The 89-kDa PARP1 cleavage fragment serves as a cytoplasmic PAR carrier to induce AIF-mediated apoptosis

Masato Mashimo,Mayu Onishi,Arina Uno,Akari Tanimichi,Akari Nobeyama,Mana Mori,Sayaka Yamada,Shigeru Negi,Xiangning Bu,Jiro Kato,Joel Moss,Noriko Sanada,Ryoichi Kizu,Takeshi Fujii

doi:10.1074/jbc.ra120.014479

Abstract

Poly(ADP-ribose) polymerase 1 (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 present study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 autopoly(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, whereas 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.

Highlights

After PAR polymers produced by Poly(ADP-ribose) polymerase 1 (PARP1) are translocated from the nucleus to the cytoplasm, they bind to apoptosis-inducing factor (AIF), which is anchored to the mitochondrial membrane [15, 19]
An apoptosis inducer, stimulated PARP1-dependent cell death
Pretreatment with PJ34 did not improve the viability of Hela cells expressing PARP1 shRNA (Fig. S1B), suggesting that PJ34 has specificity to PARP1-dependent cell death induced by staurosporine

Summary

Results

An apoptosis inducer, stimulated PARP1-dependent cell death. HeLa cells expressing PARP1 shRNA did not exhibit PAR synthesis, AIF translocation to nuclei, and nuclear shrinkage (Fig. S2, A–C). HeLa cells expressing PARP1 shRNA exhibited reduced translocation of PAR to the cytoplasm after 6-h exposure to staurosporine compared with control shRNA (Fig. S2E). Densitometric analysis indicates that approximately 20% of PARP1 was present as the cleaved form in the cytoplasm (Fig. 2E) These results indicate that the 89-kDa PARP1 fragment is translocated to the cytoplasm after PARP1mediated PAR production in nuclei. We investigated whether PARP1 is cleaved after it is recruited to DNA lesions To address this issue, we performed a 405-nm laser microirradiation to induce DNA lesions in HeLa cells expressing PARP1 constructs.

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15 Histone H3 37 GAPDH

Discussion

Experimental procedures