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

doi:10.1074/jbc.270.32.18715

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

From theFrom Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211

^§ To whom correspondence should be addressed: Vertex Pharmaceuticals Incorporated, 40 Allston St., Cambridge, MA 02139-4211. Tel.: 617-576-3111; Fax: 617-499-7315; su{at}vpharm.com.

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-1β 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-1β 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ICE

interleukin-1β converting enzyme

PARP

poly(ADP-ribose) polymerase

PARP(F)

T7-epitope-tagged, full-length poly(ADP-ribose) polymerase

PARP(T)

T7-epitope-tagged, C-terminally truncated polypeptide of poly(ADP-ribose) polymerase

pre-IL-1β

interleukin-1β precursor

prICE

protease resembling ICE

PCR

polymerase chain reaction

kb

kilobase(s)

PAGE

polyacrylamide gel electrophoresis

IVTT

in vitro transcription-translation

Ac-YVAD-CHO

Ac-Tyr-Val-Ala-Asp-aldehyde

Cbz-VAD-CH₂-DCB

benzyloxycarbonyl-Val-Ala-Asp-[(2,6-dichlorobenzoyl)oxy]methyl ketone.
↵²Y. Gu, unpublished results.
- Received May 15, 1995.
- Revision received June 12, 1995.