ADP-ribosylation Cycles

National Heart, Lung, And Blood Institute

Investigators

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Abstract

Explanation 1. Clinical PARP-1 inhibitor improves cardiac dysfunction in ADP-ribosyl Acceptor Hydrolase 3 (Arh3)-deficient mice. Poly(ADP-ribosylation) is a post-translational modification of protein, catalyzed by poly(ADP-ribose)polymerases (PARPs), in which the ADP-ribose moiety of NAD+ is transferred to protein, forming branched, long-chain polymers. The primary enzyme responsible for the reaction is PARP-1, which is activated by oxidative stress conditions that lead to DNA strand breaks. The reaction is reversible, with polymers degraded primarily by two enzymes, poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor 3 (ARH3). PARG primarily acts by endocytic cleavage of the polymer, while ARH3 acts through exocidic cleavage. Patients with a deficiency in ARH3 develop a neurodegenerative phenotype, resulting in many instances in an early death. Other ARH3-deficient patients appear to show cardiac abnormalities. It is unclear whether these cardiac manifestations results from a deficiency in ARH3 gene. To determine whether ARH3 deficiency would result from defects in the ARH3 gene, leading to cardiac manifestations of disease, we developed and phenotyped an Arh3-deficient mouse, looking at potential cardiac or motor manifestations of disease. In the present study, Arh3-deficient mice showed increased motor dysfunction, assessed physiologically by open field and rotarod tests. Cardiac dysfunction in the Arh3-deficent mice was supported by the findings showing abnormalities in the dobutamine stress test and enhanced vulnerability to myocardial ischemia/reperfusion (I/R) injury with increased area of infarction. Treatment of the mice with a PARP inhibitor improved cardiac contractility in the dobutamine stress test and reduced the size of the myocardial ischemia-reperfusion infarcts. In parallel studies, to better understand the role of Arh3, we used CRISPR-Cas9 to generate Arh3-deficient C2C12 murine myocytes. Incubation with H2O2 to induce oxidative damage showed enhanced cytotoxicity and led to poly(ADP-ribose)-dependent cell death that was reduced by PARP inhibitors or by transfection with the Arh3 gene. Addition of the Arh3 gene, even to the wild-type cell, improved viability. Thus, excess ARH3 appears to overcome oxidative stress induced by H2O2. These studies suggest that, Arh3 regulates PAR homeostasis in myocardium and that PARP inhibitors might have a therapeutic effect on the myocardial dysfunction seen with ARH3 deficiency. 2. Poly(ADP-ribose) Polymerase 1 Mediates Rab5 Inactivation after DNA Damage Parthanatos is a form of programmed cell death, mediated by poly(ADP-ribose) polymerase 1 (PARP1) after DNA damage. PARP1 acts by catalyzing the NAD-dependent formation of poly(ADP-ribose) (PAR) polymers linked to various nuclear proteins. PAR is subsequently cleaved, generating protein-free PAR polymers, which are translocated to the cytoplasm where they associate with cytoplasmic and mitochondrial proteins, altering their functions and leading to cell death (Parthanatos). We previously defined the interaction of PAR with mitochondrial Apoptosis Inducing Factor (AIF) as a mechanism of Parthanatos. In the current study, proteomic studies revealed that several proteins involved in endocytosis bind PAR after PARP1 activation, suggesting that endocytosis may be involved in Parthanatos. Endocytosis is a mechanism for cellular uptake of membrane-impermeant nutrients. Rab5, a 23.7 kDa guanine nucleotide binding protein, is associated with the plasma membrane and early endosomes. Rab5 is activated in the GTP-bound state and inactive with bound GDP. Once activated by binding GTP, Rab5 recruits its effectors to early endosomes and regulates endosomal fusion. It was observed in this study, headed by a former post-doc, Masato Mashimo, that after DNA damage, PARP1-generated PAR binds to Rab5, suppressing its activity. As a result, Rab5 is dissociated from endosomal vesicles, inhibiting the uptake of membrane-impermeant nutrients. This PARP1-dependent inhibition of nutrient uptake leads to cell starvation and death. This Rab5-mediated pathway appears to be another mechanism underlying a novel parthanatos pathway. 3. COVID 19-related studies: The SARS CoV2 NSP3 protein contains three macrodomain domains, Mac 1, Mac 2, and Mac3. Prior studies from our lab as well as others have shown that macrodomains are similar enzymatically in some ways to the ARH proteins and catalyze the hydrolysis ADP-ribose-acceptor linkages. We currently are characterizing the potential reactions catalyzed by the macrodomains of the SARS CoV2 Nsp3 protein. As found by others, Mac2 and Mac2 do not appear to hydrolyze the ADP-ribose-acceptor linkage. Mac1, however, is enzymatically active. The nature of the Mac1-catalyzed reactions is under investigation.