Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis

J Biol Chem. 2001 Jan 26;276(4):2935-42. doi: 10.1074/jbc.M007269200. Epub 2000 Oct 25.

Abstract

Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the catabolism of poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerase (PARP-1) and other PARP-1-like enzymes. In this work, we report that PARG is cleaved during etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. This cleavage is concomitant with PARP-1 processing and generates two C-terminal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic cell extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at nanomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the involvement of this protease in PARG processing in vivo. Furthermore, caspase-3-deficient MCF-7 cells did not show any PARG cleavage in response to staurosporine treatment. The cleavage sites identified by site-directed mutagenesis are DEID(256) downward arrow V and the unconventional site MDVD(307) downward arrow N. Kinetic studies have shown similar maximal velocity (V(max)) and affinity (K(m)) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic activity. The early cleavage of both PARP-1 and PARG by caspases during apoptosis suggests an important function for poly(ADP-ribose) metabolism regulation during this cell death process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis*
  • Caspase 3
  • Caspases / metabolism*
  • Cattle
  • Cells, Cultured
  • Etoposide
  • Glycoside Hydrolases / isolation & purification
  • Glycoside Hydrolases / metabolism*
  • Humans
  • Mice
  • Oligopeptides
  • Peptide Fragments / isolation & purification
  • Poly Adenosine Diphosphate Ribose / metabolism
  • Protein Processing, Post-Translational*
  • Staurosporine
  • fas Receptor

Substances

  • Oligopeptides
  • Peptide Fragments
  • aspartyl-glutamyl-valyl-aspartal
  • fas Receptor
  • Poly Adenosine Diphosphate Ribose
  • Etoposide
  • Glycoside Hydrolases
  • poly ADP-ribose glycohydrolase
  • CASP3 protein, human
  • Casp3 protein, mouse
  • Caspase 3
  • Caspases
  • Staurosporine