The 45,000 Mr protein ASP (Apoptosis Specific Protein) was observed at high levels in both human and rat cells undergoing apoptosis but not in viable or ...
572s Biochemical SocietyTransactions ( 1 996) 24 E53
CLONING OF AN APOPTOSIS SPECIFIC GENE
E.M.Hammond and R.J.A.Grand The CRC Institute for Cancer Studies, The University of Birmingham, Edgbaston, Birmingham, B 15 2TJ, U.K.
The 45,000 Mr protein ASP (Apoptosis Specific Protein) was observed at high levels in both human and rat cells undergoing apoptosis but not in viable or necrotic cells. The protein was identified by means of a cross reaction with a c-jun antibody. The gene has been cloned by means of screening a human foetal liver expression library with the c-jun antibody. The gene identified is 2.9 Kb and encodes a protein of approximately 33 KD. The difference between this and the expected 45 KD molecule has been attributed to post-translational modification. Northern blot analysis shows products of 2.9 Kb and 1.7 Kb to be present in viable human tissue suggesting the presence of splice variants. RT-PCR has shown the message to be present in all the cell types tested (human and rat) including viable cells. The protein encoded by the gene has been isolated after cloning into the pQE-31 His tag expression vector and expression in E. coli. Polyclonal antibodies are being raised.
E54
E55 STAUROSPORINE INDUCES APOPTOSIS IN PRIMARY HEPATOCYTES BY THE ACTIVATION OF INTERLEUKIN18 CONVERTING ENZYME (ICE)-LIKE PROTEASES. Carole Couet Salmaan H Inayat-Hussain, Kelvin Cain. MRC Toxicology Unit, Centre for Mechanisms of Human Toxicity, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Rd, Leicester, LEI 9HN, U.K. Interleukin-lp converting enzyme (ICE)-like (e.g. CPP-32, Mch3a) proteases are believed to play a key role in apoptosis and recently we have shown that benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.FMK) a selective ICE-like p r o m e inhibitor blocks apoptosis in hepatocyte cultures. In this study we show that staurosporine induces both necrosis and apoptosis in mono-layer cultures of hepatocytes, when cells are cultured for 4 hours before addition of staurosporine. Only the apoptotic effects of staurosporine are blocked by ZVADFMK, indicating that staurosporine induces necrosis and apoptosis by two separate biochemical pathways. Further evidence for this conclusion comes from experiments in which the cells were cultured for 8 hours instead of 4 hours before adding staurosporine. Under these conditions, staurosporine induced apoptosis without necrosis and the apoptotic effect was blocked by Z-VAD.FMK. To further investigate the role of ICE-like proteases in hepatocyte apoptosis we assayed the proteolytic activity of lysates isolated from apoptotic hepatocytes using the model substrate benzoyloxy~nyl-AspGlu-ValAsp-amino-trifluoromethylcoumarin(Z-DEVD.AFC) which is cleaved by CPP-32 like proteases. Lysates from staurosporine treated hepatocytes expressed a marked and time-dependent increase in cleavage activity which was totally absent in lysates isolated from hepatocytes which had been pretreated with ZVAD.FMK before the addition of staurosporine. In addtion the increase in cleavage activity preceded the morphological characteristics of apoptosis. In conclusion, staurosporine induces necrosis and apoptosis by two separate biochemical pathways and in the case of apoptosis involves the activation of ICE (CPP32)-lie proteases.
CHARACTERISATION OF ICE-LIKE P R O T E O L Y T I C ACTIVITY I N A P O P T O T I C HUMAN M O N O C Y T I C T U M O U R (THP.l) CELLS.
Kelvin Cain, Marion MacFarlane and Gerald M Cohen MRC Toxicology Unit, Centre for Mechanisms of Human Toxicity, Hodglan Building, University of Leicester, P.O. Box 138, Lancaster Rd, Leicester, LEI 9HN, U.K Several interleukin-1 p converting enzyme (ICE)-like proteases are believed to play a key role in apoptosis. Kinetically, only ICE, CPP32 and Mch3a have been wholly/partially characterised and we have shown that in THP1 cells both CPP32 and Ich-1 proforms are processed to the active enzymes when apoptosis IS induced. We now demonstrate that lysates from these cells have a high level of CPP32 proteolflc cleavage as measured with the model substrate, benzoyloxycarbonyl-Asp-Glu-Val-Aspaminot~uoromethylcoumann (Z-DEVD.AFC) which mimics the poly (ADP-ribose) polymerase (PARP) cleavage site. Apoptotic cells lysates exhibited Michaelis-Menten kinetics when cleaving ZDEVD.AFC with a Km value of 41 pM which is 4-fold greater than that of purified W . 1 CPP32 and is similar to the 5 1 pM value reported for bacterially expressed recombinant Mch3a. Cleavage activity was inhibited by a variety of peptide site directed inhibitors with the most active, acetyl-Asp-Glu-Val-AH0 (Ac-DEVD. CHO) inhibiting at low nM concentrations (Ic50=3nM), benzyloxycarbonylVal-alal-asp(0Me)fluoromethylketone (Z-VAD.FMK) acting at low pM concentrations (Ic50= 1.2pM) and acetyl-Tyr-Val-Ala-Asp-CHO(AcYVAD.CH0) the specific ICE inhibitor was a relatively weak inhibitor with an IcSO value of 38pM). The kinetic data suggests that the proteolflc activity of TI%. 1 cell lysate is due to both CPP32 and Mch3a, although we cannot rule out the possibility of other as yet unidentified proteases. Increased cleavage actlvity in lysates isolated from cells committed to apopotosis preceded the morphological appearance of apoptosis. Control and apoptotic lysates underwent a limited amount of auto processing when the lysates were incubated at 37°C . This was maximal at 30-60min and did not increase even if the lysates were incubated for 3h. It was not possible to heat activate control lysates to levels of activity which were comparable with those of apoptotic lysates. Also, co-incubation of apoptotic and control lysates did not lead to further processing of the inactive control, pro-ICElike proteases and shows that there was a pool of inactive proteases which did not autoprocess. Thus,autoprocessing was not the dominant mechanism for actlvaung the CPP32-like proteases. However, coincubation of purified ICE with Wl lysates resulted in maximal stimulation of ZDEVD.AFC cleavage and complete processing of CPP32 and Ich-I. These results suggest that some ICE-like proteases are processed by a different protease and that this process is tightly regulated in the non-apoptotic cell.
ES6
TGF-PI INDUCED APOPTOSIS IN HEPATOCYTES INVOLVES ACTIVATION OF INTERLEUKIN-Ip CONVERTING ENZYME LIKE PROTEASE(S) Salmaan H. Inavat-Hussriin, Carole Couet. Gerald M. Cohen and Kelvin Cain MRC Toxicology Unit. Centre for Mechanisms of Human Toxicity, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Rd. Leicester. LEI 9HN, U.K.
Mammalian interleukin-I Pconverting enzyme (ICE)-like cysteine proteases, (e.g. CPP32, Mch3a) play a key role in the induction of apoptosis. We have shown previously that TGF-PI induced apoptosis in hepatocytes was blocked by the ICE-like inhibitor. beazyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (Z-VAD.FMK). suggesting the involvement of these enzymes in liver apoptosis. We now show that benzyloxycarbonyl-Asp-GluVal-Asp-(OMe) fluoromethyl ketone (Z-DEVD.FMK), an inhibitor of CPP32 containing the PI-P, amino acid sequence of the p l y (ADP-ribose) polymerase (PARF') cleavage site (DEW), inhibits TGF-Pl induced apoptosis as assessed by morphological changes and DNA degradation. This suggested that activation of CPP32-like protease activity WBS a key step in hepatocyte apoptosis and further characterisation of this proteolytic activity was performed using a fluorogenic peptide substrate benzyloxycarbonyl-Asp-GluVal-Asp-amino-trifluoromethyl-coumarin (Z-DEVD.AFC). This substrate is known to be cleaved by CPP32 and Mch3a. Lysates isolated from TGF-pI treated hepatocytes showed a timedependent increase in this proteolytic activity. This activity was abolished when hepatocytes were pretreated with either Z-VAD.Fh4K or Z-DEVD.FMK. These compounds were potent inhibitors of activated lysate from TGF-PI treated hepatocytes, confirming that the protease inhibitors were blocking a CPP32-like protease(s). In contrast, an (Ac-YVAD.CH0) was a ICE specific inhibitor. acetyl-Tyr-Val-Ala-Asp-CHO very weak inhibitor of the proteolytic activity in apoptotic lysates. Furthermore. cycloheximide (a protein synthesis inhibitor) also blocked TGFPI induced apoptosis and totally inhibited the increase of the proteolytic activity responsible for Z-DEVD.AFC cleavage. However, cyclolieximide incubated with activated lysate did not block the proteolytic activity, thereby demonstrating that cycloheximide was not a direct inhibitor of the CPP32-like protease(s). In conclusion, our data show for the first time that TGF-PI induced apoptosis in hepatocytes is associated with the activation of CPP32like protease(s) and that cyclolieximide inhibits apoptosis by blocking upstream of the processing of the CPP32-like protease(s).
