ADP-ribosylation Cycles

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Explanation A.Effects of clinical PARP-1 inhibitors on ARH3-deficient myocytes and mouse heart upon oxidative stress caused respectively by H2O2 or ischemia-reperfusion injury. Poly(ADP-ribosyl)ation is a post-translational modification, in which poly(ADP-ribose) (PAR) polymerases (PARPs), using NAD+ as a substrate, catalyze the formation of PAR on a target protein, and PAR glycohydrolase (PARG) and ADP-ribosyl-acceptor hydrolase 3 (ARH3) hydrolyze the covalently attached PAR to generate free PAR and ADP-ribose. PAR is rapidly and abundantly synthesized by PARPs, especially PARP-1, after activation by DNA breaks with PAR synthesis tightly linked to DNA excision repair mechanisms. Activation of PARPs by oxidative DNA damage is seen in cardiovascular diseases and during heart transplantation. ARH3 deficiency, which catalyzes PAR hydrolysis, induced neurodegeneration in an ARH3-deficient family. Arh3-deficient mice and fibroblasts from an ARH3-deficient patient were sensitive to cytotoxicity caused by DNA-damaging agents, e.g., H2O2, leading to PAR-dependent cell death or parthanatos. Here, we focused on the mechanism of action of ARH3 in cardiac injury and the effects of treatment with FDA-approved PARP inhibitors, e.g., rucaparib, olaparib. To understand better the roles of PARP and ARH3 we used several model systems and investigated the effects of PARP inhibition in Arh3-deficient C2C12 mouse myocytes, mouse embryonic fibroblasts (MEFs), and mice, on oxidative stress caused, respectively, by hydrogen peroxide (H2O2) in cells or ischemia/reperfusion (I/R) injury in mice. Of note, the standard treatments for ameliorating myocardial infarction and improving heart transplantation are reperfusion strategies, which paradoxically result in I/R injury. Cardiac transplantation is a treatment for severe atherosclerotic disease; however, oxidative stress during I/R may affect the outcome of the transplantation. Under physiological conditions, PARP-1 activation regulates many cellular processes (e.g., DNA repair, cell cycle, gene transcription, chromatin remodeling). However, overactivation of PARP-1 by oxidative stress causes uncontrolled DNA damage, leading to energy depletion and altered regulation of kinase cascades, which increase vascular cell apoptosis and necrosis, eventually resulting in the functional impairment or death of the endothelial cells and cardiomyocytes. Hypoxia/reperfusion promotes PARP-1 activation, mitochondrial damage, and dysfunction in myocytes and in endothelial cells by decreasing the mitochondrial transmembrane potential. PARP-1 overexpression was observed in human atherosclerotic plaques, with increased circulating levels of proinflammatory cytokines in patients with unstable angina. Activation of PARP-1 and beneficial effects of PARP-1 inhibition have been demonstrated after heart transplantation or cardiopulmonary bypass. Finally, PARP-1 inhibitors improve myocardial contractility, preserve myocardial ATP/ NAD+ pools during reperfusion, and significantly diminish infarct size and mortality in animal models. Thus, activation of PARP-1 has been involved in various cardiovascular diseases (e.g., myocardial I/R injury, atherosclerosis, plaque disruption, diabetic cardiovascular complications, heart transplantation). However, the function of PAR hydrolyzing enzymes is not well known, even though it is a key regulatory component in the PAR pathway. Recently, we reported that ARH3, a PAR-hydrolyzing enzyme discovered in our laboratory, confers protection against oxidative stress such as H2O2 exposure by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) translocation to the nucleus and parthanatos or PAR-mediated cell death. Subsequently, we identified a family with an ARH3 gene mutation that resulted in a neurological phenotype. The patient's fibroblasts and Arh3-deficient mice were more sensitive, respectively, to H2O2 stress and cerebral I/R-induced PAR accumulation and cell death. Thus, ARH3 deficiency increased cell sensitivity to oxidative stress and has the potential to increase myocardial damage, leading to CAD and other myocardial diseases. Based on these findings, we are investigating the mechanism of ARH3 action in myocytes and mouse heart upon oxidative stress caused respectively, by H2O2 stress and myocardial I/R, examining the potential of PARP inhibitors to serve as a treatment for cardiovascular diseases, as well as ARH3-deficient patients. We observed that Arh3-deficient myocytes and mouse heart are more sensitive to oxidative stress caused by H2O2 exposure and myocardial I/R, respectively, compared to wild-type (WT). The cell death pathway caused by ARH3 deficiency resulted in PAR-dependent cell death and caused necrosis, apoptosis, and AIF-dependent cell death (parthanatos). The PARP inhibitors, rucaparib and olaparib, decreased the extent of injury during ischemia/reperfusion injury in ARH3-deficient mouse hearts and decreased the effects of H202 on viability of C2C12 myocytes. B. In collaboration with Masato Mashimo, a former post-doctoral fellow, we examined the mechanism of PAR translocation from the nucleus to the cytoplasm and the resulting decrease in cell viability. Poly(ADP-ribose) polymerase 1 (PARP1) catalyzes poly(ADP-ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, and thus recruits DNA repair machinery to sites of DNA damage. With excessive DNA damage, 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. PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis, with PARP cleavage leading to decreased activity. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in 24-kDa and 89-kDa fragments. 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, 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. C. In collaboration with In-Kwon Kim, we examined the structural and enzymatic properties of ARH3. ARH3 is also metalloenzyme with strong metal selectivity. While coordination of two magnesium ions (MgA and MgB) significantly enhances its catalytic efficiency, calcium binding suppresses function. Here, we report a new crystal structure of ARH3 complexed with its product ADP-ribose and calcium. Calcium coordination significantly distorted the binuclear metal center of ARH3, with decreased binding affinity to ADP-ribose, and suboptimal substrate alignment, leading to impaired hydrolysis of PAR and mono(ADP-ribosyl)ated serines. Combined structural and mutational analysis of the metal-coordinating acidic residues revealed that MgA is crucial for optimal substrate positioning for catalysis, whereas MgB plays a key role in substrate binding. D. COVID 19-related studies: The SARS CoV2 NSP3 protein contains three macrodomain domains. Prior studies from our lab showed that macrodomains are similar enzymatically to the ARH proteins and catalyze the hydrolysis ADP-ribose and thus in their involvement in biological pathways. We currently are characterizing the reactions and possible acceptors.