cycloheximide, boc-Asp-CH2F did not inhibit protein synthesis, hence excluding this as a rescuing mechanism. Although ICE was not detected by northern blot analysis, both CPP32 and Nedd2 expression were found to increase during apoptosis. Kinetic assays with cell extracts from boc-Asp-CH2F-treated neurons measured reduced rates of cleavage for DEVD-pNA and LEVD-pNA. At present, ICE-like proteases remain viable candidates for mediating neuronal death.
Lynch
T
T
Department of Neurology, University of Michigan, Ann Arbor 48109, USA.
Vasilakos
J P
JP
Raser
K
K
Keane
K M
KM
Shivers
B D
BD
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D020170:Caspase 1; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; C106871:benzyloxycarbonyl-aspartyl-fluoromethane; D001224:Aspartic Acid; D003546:Cysteine Endopeptidases; D020170:Caspase 1