First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway. First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway.

The nicotinamide riboside (NR) form of vitamin B3and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B3are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD+decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD+levels. BHB is naturally produced by the body when individuals fast or consume a ketogenic (KD) or calorically restricted (CR) diet. These diets have been shown to extend lifespan in mice, while they are also protective in many disease models. Caenorhabditis elegans, a roundworm with a short mean lifespan of roughly 2 to 3 weeks depending upon the temperature, is used as a model system to study aging. BHB has been previously shown to increase lifespan by roughly 20% when administered to C. elegans.We administered NR and BHB individually and together to C. elegans starting at two different developmental stages (larval stages 1 and 4) and measured lifespan. We found that administration of 20 mM DL-BHB decreased lifespan when first given at the L1 stage, while it robustly increased lifespan when first given at the L4 stage. Administration of 0.5 mM NR increased lifespan when first given at L1, with only a very slight increase when first given at L4. When initiating administration at L1, NR greatly mitigated the BHB-mediated decline in longevity, however, NR did not increase BHB-mediated lifespan extension when first administered at L4.

Environmental conditions imposed onto organisms during certain phases of their life cycles such as embryogenesis or puberty can not only impact the organisms’ own health, but also affect subsequent generations. The underlying mechanisms causing intergenerational phenotypes are not encoded in the genome, but the result of reversible epigenetic modifications. This work investigates in a mouse model the impact of paternal nicotine exposure on the next generation regarding addictive behavior modulation, metabolic changes, and molecular mechanisms. It provides evidence that male offspring from nicotine-exposed fathers (NIC offspring) is more resistant to lethal doses of nicotine. This phenotype is gender-specific and depends on short-term environmental challenges with low doses of nicotine prior to the LD50 application. The observed survival phenotype is not restricted to nicotine as drug of abuse, but also presents itself, when NIC offspring is challenged with a cocaine LD50 after acclimatization to low doses of either nicotine or cocaine. Functionally, NIC offspring metabolizes nicotine faster than control. Mechanistically, NIC offspring livers show global up-regulation of xenobiotic processing genes (XPG), an effect that is even more pronounced in primary hepatocyte cultures. Being known targets of Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR), these XPGs show higher baseline expression in naïve NIC offspring livers. Nicotine’s action on the brain’s reward circuitry does not appear to be of biological significance in our model system. Taken together, paternal nicotine exposure leads to a non-specific and conditional phenotype in male NIC offspring that may provide a general survival advantage against xenobiotic challenges.

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.

KCNE1 (E1) peptide is the founding member of the KCNE family (1-5), which is a class of type I transmembrane ß-subunits. KCNE1 peptides assemble with and modulate the gating, ion conducting properties and pharmacology of a variety of voltage-gated K+ channel a-subunits, including KCNQ1 (Q1). Mutations that interfere with the function of either E1 and/or Q1 and disrupt the assembly and trafficking of KCNE1- KCNQ1 channel complexes give rise to diseases such as Romano-Ward (RW) and Jervell Lange Nielsen Syndrome (JLNS), two different forms of Long QT Syndrome (LQTS). Using enzymatic deglycosylation assays, immunofluorescence techniques and quantitative cell surface labeling, we showed that KCNE1 peptides are retained in the early stages of the secretory pathway as immaturely N-linked glycosylated proteins. KCNE1 co-assembly with KCNQ1 leads to E1 progression through the secretory pathway and glycan maturation, resulting in cell surface expression. N-linked glycosylation of some membrane proteins is critical for proper folding, co-assembly and subsequent trafficking through the biosynthetic pathway. Previous studies have shown that genetic mutations that disrupt one of the two N-linked glycosylation sites on KCNE family members lead to LQTS (T7I, KCNE1 and T8A, KCNE2) (Schulze-Bahr et al., 1997; Sesti et al., 2000a; Park et al., 2003). Having confirmed that KCNE1 proteins acquire N-linked glycans, we examined the kinetics and efficiency of N-linked glycan addition to KCNE1. We showed that KCNE1 has two distinct N-linked glycosylation sites. The N-terminal sequon is a traditional co-translational site. The internal sequon (which is only ~ 20 residues away from the N-terminal sequon) acquires N-linked glycans primarily after protein synthesis (post-translationally). Surprisingly, mutations that prevent N-glycosylation at the cotranslational site also reduce the glycosylation efficiency of post-translational glycosylation at the internal sequon, resulting in a large population of unglycosylated KCNE1 peptides that are retained in the early stages of the secretory pathway and do not reach the cell surface with their cognate K+ channel. We showed that KCNE1 post-translational N-glycosylation in the endoplasmic reticulum is a cellular mechanism that ensures E1 proteins acquire the maximal number of glycans needed for proper channel assembly and trafficking. Our findings provide a new biogenic mechanism for human disease by showing that the JLNS mutation, T7I, not only inhibits glycosylation of the N-terminal sequon, but also indirectly prevents the glycosylation of the internal sequon, giving rise to a large population of assembly incompetent hypoglycosylated KCNE1 peptides. To further investigate the two N-linked glycosylation sites on KCNE1, we generated structure-function deletion scans of KCNE1 and performed positional glycosylation scanning mutagenesis. We examined the glycosylation pattern of glycosylation mutants in an effort to define the glycosylation window important for proper KCNE1 assembly and trafficking. Our findings suggested a nine amino acid periodicity to serve as a desirable glycosylation site and a better substrate for N-glycosylation. Appendix II shows work on the characterization of the C-terminally HA-tagged KCNE1 protein, which was used throughout the experiments presented in Chapter II, Chapter III and Chapter IV. Analysis of the C-terminally HA-tagged KCNE1 protein revealed that in heterologous expression systems KCNE1 had an internal translational start site, a methionine at position 27. A proteolytic cleavage site was also identified at the arginine cluster spanning residues 32 through 38 bearing the two known Long QT mutations (R32H and R36H) (Splawski et al., 2000; Napolitano et al., 2005). My work in Professor Craig C. Mello’s lab during the first four years of my graduate study is presented in Appendix I. The highly conserved Wnt/Wingless glycoproteins regulate many aspects of animal development. Wnt signaling specifies endoderm fate by controlling the fate of EMS blastomere daughters in 4-cell stage Caenorhabditis elegans embryos. A suppressor genetic screen was performed using two temperature sensitive alleles of mom-2/Wnt to identify additional regulators of the Wnt/Wingless signaling pathway during C. elegans endoderm specification. Five intragenic suppressors and three extragenic suppressors of mom-2/Wnt embryonic lethality were identified. We cloned ifg-1, eIF4G homologue, as one of the extragenic suppressors suggesting an intriguing connection between the Wnt signaling pathway and the translational machinery.

In multicellular organisms, determining when and where genes will be expressed is critical for their development and physiology. Transcription factors (TFs) are major specifiers of differential gene expression. By establishing physical contacts with the regulatory elements of their target genes, TFs often determine whether the target genes will be expressed or not. These physical and/or regulatory TF-DNA interactions can be modeled into gene regulatory networks (GRNs), which provide a systems-level view of differential gene expression. Thus far, much of the GRN delineation efforts focused on metazoan development, whereas the organization of GRNs that pertain to systems physiology remains mostly unexplored. My work has focused on delineating the first gene regulatory network of the nematode Caenorhabditis elegans metabolic genes, and investigating how this network relates to the energy homeostasis of the nematode. The resulting metabolic GRN consists of ~70 metabolic genes, 100 TFs and more than 500 protein–DNA interactions. It also includes novel protein-protein interactions involving the metabolic transcriptional cofactor MDT-15 and several TFs that occur in the metabolic GRN. On a global level, we found that the metabolic GRN is enriched for nuclear hormone receptors (NHRs). NHRs form a special class of TFs that can interact with diffusible biomolecules and are well-known regulators of lipid metabolism in other organisms, including humans. Interestingly, NHRs comprise the largest family of TFs in nematodes; the C. elegans genome encodes 284 NHRs, most of which are uncharacterized. In our study, we show that the C. elegans NHRs that we retrieved in the metabolic GRN organize into network modules, and that most of these NHRs function to maintain lipid homeostasis in the nematode. Network modularity has been proposed to facilitate rapid and robust changes in gene expression. Our results suggest that the C. elegans metabolic GRN may have evolved by combining NHR family expansion with the specific modular wiring of NHRs to enable the rapid adaptation of the animal to different environmental cues.

Breast cancer is the most common malignancy among women in the U.S. While many successful treatments exist for primary breast cancer, very few are available for patients with metastatic disease. The purpose of this study was to understand the role of Insulin Receptor Subtrate-2 (IRS-2) in breast cancer metastasis. IRS-2 belongs to the IRS family of cytoplasmic adaptor proteins that mediate signaling from cell surface receptors, many of which have been implicated in cancer. Although the IRS proteins are highly homologous in structure and have some complementary functions, growing evidence supports that the IRS proteins have unique roles in cancer. IRS-1 has been shown to promote tumor cell proliferation, while IRS-2 has been positively associated with cancer cell invasion, glycolysis and tumor metastasis. In the current work, we identified IRS-2 as a novel hypoxia-responsive gene in breast carcinoma cells. In contrast, IRS-1 expression does not increase in response to hypoxia, supporting the notion of their non-overlapping functions. Hypoxia promotes the adaptation and resistance of cancer cells to chemo- and radiation therapy, and also promotes tumor cell survival, invasion and metastasis by selecting for aggressive tumor cells that can survive under stressful low oxygen conditions. We have shown that IRS-2 upregulation in response to hypoxia promotes Akt signaling and tumor cell viability and invasion. We identified a cell context-dependent role for Hypoxia Inducible Factor (HIF) in the regulation of IRS-2 expression in hypoxia, with HIF-2 playing a more dominant role than HIF-1. We also demonstrate that binding of Snail, a regulator of the EMT, to the IRS-2 promoter keeps the chromatin in an open conformation that is permissive for HIF-dependent transcription of IRS-2 in hypoxia. IRS-2 is not upregulated by hypoxia in well-differentiated epithelial-like carcinoma cells that do not express Snail, implicating IRS-2 gene expression as part of the EMT programming. In summary, we have identified an endogenous mechanism by which cancer cells can shift the balance of IRS-1 and IRS-2 to favor IRS-2 expression and function, which promotes survival, invasion, and ultimately metastasis. Understanding the mechanism of IRS-2 regulation by hypoxia may reveal new therapeutic targets for metastatic breast cancer.

Human papillomavirus infection is the cause of more than 99% of cervical cancer cases. The current vaccine is ineffective therapeutically; highlighting the need for continued papillomavirus research. One avenue that could be explored in this regard is the function of the papillomavirus E2 regulatory proteins. HPV E2 represses expression of the viral E6 and E7 oncoproteins. Reintroduction of E2 into cervical carcinoma cells results in growth arrest and cellular senescence. Understanding the mechanism of how E2 regulates the early promoter may be key to developing new therapeutic and prophylactic vaccines. Here, we describe regulation of E2 through acetylation and possibly through direct interaction with a novel cellular interacting protein, RINT1. Histone acetyltransferase (HAT) proteins have been demonstrated to interact with Bovine Papillomavirus (BPV) and Human Papillomavirus (HPV) E2 proteins as well as enhance E2 dependant transcription luciferase reporter plasmid containing E2 binding sites. We demonstrate that HATs p300, CBP, and pCAF are limiting for E2 dependant transcriptional activation and that each protein functions independently. We have also identified that BPV-1 E2 is a substrate for acetylation by p300. Mutants of E2 that cannot be acetylated on lysines 111 or 112, display abnormal transcriptional phenotypes. Cells deficient in p300 display similar transcriptional defects that are intensified by CBP depletion. We propose that acetylation of BPV-1 E2 is necessary for transcriptional activation. Acetylation generates a binding site through which a co-factor may interact via a bromodomain. Regulation of E2 dependent transcriptional activation through a post-transcriptional modification represents a novel method through which BPV-1 controls gene expression. We also present evidence for a direct interaction between BPV-1 E2 and the cellular factor RINT1. This interaction does not appear to be critical for transcriptional regulation; however, several other functional pathways are indicated by the cellular complexes in which RINT1 functions. Some of these, such as ER/Golgi vesicular transport and hTERT independent telomere maintenance, are pathways in which E2 has no known role. Further investigation into regulation and consequences of E2 acetylation and the biological significance of the interaction between E2 and RINT1 could prove important in understanding the complex role of E2 in papillomavirus infection.

Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to traditional, synthetic organic chemistry alkylation protocols. The full potential of this technology is stifled by several impediments largely deriving from the AdoMet-based reagents, including the instability, membrane impermeability, poor synthetic yield and resulting diastereomeric mixtures. To circumvent these main liabilities, novel chemoenzymatic strategies that employ methionine adenosyltransferases (MATs) and methionine (Met) analogs to synthesize AdoMet analogs in vitro were advanced. Unstable AdoMet analogs are simultaneously utilized in a one-pot reaction by MTs for the alkylrandomization of NP scaffolds. As cell membranes are permeable to Met analogs, this also sets the stage for cell-based and, potentially, in vivo applications. In order to further address the instability of AdoMet and analogs thereof, MAT-catalyzed reactions utilizing Met and ATP isosteres generated highly stable AdoMet isosteres that were capable of downstream utilization by MTs. Finally, the development, use, and results of a high-throughput screen identified mutant-MAT/Met-analog pairs suitable for postliminary bioorthogonal applications.

Liver disease is now a leading cause of non-AIDS related morbidity and mortality in people living with HIV (PLWH). The present study investigated the interplay between adverse lifestyle factors that are prevalent in PLWH, biological mediators of liver pathogenesis, and a non-invasive measure of liver fibrosis (FIB-4 index) in HIV mono- and HIV/HCV co-infected individuals. The results of this investigation in the Miami Adult Studies of HIV (MASH) cohort show that the odds of liver fibrosis progression significantly increased over two years for HIV mono-infected participants who drank alcohol hazardously (OR 3.038, P=0.048), and had BMI ≥ 28kg/m2 (OR 2.934, P=0.027). Cocaine use reduced the odds of advancing one stage of liver fibrosis (OR 0.228, P=0.038), but an interaction between high BMI and cocaine use slightly raised the odds by 4.8% of liver fibrosis progression (P=0.072). HIV/HCV co-infected participants showed interactions between cocaine use and high BMI with increased FIB-4 stage (OR 4.985, P= 0.034), however no lifestyle factors could independently predict FIB-4 stage in this group. Biological mediators previously associated with liver pathogenesis were associated with higher FIB-4 index over 2 years in a subset of (n=65) HIV mono-infected participants. Plasma measures of oxidative stress (% oxidized glutathione: OR 4.342, P= 0.046), hepatocyte-specific apoptosis (Cytokeratin-18 (CK-18): OR 1.008, P=0.021), and microbial endotoxin (lipopolysaccharide (LPS): OR 1.098, P= 0.097) were associated with having higher odds of progressing at least one stage of FIB-4 over 2 years. The same biological mediators were also associated with liver fibrosis within HIV infected people who also had a harmful lifestyle characteristic. FIB-4 index was significantly associated with % oxidized glutathione in obese subjects (β=0.563, P=0.018), TGF-β1 in cocaine users (β=0.858, P=0.027), and CK-18 in HIV infected individuals without any adverse lifestyle factors (β=0.435, P=0.015). Taken together, the findings of these studies describe interrelationships between HIV disease status, lifestyle, and biological mediators of liver fibrosis. The results show interactions between lifestyle conditions and the mediators of liver fibrosis may account for higher rates of liver disease in HIV infection. Research is warranted to develop personalized therapeutics for PLWH to curb the burden of liver disease.