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

Abstract

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.

Relevance of AIF/CypA Lethal Pathway in SH-SY5Y Cells Treated with Staurosporine

“…To further elucidate the mechanism underpinning the AIF(370-394) protective effect on the STS-treated SH-SY5Y cells, we also inspected the amounts of cleaved caspase-3 and PARP in both the cytosolic and nuclear extracts of cells treated and untreated with 10 µM STS. According to previous reports [ 5 , 34 ], in STS-treated cells, we detected a significant increase of activated caspase-3 and cleaved PARP, detected as the p24 subunit at ~24 kDa, in the cytosol and nucleus, respectively, compared to control cells ( Figure 4 C–F). The delivery of AIF(370-394) to STS-treated cells did not alter the levels of cleaved target proteins compared to cells treated with STS alone, indicating that the presence of the peptide did not influence the cell death mediated by caspase activation.…”

PARP-1-Associated Pathological Processes: Inhibition by Natural Polyphenols

“…In turn, PARP-1 hyperactivation can initiate parthanatos—a programmed caspase-independent cell death ( Figure 2 ) [ 26 , 27 ]. PAR and PARylated proteins that are formed in large quantities migrate from a nucleus to cytoplasm and cause the release of apoptosis-inducing factor (AIF) from mitochondria [ 26 , 28 , 29 ]. Released AIF is transported to a nucleus due to a nuclear localization signal (NLS) and activates endonucleases, which cause DNA fragmentation followed by cell death.…”

Histone Acetylation Defects in Brain Precursor Cells: A Potential Pathogenic Mechanism Causing Proliferation and Differentiation Dysfunctions in Mitochondrial Aspartate-Glutamate Carrier Isoform 1 Deficiency

“…In order to exclude apoptosis and cell death, we then verified through WB analysis, the expression of the pro-apoptotic marker, caspase3 (both precursor and cleaved versions of the enzyme) ( Widmann, 2007 ), and no differences were observed in treated vs. control cells ( Figures 6C,J ). Additionally, we performed WB for the full length PARP1 (116 kDa) and the large fragment (89 kDa) of PARP1 resulting from caspase cleavage ( Mashimo et al, 2021 ; Figures 6C,I ), as well as for the anti-apoptotic protein, BCL-2 ( Chipuk et al, 2010 ) (data not shown). In parallel, in Oli-Neu cells with AGC1 downregulation, the marker of mature oligodendrocytes CNPase ( Scherer et al, 1994 ) showed higher expression as compared to the control cells and was further induced by curcumin treatment ( Figures 6C,H ).…”