Graduation Year

2018

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Stanley M. Stevens Jr, Ph.D.

Co-Major Professor

Brant Burkhardt, Ph.D.

Committee Member

Meera Nanjundan, Ph.D.

Committee Member

James Garey, Ph.D.

Keywords

Mass Spectrometry, Alcohol liver disease, Metabolic tracing, Tyrosine Nitration, Epigenetics, Histone, Acetylation

Abstract

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.

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