Caspases are cysteine peptidases involved in the cleavage of many proteins, and these cleavage events are key to many cellular processes such as apoptosis and inflammation. One of the hallmark of apoptosis is the proteolysis of the poly (ADP-ribose) polymerase 1 (PARP-1), a DNA-repair enzyme. PARP-1 is cleaved by both caspase-3 and caspase-7, but with a greater efficacy by the latter despite that it has lower intrinsic activity than caspase-3. Our laboratory discovered that the N-terminal domain of caspase-7 contains an exosite that interacts with PARP-1 to enhance its cleavage. However, the detail of this interaction remains to be characterized.
In order to better understand the caspase-7/PARP-1 interaction, we first used truncated mutant of the N-terminal domain of caspase-7 to identify the boundaries of the exosite. The ability of each truncated protein to interact with PARP-1 was evaluated using GST pull-down assays. We identified the critical region between the amino acids 34 and 47 as the minimal boundaries of the exosite. Secondly, truncation mutants of PARP-1 were generated to identify which domain interacts with caspase-7’s exosite. These mutants were tested in cleavage assays using wild-type caspase-7 and preliminary results identified the linker separating PARP’s BRCT and WGR domains as the region that binds the exosite. Finally, we tested the involvement of PARP-1 automodification (ADP-ribosylation) in the interaction with the exosite using cleavage assays and we found that the unmodified PARP-1 is cleaved more efficaciously than the automodified protein.
Our studies will provide a better understanding of the interaction between caspases and their substrates that involves more than primary structure recognition.
This work was supported by a NSERC (355388-2010) grant and FRQ-NT team grant (2014-PR-171852) to JBD.