Pál Gergely

Peroxynitrite-induced cytotoxicity: Mechanism and opportunities for intervention

Peroxynitrite is formed in biological systems when superoxide and nitric oxide are produced at near equimolar ratio. Although not a free radical by chemical nature (as it has no unpaired electron), peroxynitrite is a powerful oxidant exhibiting a wide array of tissue damaging effects ranging from lipid peroxidation, inactivation of enzymes and ion channels via protein oxidation and nitration to inhibition of mitochondrial respiration. Low concentrations of peroxynitrite trigger apoptotic death, whereas higher concentrations induce necrosis with cellular energetics (ATP and NAD) serving as switch between the two modes of cell death. Peroxynitrite also damages DNA and thus triggers the activation of DNA repair systems. A DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) also becomes activated upon sensing DNA breakage. Activated PARP-1 cleaves NAD(+) into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins. Peroxynitrite-induced overactivation of PARP consumes NAD(+) and consequently ATP culminating in cell dysfunction, apoptosis or necrosis. This cellular suicide mechanism has been implicated among others in the pathomechanism of stroke, myocardial ischemia, diabetes and diabetes-associated cardiovascular dysfunction. Here, we review the cytotoxic effects (apoptosis and necrosis) of peroxynitrite focusing on the role of accelerated ADP-ribose turnover. Regulatory mechanisms of peroxynitrite-induced cytotoxicity such as antioxidant status, calcium signalling, NFkappaB activation, protein phosphorylation, cellular adaptation are also discussed.

Detection of Poly(ADP-ribose) Polymerase Activation in Oxidatively Stressed Cells and Tissues Using Biotinylated NAD Substrate

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD+ into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD+), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide-induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD+ incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [3H]-NAD incorporation method. The bio-NAD+ assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.

Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells

Activation of poly(ADP‐ribose) polymerase‐1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP‐ribose) glycohydrolase (PARG), the enzyme responsible for polymer degradation, has been largely unexplored in the regulation of cell death. Using lentiviral gene silencing we generated A549 lung adenocarcinoma cell lines with stably suppressed PARG and PARP1 expression (shPARG and shPARP1 cell lines, respectively) and determined parameters of apoptotic and necrotic cell death following hydrogen peroxide exposure. shPARG cells accumulated large amounts of poly(ADP‐ribosyl)ated proteins and exhibited reduced PARP activation. Hydrogen peroxide‐induced cell death is regulated by PARG in a dual fashion. Whereas the shPARG cell line (similarly to shPARP1 cells) was resistant to the necrotic effect of high concentrations of hydrogen peroxide, these cells exhibited stronger apoptotic response. Both shPARP1 and especially shPARG cells displayed a delayed repair of DNA breaks and exhibited reduced clonogenic survival following hydrogen peroxide treatment. Translocation of apoptosis‐inducing factor could not be observed, but cells could be saved by methyl pyruvate and α‐ketoglutarate, indicating that energy failure may mediate cytotoxicity in our model. These data indicate that PARG is a survival factor at mild oxidative damage but contributes to the apoptosis‐necrosis switch in severely damaged cells.—Erdélyi, K., Bai, P., Kovács, I., Szabó, E., Mocsar, G., Kakuk, A., Szabó, C., Gergely, P., Virag, L. Dual role of poly(ADP‐ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells. FASEB J. 23, 3553–3563 (2009). www.fasebj.org

Efficient inhibition of muscle and liver glycogen phosphorylases by a new glucopyranosylidene-spiro-thiohydantoin

Reaction of C-(1-bromo-1-deoxy-beta-glucopyranosyl)formamide 2 with thiocyanate ions was the key step of a short synthesis of D-glucopyanosylidene-spiro-thiohydantoin 7 which proved to be a potent inhibitor of muscle and liver glycogen phosphorylases.

Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis

The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host’s response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5′-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, β-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.

Kinetic and crystallographic studies on 2‐(β‐D‐glucopyranosyl)‐5‐methyl‐1, 3, 4‐oxadiazole, ‐benzothiazole, and ‐benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site

In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of β‐D‐glucopyranose, 2‐(β‐D‐glucopyranosyl)‐5‐methyl‐1, 3, 4‐oxadiazole, ‐benzothiazole, and ‐benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc‐1‐P) with Ki values of 145.2 (±11.6), 76 (±4.8), and 8.6 (±0.7) μM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb‐methyl‐oxadiazole complex, 1.92 Å; GPb‐benzothiazole, 2.10 Å; GPb‐benzimidazole, 1.93 Å). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T‐state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (∼ 32 Å) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.

Nitric oxide‐peroxynitrite‐poly(ADP‐ribose) polymerase pathway in the skin

Abstract: In the last decade it has become well established that in the skin, nitric oxide (NO), a diffusable gas, mediates various physiologic functions ranging from the regulation of cutaneous blood flow to melanogenesis. If produced in excess, NO combines with superoxide anion to form peroxynitrite (ONOO–), a cytotoxic oxidant that has been made responsible for tissue injury during shock, inflammation and ischemia‐reperfusion. The opposite effects of NO and ONOO– on various cellular processes may explain the ‘double‐edged sword’ nature of NO depending on whether or not cellular conditions favour peroxynitrite formation. Peroxynitrite has been shown to activate the nuclear nick sensor enzyme, poly(ADP‐ribose) polymerase (PARP). Overactivation of PARP depletes the cellular stores of NAD+, the substrate of PARP, and the ensuing ‘cellular energetic catastrophy’ results in necrotic cell death. Whereas the role of NO in numerous skin diseases including wound healing, burn injury, psoriasis, irritant and allergic contact dermatitis, ultraviolet (UV) light‐induced sunburn erythema and the control of skin infections has been extensively documented, the intracutaneous role of peroxynitrite and PARP has not been fully explored. We have recently demonstrated peroxynitrite production, DNA breakage and PARP activation in a murine model of contact hypersensitivity, and propose that the peroxynitrite‐PARP route represents a common pathway in the pathomechanism of inflammatory skin diseases. Here we briefly review the role of NO in skin pathology and focus on the possible roles played by peroxynitrite and PARP in various skin diseases.

Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure

Our recently published data suggested the involvement of protein phosphatase 2A (PP2A) in endothelial cell (EC) barrier regulation (Tar et al. [2004] J Cell Biochem 92:534–546). In order to further elucidate the role of PP2A in the regulation of EC cytoskeleton and permeability, PP2A catalytic (PP2Ac) and A regulatory (PP2Aa) subunits were cloned and human pulmonary arterial EC (HPAEC) were transfected with PP2A mammalian expression constructs or infected with PP2A recombinant adenoviruses. Immunostaining of PP2Ac or of PP2Aa + c overexpressing HPAEC indicated actin cytoskeleton rearrangement. PP2A overexpression hindered or at least dramatically reduced thrombin‐ or nocodazole‐induced F‐actin stress fiber formation and microtubule (MT) dissolution. Accordingly, it also attenuated thrombin‐ or nocodazole‐induced decrease in transendothelial electrical resistance indicative of barrier protection. Inhibition of PP2A by okadaic acid abolished its effect on agonist‐induced changes in EC cytoskeleton; this indicates a critical role of PP2A activity in EC cytoskeletal maintenance. The overexpression of PP2A significantly attenuated thrombin‐ or nocodazole‐induced phosphorylation of HSP27 and tau, two cytoskeletal proteins, which potentially could be involved in agonist‐induced cytoskeletal rearrangement and in the increase of permeability. PP2A‐mediated dephosphorylation of HSP27 and tau correlated with PP2A‐induced preservation of EC cytoskeleton and barrier maintenance. Collectively, our observations clearly demonstrate the crucial role of PP2A in EC barrier protection. J. Cell. Biochem. 98: 931–953, 2006.

Heparin inhibits the activity of protein phosphatase-1.

Heparin inhibited the dephosphorylation of rabbit skeletal muscle or liver phosphorylase a by protein phosphatase‐1. Other glycosaminoglycans (chondroitin sulfates) and their constituents were found to be without effect. The chromatography of a partially purified phosphatase preparation on heparin—Sepharose CL‐6B resulted in a fraction that did not bind to the matrix and its activity was not inhibited by heparin or inhibitor‐1. The phosphatase bound to heparin—Sepharose was eluted by 0.2 M NaCl and was inhibited by heparin or inhibitor‐1.

Synthesis of 1-(d-glucopyranosyl)-1,2,3-triazoles and their evaluation as glycogen phosphorylase inhibitors

1-(D-Glucopyranosyl)-1,2,3-triazoles were prepared from per-O-acetylated alpha- and beta-D-glucopyranosyl azides as well as per-O-benzoylated (beta-D-gluco-hept-2-ulopyranosylazide)onamide and onic acid methylester by using azide-alkyne cycloaddition catalysed by in situ generated Cu(I) under aqueous conditions. The O-acyl protecting groups were removed by the Zemplén protocol. The test compounds were assayed against rabbit muscle glycogen phosphorylase b to show that the beta-D-glucopyranosyl derivatives were superior inhibitors as compared to the two other series of triazoles.