O4. Nitric oxide and metalloproteinases: An intricate relationship

Nitric Oxide 19 (2008) S20–S42

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Nitric Oxide journal homepage: www.elsevier.com/locate/yniox

Oral abstracts Plenary talks O1. Bioactivation of nitroglycerin by aldehyde dehydrogenases: New insights into the molecular mechanism of nitric oxide formation and the development of nitrate tolerance Bernd Mayer Pharmacology and Toxicology, Karl-Franzens-University Graz The organic nitrate glyceryl trinitrate (nitroglycerin; GTN) is used clinically to treat coronary artery disease, myocardial infarction and congestive heart failure. The therapeutic benefit of the drug is due to dilation of large coronary arteries and veins. GTNinduced vasodilation is mediated by release of nitric oxide (NO), which activates soluble guanylate cyclase (sGC) in vascular smooth muscle. Metabolism of GTN by mitochondrial aldehyde dehydrogenase (ALDH2), yielding 1,2-GDN and nitrite, is thought to be a key pathway of vascular GTN bioactivation in mammalian blood vessels. In search for a link between ALDH2-catalyzed GTN biotranformation and sGC activation we found that purified ALDH2 (and cytosolic ALDH1) catalyze direct 3-electron reduction of GTN to NO. NO formation was sensitive to substrate-competitive ALDH inhibitors and abolished upon mutation of the essential cysteine residue in ALDH2 (C302), suggesting that the reaction involves nucleophilic attack of GTN at C302 which is essential for all ALDH2 activities known so far. Site-directed mutagenesis of ALDH2 further indicated that clearance-based GTN denitration (yielding nitrite) and bioactivation (yielding NO) are two independent reactions, whereby clearance-based GTN denitration appears to counteract sGC activation by GTN-derived NO due to formation of superoxide. The implications of these findings for GTN bioactivation and nitrate tolerance will be discussed.

O2. What can chemistry tell us about the biology of nitric oxide? A personal perspective from a 30 year relationship Jack Lancaster Jr. Center for Free Radical Biology, U. Alabama Birmingham Nitric oxide (NO, nitrogen monoxide) has a long and distinguished history in Chemistry and Biochemistry. Its chemical and physical properties have been comprehensively delineated, and this extensive body of knowledge has provided a wealth of insight into the newly-discovered biological actions of NO. Although (contrary to some common perceptions) the reactivity of NO is limited, the immediate products of its reactions in the biological milieu spawn a complex and interacting network of more highly reactive nitrogen species. Being a radical, the dominant determinant in the reactions of NO is the stabilization of its unpaired electron. Thus, the virtually exclusive reactants biologically are (1) transition metals (which stabilize unpaired electrons through coordinate interactions with the d-orbitals of the metal) and (2) other radical species, resulting in pairing of the electron. I will provide a basic tutorial of these principles for the nonchemist, and provide examples of the utility of these insights into two major cellular actions of NO, formation of iron nitrosyl species and formation of protein nitrosothiols (and a surprising mechanistic connection between the two). doi:10.1016/j.niox.2008.06.003

doi:10.1016/j.niox.2008.06.002

Interaction of NO with Proteins O3. Redox regulation of protein kinases A and G in the cardiovascular system Philip Eaton Cardiology, King’s College London There are many mechanisms by which proteins can sense and transduce oxidant signals in cells and tissues. To identify proteins that act as redox sensors in the cardiovascular system we have undertaken several different proteomic screens, looking for proteins that become oxidised at cysteinyl thiols under oxidising conditions. A variety of different screens were needed, as thiols can be oxidised in many different ways, each needing specific methods to identify them. We have identified many proteins that are redox active, but were intrigued to find that both protein kinases A and G were oxidant modified. Specifically the RI subunit of PKA and the 1a isoform of PKG were oxidised at their redox active thiols. These kinases are particularly important in the cardiovascular system, playing crucial roles in the regulation of blood pressure and cardiac output. Both these proteins formed interprotein disulphide bonds in response to oxidant stress involving treatment with hydrogen peroxide. This activated both these kinases, and for PKG we proved that this activation was independent of the classical nitric oxide/cGMP pathway. We could prevent the peroxide activation of PKG by mutating its redox Cysteine-42 to a charge-conserved serine, which prevented disulphide formation. Disulphide formation in PKG increased the kinases affinity for its substrates, causing relocalisation of cytosolic PKG1alpha to the membrane or myofilaments compartments (i.e. sites where its substrates are located).

When PKG arrived at these locations it phosphorylated its substrates, which culminated in a vasorelaxation. These events explains how hydrogen peroxide can act as an endothelium-derived hyperpolarizing factor (EDHF), providing a molecular basis for oxidant-mediated vasorelaxation with is independent of cGMP. doi:10.1016/j.niox.2008.06.004

O4. Nitric oxide and metalloproteinases: An intricate relationship Van der Vliet Albert, Peter Bove, Milena Hristova, Sean McCarthy, Nels Olson Department of Pathology, University of Vermont The production of nitric oxide (NO) within the respiratory tract is mediated by several isoforms of NO synthase (NOS) in a number of different cell types. The airway epithelium is an important source of NO, which largely originates from persistent expression of the inducible isoform NOS2, and epithelial NOS2 expression is typically enhanced during lung infection and inflammation, giving rise to increased airway NO production. In addition to serving functions in innate host defense, epithelial NOS2 has been postulated to regulate production of inflammatory mediators, as well as epithelial injury and regeneration. In vitro studies with tracheobronchial epithelial cells indicated that low nM concentrations of NO or basal NOS2 activity enhance epithelial

Oral abstracts / Nitric Oxide 19 (2008) S20–S42 cell migration, and assist in epithelial wound repair and barrier restoration upon injury. Complementary results were obtained using tracheal epithelial cells from NOS2/ mice. These stimulatory effects of NO are in part mediated by increased activation/expression of gelatinase B (MMP-9), an important mediator of epithelial repair and remodeling, and were fully dependent of cGMP-mediated pathways. Importantly, in spite of previous claims that NO may directly activate MMP-9, cellfree experiments showed that NO was unable to activate proMMP-9 at nM concentrations. In fact, high nM concentrations of NO representative of inflammatory conditions were found to inhibit MMP-9 activity, independent of oxidation or nitrosylation of the protein. Although NO under these conditions did not induce direct injury to tracheal epithelial monolayers, it impaired epithelial cell migration and wound repair in response to injury, which was related to suppression of epithelial MMP-9 expression and activity, activation of hypoxia-inducible factor 1a (HIF-1a)mediated signaling, and activation of the tumor suppressor p53. Collectively, our findings illustrate that NO has variable roles on epithelial injury and repair, depending on local NO concentrations and other factors that regulate NO bioavailability and catabolism. Moreover, our studies reveal various modes of NO-mediated regulation of MMP-9 expression and activation, which may have implications for NO-mediated regulation of other related metalloproteinases.

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keratinocytes upon treatment with nitric oxide donors such as DETA/NO, SPER/ NO and GSNO. Translocation is independent of NO-stimulated cGMP signaling. Rather, increased nuclear translocation of CLIC4 is coincident with increased Snitrosylation of the protein on specific cysteine residues(s) and enhanced association with proteins of the nuclear import machinery like Importin a, NTF2 and Ran. Cysteine ? alanine (or serine) point mutation at residue 35 decreases protein stability by proteasome-dependent degradation and shows significantly increased S-nitrosylation compared to wildtype CLIC4, even in untreated cells. Moreover, the mutant shows enhanced nuclear residence. Mutation of Cys ? Ala (or serine) at residue 234 inhibits nitrosylation suggesting it is vital for modification of the protein. Thus, both cysteine residues, close to each other in the crystal structure, show opposing effects on S-nitrosylation of CLIC4. These results suggest that CLIC4 nuclear translocation may depend on nitric oxide synthase(s) activity in the cell and S-nitrosylation may govern the level and/or intracellular distribution of the protein. Many tissues in a growth arrested state, in vivo, have nuclear CLIC4 while matched tumors exclude CLIC4 from the nucleus or show decreased levels of the channel protein. Thus, the changed redox state of tumor cells may account for the altered levels and subcellular distribution of CLIC4 in these cells. doi:10.1016/j.niox.2008.06.007

doi:10.1016/j.niox.2008.06.005

O5. Protein S-guanylation induced by 8-nitro-cGMP

O7. Nitration and oxidation of tryptophan 372 in mitochondrial enzyme succinylCoA: 3-ketoacid CoA transferase (SCOT) during aging

Takaaki Akaike Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University

Igor Rebrin, Catherine Bregere, Timothy K. Gallaher, Rajindar S. Sohal Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033, USA

We explored the physiological formation of 8-nitroguanosine 30 ,50 -cyclic monophosphate (8-nitro-cGMP) and its chemical biology in NO signal transductions. We previously reported NO-dependent guanine nitration in several types of cells [1–5]. Here we identified physiological formation and functions of 8-nitro-cGMP [6], which is the first demonstration of a new second messenger derived from cGMP in mammals since the discovery of cGMP more than 40 years ago. Using immunocytochemical methods, we confirmed 8-nitro-cGMP formation in cultured macrophages, hepatocyte-like cells, adipocytes, and endothelial cells, depending on NO production. We further verified 8-nitro-cGMP formation via HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP is a strong electrophile that reacts efficiently with sulfhydryls of proteins to generate a novel post-translational modification, which we call protein S-guanylation. It was found that specific intracellular proteins can readily undergo S-guanylation by 8-nitro-cGMP. 8-Nitro-cGMP regulates the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. More importantly, we determined that S-guanylation of Keap1 is involved in cytoprotective actions of NO and 8-nitro-cGMP, by inducing oxidative stress– response genes such as heme oxygenase-1. Our discovery of 8-nitro-cGMP and its unique chemical properties sheds light on new areas of NO and cGMP signal transduction. Protein S-guanylation induced by 8-nitro-cGMP thus may have important implications in NO-related physiology and pathology, pharmaceutical chemistry, and development of therapeutics for many diseases.

Purpose of this study was to identify targets and elucidate mechanisms of protein nitration in mitochondria during aging. Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT), the mitochondrial matrix enzyme involved in the breakdown of ketone bodies in the extrahepatic tissues, was identified in different rat tissues as a target of a novel, nitro-hydroxy, addition to tryptophan 372, located in close proximity (10 Å) of the enzyme active site. This post-translational modification was characterized using several proteomic approaches: western blot with anti-3-nitrotyrosine monoclonal antibody, HPLC-electrochemical detection of nitrohydroxytryptophan, matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization mass spectrometry (ESI–MS). Novel finding was that tryptophan, in contrast to tyrosine, was identified to be a specific target of simultaneous nitration and oxidation in vivo [1]. Nitrohydroxytryptophan formation was demonstrated after in vitro exposure of the synthetic peptide YGDLANWMIPGK to peroxynitrite. We hypothesize that increases in tryptophan nitration of SCOT and catalytic activity in old animals constitute a plausible mechanism for the age-related metabolic shift towards enhanced ketone body consumption by mitochondria as an alternative source of energy supply in the heart.

References [1] [2] [3] [4] [5] [6]

T. Akaike et al., Proc. Natl. Acad. Sci. USA 100 (2003) 685–690. J. Yoshitake et al., J. Virol. 78 (2004) 8709–8719. R. Yasuhara et al., Biochem. J. 389 (2005) 315–323. Y. Terasaki et al., Am. J. Respir. Crit. Care Med. 174 (2006) 665–673. T. Sawa et al., Free Radic. Biol. Med. 40 (2006) 711–720. T. Sawa et al., Nat. Chem. Biol. 3 (2007) 727–735.

doi:10.1016/j.niox.2008.06.006

O6. Nitric oxide directly regulates nuclear translocation of chloride intracellar channel CLIC4 Mariam Q. Malik, Wendy Niedelman, Jessica Lee, Stuart H. Yuspa Lab of Cancer Biology & Genetics, National Cancer Institute, Bethesda, MD, USA The intracellular chloride channel protein, CLIC4, localizes mainly to the cytosol of proliferating keratinocytes but translocates to the nucleus in response to diverse stress stimuli like etoposide, TNFa and LPS. Translocation causes growth arrest or apoptosis, depending on the level of CLIC4 expression. The channel activity and structure of CLIC4 are dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. We show that nuclear translocation of CLIC4 is induced in normal

Reference [1] I. Rebrin, C. Bregere, S. Kamsalov, T.K. Gallaher, R. Sohal, Nitration of tryptophan 372 in succinyl-CoA:3-ketoacid CoA transferase during aging in rat heart mitochondria, Biochemistry 46 (2007) 10130–10144. doi:10.1016/j.niox.2008.06.008

O8. NO-induced activation of a heme-sensor, eIF2a kinase, in association with binding to cysteine and heme Jotaro Igarashi, Motohiro Murase, Jun Iwashita, Takehiko Sasaki, Toru Shimizu IMRAM, Tohoku University, Sendai 980-8577, Japan Eukaryotic cells decrease their rates of protein synthesis to survive in emergency states, such as those caused by shortage of amino acids, irradiation with UV light, virus infection, and accumulation of denatured proteins. The decrease in protein synthesis is caused by phosphorylation of eukaryotic initiation factor 2a (eIF2a) at Ser51 by eIF2a kinases that respond specifically to these emergencies. Heme-regulated eIF2a kinase or heme-regulated inhibitor (HRI) is a member of the eIF2a kinase family that controls globin synthesis in response to the heme concentration in reticulocytes. At the appropriate heme concentrations under normal conditions, HRI function is suppressed. His119/His120 in the N-terminal region and Cys409 in the catalytic domain are the heme binding sites in HRI [1,2]. Conversely, upon heme iron shortage, HRI auto-phosphorylates, and subsequently phosphorylates eIF2a, leading to the termination of protein synthesis. NO significantly enhances the catalytic actions of both heme-bound and heme-free HRI enzymes. We demonstrate that NO-stimulated catalytic enhancement of the heme-bound form is exerted by the formation of a