Caspase-3-mediated processing of poly(ADP-ribose) glycohydrolase during apoptosis.
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.
Affar
E B
EB
Health and Environment Unit, Laval University Medical Research Center, Centre Hospitalier Universitaire de Québec, Canada.
Germain
M
M
Winstall
E
E
Vodenicharov
M
M
Shah
R G
RG
Salvesen
G S
GS
Poirier
G G
GG
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D005047:Etoposide; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009842:Oligopeptides; D010446:Peptide Fragments / Q000302:isolation & purification; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D019311:Staurosporine; D019014:fas Receptor
D009842:Oligopeptides; D010446:Peptide Fragments; C108637:aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; D011064:Poly Adenosine Diphosphate Ribose; D005047:Etoposide; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine