Cleavage of Poly(ADP-ribose) Polymerase by Interleukin-1β Converting Enzyme and Its Homologs TX and Nedd-2

Abstract

The proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) is an early biochemical event, which occurs during apoptosis. A recent study suggested that PARP cleavage can be mediated by a novel cytosolic protease (prICE) that resembles interleukin-1 beta converting enzyme (ICE), but cannot be mediated by ICE itself (Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G., and Earnshaw, W.C. (1994) Nature 371, 346-347). We have used a COS cell co-transfection assay to investigate if ICE or any known ICE-like protease is active in PARP cleavage within the cell. Here we report that co-expression of human PARP with human ICE, or the ICE homologs TX and Nedd-2, resulted in a cleavage of PARP identical to that observed in apoptotic cells. Experiments with purified recombinant human ICE indicated that PARP polypeptide can be specifically cleaved in vitro by ICE in a time- and enzyme concentration-dependent manner. PARP cleavage, however, requires a 50-100-fold higher ICE concentration than does processing of the interleukin-1 beta precursor at an equivalent substrate concentration. The abilities of ICE, TX, and Nedd-2, when expressed at high intracellular concentrations, to cleave PARP are consistent with their induction of apoptosis in transfected cells.

Bcl-2 prevents topoisomerase II inhibitor GL331-induced apoptosis is mediated by down-regulation of poly(ADP-ribose)polymerase activity

“…In addition, this proteolytic cleavage decreases the activity of PARP and the 85-kDa fragment retains only basal PARP activity. The ICElike cysteine proteases are the executors for this PARP cleavage (Gu et al, 1995). However, in this study, the apparent PARP cleavage during the course of apoptosis is not observed, suggesting that ICE-like cysteine protease is not involved in GL331-mediated cell death.…”

Caspase substrates

“…58 PARP was one of the first identified examples of a substrate processed by an executioner caspase much more efficiently than the inflammatory caspase-1. 59,60 The PARP cleavage site perfectly matches the substrate specificity of executioner caspases-3 and -7. Interestingly, secondary interactions seem to be at work because caspase-7, which is less catalytically active than caspase-3 on short synthetic substrates, processes PARP molecules modified with long, branched poly(ADP-ribose) chains more efficiently than its close homolog caspase-3.…”

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8

“…At higher concentrations (3 mg/ml), the PARP cleavage site peptide showed broad spectrum inhibition of all caspases (Table 2). This is reminiscent of the ability of caspases other than caspases-3 and -7/a to cleave PARP, albeit with lower e ciency (Fernandes- Alnemri et al, 1995a;Gu et al, 1995;Duan et al, 1996b;Muzio et al, 1996). The lamin A cleavage site peptide preferentially inhibited F19 and S19, with other caspases showing much lower sensitivities (F19»F20&F174F22; S19»S20&S174S22; Figure 3c, lane 2).…”