Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes.
Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.
Inayat-Hussain
S H
SH
MRC Toxicology Unit, University of Leicester, United Kingdom.
Couet
C
C
Cohen
G M
GM
Cain
K
K
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004790:Enzyme Induction; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008297:Male; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D003374:Coumarins; D009842:Oligopeptides; D011500:Protein Synthesis Inhibitors; D016212:Transforming Growth Factor beta; C106566:benzyloxycarbonyl-aspartyl-glutamyl-valyl-aspartyl-7-amino-4-trifluoromethylcoumarin; D003513:Cycloheximide; D011065:Poly(ADP-ribose) Polymerases; D010450:Endopeptidases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases