PARP-1 is a GzmA substrate. (A) Schematic of functional domains of PARP-1, indicating the caspase and GzmB cleavage site within the nuclear localization signal and GzmA cleavage site within the automodification domain (site of PARylation). (B) GzmA treatment of isolated nuclei produces a 55-kDa C-terminal fragment of PARP-1. HeLa nuclei were incubated with GzmA at the indicated concentrations for the indicated times and analyzed by immunoblot probed with PARP-1 C-terminal antiserum or for C23, a loading control. (C) When recombinant PARP-1 was incubated with GzmA, a 55-kDa C-terminal cleavage product is again seen by immunoblot. N-terminal sequencing of the 55-kDa fragment identified the cleavage site after Lys⁴⁹⁸. K498A PARP-1 is less susceptible to cleavage, but at longer times (top) or higher GzmA concentrations (bottom) it is also cleaved to a similarly sized fragment. (D) Treatment of K562 cells with GzmA and PFN leads to PARP-1, but not PARP-2, degradation. β-Actin was probed as a loading control, and the GzmA substrate SET was probed as a positive control. GzmB cleaves PARP-1 to generate an 89-kDa C-terminal fragment; S-AGzmA does not cleave either PARP (top). GzmA and GzmB (0.5 μM) cleave PARP-1 with similar kinetics (bottom). (E) PARP-1 is degraded within 40 minutes of CTL attack, with similar kinetics as SET. The serine protease inhibitor diisocoumarin (DCI) blocks PARP-1 and SET cleavage. (B-E) These are immunoblots; the PARP-1 blots are probed with an antibody that recognizes the C-terminal region of PARP-1.