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Poly(ADP-ribose) polymerase (PARP1) is a nuclear protein that is activated by binding to DNA lesions and catalyzes poly(ADP- ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, to recruit DNA repair machinery to DNA lesions. When excessive DNA damage occurs, poly(ADP-ribose) (PAR) produced by PARP1 is translocated to the cytoplasm, changing the activity and localization of cytoplasmic proteins e.g. apoptosis-inducing factor (AIF), hexokinase and resulting in cell death. This cascade, termed parthanatos, is a caspase-independent programmed cell death distinct from necrosis and apoptosis. In contrast, PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis. Once cleaved, PARP1 loses its activity, thereby suppressing DNA repair. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in the formation of 24-kDa and 89-kDa fragments. In the current study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 auto-poly(ADP-ribosyl)ation and fragmentation, generating poly(ADP-ribosyl)ated 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments with covalently attached PAR polymers were translocated to the cytoplasm, while 24-kDa fragments remained associated with DNA lesions. In the cytoplasm, AIF binding to PAR attached to the 89-kDa PARP1 fragment facilitated its translocation to the nucleus. Thus, the 89-kDa PARP1 fragment is a PAR carrier to the cytoplasm, inducing AIF release from mitochondria. Elucidation of the caspase-mediated interaction between apoptosis and parthanatos pathways extend the current knowledge on mechanisms underlying programmed cell death and may lead to new therapeutic targets.

J. Neurochem. (2011) 117, 346–356. Abstract Hypoxic preconditioning (HPC) initiates intracellular signaling pathway to provide protection against subsequent cerebral ischemic injuries, and its mechanism may provide molecular targets for therapy in stroke. According to our study of conventional protein kinase C βII (cPKCβII) activation in HPC, the role of cPKCβII in HPC‐induced neuroprotection and its interacting proteins were determined in this study. The autohypoxia‐induced HPC and middle cerebral artery occlusion (MCAO)‐induced cerebral ischemia mouse models were prepared as reported. We found that HPC reduced 6 h MCAO‐induced neurological deficits, infarct volume, edema ratio and cell apoptosis in peri‐infarct region (penumbra), but cPKCβII inhibitors Go6983 and LY333531 blocked HPC‐induced neuroprotection. Proteomic analysis revealed that the expression of four proteins in cytosol and eight proteins in particulate fraction changed significantly among 49 identified cPKCβII‐interacting proteins in cortex of HPC mice. In addition, HPC could inhibit the decrease of phosphorylated collapsin response mediator protein‐2 (CRMP‐2) level and increase of CRMP‐2 breakdown product. TAT‐CRMP‐2 peptide, which prevents the cleavage of endogenous CRMP‐2, could inhibit CRMP‐2 dephosphorylation and proteolysis as well as the infarct volume of 6 h MCAO mice. This study is the first to report multiple cPKCβII‐interacting proteins in HPC mouse brain and the role of cPKCβII‐CRMP‐2 in HPC‐induced neuroprotection against early stages of ischemic injuries in mice.

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