Maintaining the balance between dynamic DNA methylation and demethylation is crucial to mammalian development and pathogenesis. In vitro methylation at the C-5 position of cytosine enhances cyclobutane pyrimidine dimer (CPD) formation and promotes transition mutations. While the loss of 5-hydroxymethylcytosine (5hmC) and inactivation of the ten-eleven translocation (TET) family have been implicated in cancers, and repeated exposure to UV radiation is a known risk factor for developing skin cancers, the link between DNA demethylation and UV damage has not yet been illustrated. We report that hydroxylation and carboxylation of 5-methylcytosine mitigate methylation-induced CPD enhancement upon UV irradiation. However, 5hmC also increases UV induction of (6-4) photoproducts. In a melanoma cell model, this duality by 5hmC in modulating the UV response is accentuated through TET2 overexpression. These findings implicate the DNA demethylation intermediates 5-hydroxymethylcytosine (5hmC) and 5-carboxylcytosine (5caC) as selective epigenetic shields against UV induction of DNA photoproducts.

<p>The estrogen-related receptor alpha (ERR&alpha;) is an orphan nuclear receptor (NR) with no natural ligand identified. Recent studies report that ERR&alpha; expression and activity correlate with poor prognosis in breast cancer. It is also suggested that ERR&alpha; is involved in tumor growth and progression, thus this receptor may be a therapeutic target in the treatment of breast cancer. However, the specific role of ERR&alpha; in breast cancer is not fully understood. Similar to other nuclear receptors, ERR has been suggested to regulate target gene transcription through both classical (direct DNA binding) and non-canonical (tethering mechanisms) to effect various aspects of tumor pathogenesis, such as angiogenesis, regulation of hypoxic response, tumor growth, and migration. Thus, the objective of this dissertation research is to explore the roles of ERR&alpha; in breast cancer by (a) identifying novel ERR&alpha; target genes important for tumor pathogenesis, (b) characterizing the molecular mechanism of non-canonical actions of ERR&alpha;-mediated gene transcription, and (c) examining the structure basis of ERR&alpha; antagonism for future pharmaceutical exploitation. First, we identified an ERR&alpha; target gene, ECM1, which is relevant to breast cancer angiogenesis. The role of ECM1 in angiogenesis was confirmed by endothelial tube formation assay. We further showed that knocking down ECM1 has a dramatic inhibitory effect on tumor xenograft growth. This result, for the first time, directly demonstrates the role of ECM1 in tumor environment and further sheds light on the significance of ERR&alpha-regulated genes in tumors angiogenesis. Next, we explored the molecular mechanism of ERR&alpha; non-canonical pathways using transcriptional reporter assay and ERR&alpha; DNA-binding domain (DBD) mutants. We discovered that the expression of carbonic anhydrase 9 (CA9), a target gene of one of the ERR&alpha; tethering partner hypoxia inducible factor-1 (HIF-1), does not require direct binding of ERR&alpha; to DNA but its DBD is indispensible. These results reflect on the importance of ERR&alpha; DBD even in the non-canonical signaling of ERR&alpha;, which brings challenges to dissecting ERR&alpha; canonical/non-canonical pathways in the future. Finally, to determine the molecular mechanisms underlying ERR&alpha; antagonism, we probed the conformations of ERR&alpha; upon antagonist treatments. M13 phage display was used to screen for ERR&alpha;-interacting peptides. We identified peptides that interact with ERR&alpha; in the activation function 2 (AF2) domain, some of which are able to distinguish the binding of different classes of ERR&alpha; antagonists. Cumulatively, these studies have explored the biological functions of ERR&alpha; and the molecular basis ERR&alpha;-mediated signaling pathways.</p> / Dissertation

Queuosine (Q) is a hypermodified nucleoside present at the wobble position in the 5'-GUN-3' anticodon loop of asparagine, aspartic acid, histidine, and tyrosine encoding tRNAs. This hypermodified base contains a 7-deazapurine structure common to many antibiotics, antivirals, and antineoplastic secondary metabolites. It is synthesized de novo in prokaryotes from GTP, whereas in eukaryotes it is ingested from dietary sources as the free-base queuine and exchanged for guanine in mature tRNA. Queuosine has been associated with many physiological phenomena such as cancer pathology, neoplasia, and virulence; although a discrete physiological relevance of this modification remains to be determined due to the lack of observable phenotypes associated with its respective loss. However, conservation of this modification across almost all domains of life suggests that it confers a selective advantage to its host. CPH₄ synthase (QueD) catalyzes the second step in the queuosine biosynthetic pathway entailing conversion of 7,8-dihydroneopterin triphosphate to 6-carboxy-5,6,7,8-tetrahydropterin. By contrast, the almost structurally identical mammalian homolog catalyzes the conversion of the same substrate to 6-pyruvoyltetrahydropterin, which is an intermediate in the tetrahydrobiopterin biosynthetic pathway. Herein, we present multiple X-ray crystal structures coupled with biochemical studies that reveal an additional active site catalytic dyad in QueD responsible for the differing activity. Prior to the studies detailed in this dissertation, the enzyme responsible for catalyzing the final step in the pathway, conversion of epoxyqueuosine to queuosine, had yet to be elucidated. To search for this enzyme, we screened a library of isogenic variants of Escherichia coli where all of the nonessential genes are sequentially inactivated. RNA was extracted from each strain and analyzed using LC-MS methods, which led to the identification of a mutant strain that accumulates epoxyqueuosine and contains no queuosine. The enzyme, epoxyqueuosine reductase (QueG), has been subjected to extensive biochemical analyses both in vitro and in vivo. From these studies, we have shown QueG to contain two [4Fe-4S] clusters and cobalamin as cofactors that are absolutely required for catalysis. In addition, we have identified conserved residues that affect activity and modulate the coordination sphere around the cobalamin cofactor.

Increased emergence of antibiotic resistance in bacterial pathogens has posed a serious threat to human health. Due to similar structural and functional characteristics of metal and antibiotic resistance systems in gram-negative bacteria, there is a growing concern that metal contamination functions as a selective agent in the proliferation of antibiotic resistance. The CusCFBA copper/silver resistance system of Escherichia coli forms a protein complex that spans the inner and outer membranes and functions in the efflux of metal from the periplasm to the extracellular space. In order to understand the molecular details of metal resistance by the Cus system and more specifically, to define the role of the periplasmic components in CBA type metal transporters, I characterized CusB and probed its interactions with CusF using various structural and biochemical tools. CusB was previously thought to play a relatively passive role as an adaptor protein that stabilized the association of the inner and outer membrane proteins. Through isothermal titration calorimetry (ITC), X-ray absorption spectroscopy (XAS), and mutagenesis, I have shown that CusB binds Cu(I)/Ag(I) with high affinity using three conserved methionines. Gel filtration chromatography experiments showed that upon binding Ag(I), CusB undergoes a substantial conformational change. Importantly, functional metal binding by CusB is essential for cell survival in environments with elevated copper concentrations. The small periplasmic metal binding protein CusF is a unique component of monovalent metal resistance systems serving an unknown function. To determine the nature and specificity of interaction between CusF and CusB, ITC and NMR were used to show that the interaction between CusF and CusB is metal-dependent and specific for the components of Cus system. From NMR chemical shift perturbations, the CusB interaction face on CusF was determined to overlap with the metal binding site. XAS experiments demonstrate metal transfer between CusB and CusF, which supports the role of CusF as a metallochaperone. In summary, these findings demonstrate an active role for CusB in metal resistance, and suggest that the plausible role for CusF is that of a metallochaperone for CusB.

Presented in this dissertation are proteomic analysis studies focused on identifying proteins to be used as vaccine candidates against Coccidioidomycosis, a potentially fatal human pulmonary disease caused by inhalation of a spore from the soil-dwelling pathogenic fungi Coccidioides posadasii and C. immitis. A method of tandem mass spectrometry data analysis using dual protein sequence search algorithms for increasing the total protein identifications from an analysis is described. This method was utilized in a comprehensive proteomic analysis of cell walls isolated from the dimorphic fungal pathogen C. posadasii. A strategy of tandem mass spectrometry-based protein identification coupled with bioinformatic sequence analysis was used to produce a list of protein vaccine candidates for further testing. A differential proteome analysis using stable isotope protein labeling was undertaken to identify vaccine candidate proteins that are more highly expressed in the spherule, or pathogenic phase, of C. posadasii. The results of these analyses are 9 previously undescribed protein vaccine candidates isolated from spherule cell walls that have sequence indications of extracellular association such as GPI anchors and N-terminal signal sequences and antigen potential based on homology to known antigenic or secreted proteins. An additional 14 proteins identified from spherule cell walls are potential vaccine candidates based on extracellular sequence predictions without any indications of antigenic potential. The stable isotope labeling study has identified 3 more proteins that are preferentially expressed in spherules and exhibit antigenic potential based on extracellular localization or homology to known antigenic proteins. Additionally, there were 5 unknown function proteins identified by stable isotope labeling that are more highly expressed in spherules that may be good vaccine candidates but cannot be identified or localized by sequence analysis.The dual algorithm protein identification method presented here is a new technique to address some common shortcomings associated with a proteomic analysis. The comprehensive proteomic analyses of Coccidioides posadasii presented here have provided new targets for Coccidioidomycosis vaccine development as well as insights into the proteome of this pathogen, such as the sequence comparison of C.posadasii proteins to human proteins, as well as a comprehensive analysis of predicted protein function in the Coccidioides proteome.

Insects such as Manduca sexta must be efficient in obtaining energy stores in order to survive. The main goal of the M. sexta larva is to eat and store enough energy to reach the adult stage, produce eggs (in the case of females), and reproduce. Triacylglycerol (TAG), the most important energy storage molecule, is stored mainly in adipose tissues in lipid droplets, although other tissues are also able to store TAG in similar organelle structures but to a lesser degree. The phosphatidic acid (PA) pathway and the 2-monoacylglycerol (MAG) pathway are both energy-dependant acyl-CoA processes and are the main synthetic pathways by which TAG is synthesized in adipose and other tissues. My research led to the discovery of an energy-independent pathway for the synthesis of TAG that was present in the M. sexta midgut. Based on partial purification, a transacylase/lipase enzyme is present and responsible for DAG and TAG synthesis in the M.sexta midgut. Lipogenesis and lipolysis in adipose tissue involves a series of enzymes. Adiponutrin and desnutrin, two proteins involved in fat homeostasis in humans and mice, have received a lot of attention since their activities are dependent on the fed or unfed state of the animal. In this study, bioinformatics analyses were performed, which allowed the identification of an insect gene that has an ortholog in human and mice that plays an important role in adipose tissue TAG hydrolysis and synthesis. Only one insect gene ortholog was found to be present in the Aedes aegypti (mosquito), Drosophila melanogaster (fruit fly), Anopheles gambiae (mosquito), Bombyx mori (silk worm), and Tribolium castaneum (red flour beetle) genomes corresponding to genes involved in the regulation of TAG metabolism in mice (adiponutrin, desnutrin) and humans (iPLA-epsilon, iPLA-zeta, and iPLA-eta). Expression of the M. sexta calcium-independent phospholipase A2 (iPLA) ortholog has demonstrated that the protein is able to transfer acyl groups between MAGs in an energy-independent manner, similar to that in human iPLAs. This is the first example of a transacylase identified in insects.

A study of the metabolism of steroid hormones is beset with many difficulties if the usual biochemical methods are to be applied. One of the most important difficulties, perhaps, is that the quantities of material present in animal tissues are usually extremely small. With the availability of radioactive isotopes, the problem was considerably simplified. In fact, if a steroidal hormone is made radioactive, it becomes a simple matter to determine its distribution in tissues and therefore much easier to obtain extracts indicative of its metabolic pathway.

An extensive investigation of blood preservation, plasma 'substitutes' and the survival of blood cells after transfusion has been carried out in the Department of Biochemistry, McGill University, since the first months of World War II, under the direction of Dr. O.F. Denstedt and with the financial support of the National Research Council of Canada and, more recently, of the Defence Research Board of Canada.

Cancer has developed into one of the most complex of biological problems and research into its causes, development, manifestations and control is the focal point of a large number of scientific disciplines. It is unlikely that any investigator can completely confine his work or interest within the boundaries of only one of its component disciplines. Cowdry, in a discussion on modern trends in cancer research, has remarked "it is said that in cancer the most productive research is between the sciences, as in the cell itself very fundamental processes take place at the surfaces between different materials". [...]

The experiments to be presented consist of an attempt to extend the observations of Dickens and Greville on the effects of anoxia and lack of substrate on the subsequent carbohydrate metabolism of brain tissue. Dickens and Greville (1933) working with brain slices, and Elliott and Henry (1946) with suspensions, showed that deprivation of both oxygen and substrate for relatively short periods caused a marked inhibition of the subsequent anaerobic glycolysis, while respiration was inhibited to a lesser degree.