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 o ther PARP-1-like enzymes. In this work, we report that PARG is cleaved duri ng etoposide-, staurosporine-, and Fas-induced apoptosis in human cells. Th is cleavage is concomitant with PARP-1 processing and generates two C-termi nal fragments of 85 and 74 kDa. In vitro cleavage assays using apoptotic ce ll extracts showed that a protease of the caspase family is responsible for PARG processing. A complete inhibition of this cleavage was achieved at na nomolar concentrations of the caspase inhibitor acetyl-Asp-Glu-Val-Asp-alde hyde, suggesting the involvement of caspase-3-like proteases. Consistently, recombinant caspase-3 efficiently cleaved PARG in vitro, suggesting the in volvement of this protease in PARG processing in vivo, Furthermore, caspase -3-deficient MCF-7 cells did not show any PARG cleavage in response to stau rosporine treatment. The cleavage sites identified by site-directed mutagen esis are DEID256 down arrow V and the unconventional site MDVD307 down arro w N. Kinetic studies have shown similar maximal velocity (V-max) and affini ty (K-m) for both full-length PARG and its apoptotic fragments, suggesting that caspase-3 may affect PARG function without altering its enzymatic acti vity. The early cleavage of both PARP-1 and PARG by caspases during apoptos is suggests an important function for poly(ADP-ribose) metabolism regulatio n during this cell death process.