Nitric oxide (NO) is a short-lived free radical that plays a critical role in the regulation of cellular signalling. Mast cell (MC) derived NO and exogenous NO regulate MC activities including the inhibition of MC degranulation. At a molecular level the intermediate metabolites of NO modify protein structure and function through several mechanisms including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MC, we investigated protein tyrosine nitration in human mast cell lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione (SNOG). Using two dimensional gel western blot analysis with an anti-nitrotyrosine antibody together with mass spectroscopy we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MC. S-nitrosoglutathione treatment also reduced the Vmax of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance (NMR) analysis showed that despite these changes in activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that SNOG treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose-1,6-bisphosphate (FBP). Interestingly, FBP inhibited IgE-mediated MC degranulation and intracellular Ca2+ levels in LAD2 cells. In addition to aldolase, 15-hydroxy prostaglandin dehydrogenase (PGDH), a critical enzyme in the metabolism of PGE2, was identified as a prominent target for tyrosine nitration in LAD2 cells. Thus for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target in HMC-1 and LAD2; and PGDH in LAD2 cells. The post translational nitration of aldolase A and PGDH may be important pathways that regulate MC phenotype and function. / Experimental Medicine

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Alcohol liver disease (ALD) is a major health concern throughout the world. Currently, in the United States, 17 million people suffer from alcoholism, of which 1.4 million people are receiving treatment [1, 2]. The link between ethanol metabolism, reactive oxygen species (ROS) and liver injury in ALD has been well characterized over the last couple decades [3-10]. Ethanol metabolism relies on the availability of the cofactor NAD+ for the oxidation of ethanol into acetate, consequently causing alterations in redox potential. Redox dysfunction within the mitochondria can affect multiple pathways important in maintaining cellular homeostasis. Chapter 1 provides an introduction to the role of ethanol metabolism in oxidative stress and alcohol liver injury (ALI). During ethanol metabolism, both the cytochrome bc1 and NADH dehydrogenase complexes within the mitochondria have been demonstrated to be major contributors to ROS formation and “leak” free radicals [11-13]. As a result, the free radicals superoxide (O2-) and hydrogen peroxide (H2O2) is diffused into the cytoplasm where they can react with other molecules, proteins and DNA and cause tissue injury [4, 14]. Chapter 1 aims to introduce the link between ethanol metabolism and histone post-translational modifications (PTM) such as tyrosine nitration and lysine acetylation using proteomics techniques. Chapter 2 uses a global proteomic study to identify links between gender and ALI. A 10-day chronic-binge mouse model was employed in order to identify gender-specific proteins that may influence the development of ALD. It has previously been established that females are more susceptible to developing ALD, however, the cause is still unknown. This study identifies gender differences in the family of cytochrome P450 proteins using a mouse model for chronic-binge alcohol exposure. The cytochrome P450 family of proteins are important in the metabolism of toxic compounds, such as acetaldehyde, a byproduct of ethanol metabolism. Interestingly, I also identified that female mice expressed naturally higher levels of histone acetylation prior to alcohol exposure when compared to males. Following alcohol exposure, the female mice did not show much change in acetylation, whereas male acetylation levels were raised to similar levels of the female mice. These acetylation changes raised the question, how does alcohol influence epigenetic marks on histone proteins? Recently, new evidence has emerged that supports the role of epigenetics in the pathophysiology of ALD [4, 14-27]. Ethanol metabolism will promote shifts in redox potential and mitochondrial dysfunction, the result is the formation of reactive oxygen and/or nitrogen species (ROS/RNS) [4, 5, 7, 10, 14, 28]. As ethanol is metabolized, the accumulation of ROS/RNS species such as NO- and O2- can induce the post-translational modification nitrotyrosine. Shifts in redox potential will cause the electron transport chain to “leak” the free radical O2-. Another free radical known as nitric oxide (NO-) has been shown to be elevated during times of ethanol consumption [29, 30]. Traditionally, NO has a protective role within the cell at low concentrations, however, in surplus can lead to tissue damage. Ethanol-induced increases in NO- and O2- can instigate to peroxynitrite (ONOO-) formation; a potent oxidant and nitrating agent of tyrosine residues [29, 31-34]. Chapter 3 examines the indirect effect of alcohol metabolism and ROS/RNS formation on histone tyrosine nitration. This project used mass-spectrometry to identify novel targets of histone tyrosine nitration using a mouse-model of chronic-binge alcohol exposure. Interestingly, histone H3 was found to be nitrated on the hinge-region of the N-terminal tail at tyrosine 41. Molecular dynamics of the nitrated and unmodified proteoforms revealed that the DNA prefers a change in conformation upon H3Y41 nitration. Further studies using an antibody synthesized against the nitrated H3y41 region of the protein revealed potential targets within the genome important in fatty acid synthesis and metabolism. Chapter 4 looks at the direct influence of alcohol metabolism and its contribution to histone acetylation via acetate production and acetyl-CoA. Alcohol metabolism has traditionally been thought influence acetylation through the sirtuin family of deacetylase proteins. Sirtuin deacetylases are NAD+-dependent and have been shown to be a regulate protein acetylation within the mitochondria, cytoplasm, and nucleus during times of ethanol exposure [35-37]. Shifts in redox potential attributed to ethanol metabolism can inhibit sirtuin deacetylase activity by out-competing the enzymes for available NAD+, ultimately leading to mitochondrial and nuclear hyperacetylation [17, 28, 38-42]. Currently, there is evidence that ethanol increases acetylation of histone 3 lysine 9, which then targets activation of the alcohol dehydrogenase gene (ADH) [17, 18, 43]. Moreover, Shukla et.al. (2008) support the idea that ethanol can alter epigenetic transcriptional activation based on which modification is selected for a site during times of stress when it can be occupied by more than one modification [22]. Chapter 4 demonstrates the use of mass-spectrometry to metabolically trace 13C2-labeled ethanol in vivo. These new data show clear evidence of 13C2 heavy-labeled ethanol being incorporated into known sites of acetylation on the N-terminal tails of histone H3 and H4. Incorporation of heavy-label was calculated using extracted ion chromatograms (XIC) for the double and singly acetylated and unmodified peptides belonging to H3K9-R17 and H3K18-R23. Total change in acetylation was also assessed for each peptide using the ratio of ratios of total acetylation to unmodified peptide over the fold change in ethanol- to control-fed groups. An interesting observation was observed in that the incorporation of heavy-label suggests site-selectivity of lysine residues over time. Histone 4 contains multiple sites of acetylation on the peptide H4K5-R17, making it hard to quantify manually. MaxQuant evidence files in conjunction with R were used to calculate the 13C2 incorporation on the multiple H4 acetyl-sites over 24-hours. Ethanol-heavy label incorporation at multiple acetyl-sites occurred as a mixture suggesting a role in transcriptional regulation. These new data establish a link between alcohol metabolism and known epigenetic marks on histone proteins. These studies have now established that alcohol metabolism is indirectly linked to histone tyrosine nitration through increased ROS/RNS and directly through acetate production. Understanding how these epigenetic marks fluctuate as ALD progresses will provide potential targets for the development of new drug therapies. The epigenetic marks identified in these studies have previously been established to be important activators in transcription. These data provide novel techniques using proteomics-based metabolic tracing in vivo. Future studies will assess how these marks change after chronic ethanol exposure and whether the changes in epigenetics are heritable. Understanding hereditary of alcoholism will provide insight to those predisposed to the disease.

Reactive oxygen species (ROS) such as superoxide radical anion (O2•¯), hydroxylradical (OH•), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) that are produced during the metabolism of oxygen under oxidative stress in aerobic organisms destroy several key biomolecules and lead to a number of disease states. Mammalian systems possess several effective defense mechanisms including antioxidant enzymes to detoxify these ROS. The selenocysteine-containing Glutathione peroxidase (GPx) is particularly an efficient enzyme in the detoxification of H2O2 and other hydroperoxides by using glutathione (GSH) as cofactor. The chemistry at the active siteof GPx has been extensively investigated with the help of synthetic selenium compounds. Although the anti-inflammatory compound ebselen(2-phenyl-1,2-benzoisoselenazol-3(2H)-one) is undergoing phase III clinical trial as antioxidant, the chemistry of ebselen is still not understood. The present study on a number of ebselen derivatives with various N-substitutions reveals that the substitution at the N atom is important for the antioxidant activity. This study also suggests that the nature for thiol cofactor has a dramatic effect on the GPx activity of ebselen derivatives. It has been shown that ebselen exhibits very poor catalytic activity in the presence of aromatic thiols mainly due to strong Se….O nonbonded interactions that lead to extensive thiol exchange reactions in the selenenyl sulfide intermediate. To prevent the se….O interactions, a series of tertiary amide-based diselenides have been synthesized along with their secondary amide counterparts. Detailed structure-activity correlation studies reveal that the GPx-like activity of the sec-amide-based compounds can be significantly enhanced by the substitution at the free-NH group of sec-amide functionality. The N,N-dialkylbenzylamine-based diselenides exhibit their catalytic activities via the generation of selenols which was confirmed by the reaction with anti-arthritic gold(I) compounds. Interestingly, the replacement of the hydrogen atom at the 6th position of the benzene ring of N,N-dialkylbenzylamine-based diselenides by a methoxy group prevents the thiol exchange reactions mainly be weakening the Se…N interactions and thus enhances the GPx activity. On the other hand, the catalytic activity of the tert-amine-based diselenides can also be increased by replacing the tert-amino groups with the corresponding sec-amine moieties. It has been observed that the basic amino group in the amine-based diselenides deprotonates the selenol and also the thiol cofactor, which is crucial for the higher catalytic activities of the amine-based compounds. Peroxynitrite (PN, ONOO), a strong nitrating agent, is known to inactivate a number of proteins, enzymes and other biomolecules by nitration of tyrosine residues. In this study, we have shown that the commonly used antithyroid drugs and their analogues inhibit protein tyrosine nitration. This study reveals that antithyroid agents having PN scavenging activity may be beneficial of hyperthyroidism as these compounds may protect the thyroid gland from nitrative or nitrosative stress.