Thesis (Ph. D.)--University of Iowa, 2007. / Thesis supervisor: Amnon Kohen. Includes bibliographical references (leaves 108-114).

Background: Significant interest in homocysteine exists due to its established role in embryogenesis, cardiovascular disease and neurotoxicity. This research investigates total plasma homocysteine in the context of carcinogenesis among healthy individuals aged 20 to 50 years. It is hypothesized that during this timeframe, elevated total plasma homocysteine (tHcy) concentration implicates a breakdown of the methionine-homocyteine biosynthesis pathway, in which folate deficiency, oxidative stress and altered DNA methylation capacity are potential consequences relevant to cancer etiology. Purpose: The overall purpose of this research is to identify novel genetic predispositions to a biomarker of cancer risk (tHcy). Interactions with dietary and genetic factors that act on this pathway are explored. Methods: The study population consisted of 284 healthy male and female volunteers recruited in Kingston, Ontario and Halifax, Nova Scotia between 2006 and 2008. Specifically, polymorphisms under consideration included: i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and ii) the GC single nucleotide polymorphism (G/C SNP) both found on the 5’untranslated region (UTR) of the TS gene, and iii) the 6 base pair deletion at base pair 1494 (TS1494del6) found on the 3’UTR. TS polymorphisms were categorized based on either 5’ or 3’ location and were dichotomized to either high or low TS expression. Gene-gene interactions between polymorphisms in TS and methylenetetrahydrofolate reductase (MTHFR C677T) on tHcy concentration were also analyzed. In addition, gene-diet interactions between serum folate and vitamin B12 status were examined. Results: Mean tHcy concentration for this study population was 8.65 µmol/L (standard deviation=1.96 µmol/L). After adjustment for confounders, higher mean tHcy levels of 0.48 μmol/L and 0.46 μmol/L were observed for the main effects of 5’polymorphisms (5’High) (p=0.04) and 3’polymorphism (3’High) (p=0.05), respectively. The largest difference in mean tHcy concentration was observed for the joint effects of TS polymorphisms (µ=0.74 μmol/L, p=0.11). Gene-gene interaction was observed between TS and MTHFR polymorphisms on tHcy concentrations (p<0.01). Conclusions: The findings of this research provide evidence of an association between TS polymorphisms and tHcy concentrations. These results suggest that TS polymorphisms, independent of dietary factors, may lead to elevated tHcy levels and potentially contribute to cancer development. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2008-08-21 16:05:02.797

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Thesis (Ph. D.)--Cornell University, August, 1999. / Vita. Includes bibliographical references (leaves 141-168).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 141-154).

Antibiotic resistance represents a real threat in the modern world. The problem of resistance is brought about by the fast evolution of bacteria, accelerated by misuse and over-prescription of antibiotics and compounded by the decline in the discovery and development of new classes of antibiotics. Consequently, new targets for antibiotics are in high demand. Flavin-dependent thymidylate synthase (FDTS), which is not present in humans and is responsible for the biosynthesis of a DNA building block in several human pathogens (e.g., M. tuberculosis, B. anthracis, H. pylori), is one such novel target. FDTS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to produce 2'-deoxythymidine-5'-monophosphate (dTMP), with N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄fol) serving as the carbon source and a nicotinamide cofactor as the electron source. No efficient inhibitors of FDTS are known, despite high-throughput screening attempts to find them. Intermediate and transition-state mimics are likely to bind the enzyme with greater affinity and hence have a better chance at inhibiting FDTS. Therefore, the understanding of the chemical mechanism of FDTS is critical to the informed design of compounds capable of disrupting its function in bacteria. We utilized various techniques, including chemical trapping of reaction intermediates, substrate isotope exchange and stopped-flow, to investigate the FDTS mechanism and determine what sets it apart from other pyrimidine methylases. We found that at least two different intermediates kinetically accumulate in the FDTS-catalyzed reaction. Both of these intermediates are trapped in acid in the form of 5-hydroxymethyl-dUMP, which has never been isolated in other uracil-methylating enzymes. Under basic conditions, however, the earlier intermediate is converted to a species with an unusual flavin-derived adduct, while the later intermediate is converted to dTMP product. Our experiments also suggest that dUMP is activated for the reaction by the reduced flavin - a substrate activation mechanism distinct from the one employed by the classical pyrimidine-methylating enzymes.

The relationship between protein structure, motions, and catalytic activity is an evolving perspective in enzymology. An interactive approach, where experimental and theoretical studies examine the same catalytic mechanism, is instrumental in addressing this issue. We combine various techniques, including steady state and pre-steady state kinetics, temperature dependence of kinetic isotope effects (KIEs), site-directed mutagenesis, X-ray crystallography, and quantum mechanics/molecular mechanics (QM/MM) calculations, to study the catalytic mechanisms of thymidylate synthase (TSase). Since TSase catalyzes the last step of the sole intracellular de novo synthesis of thymidylate (i.e. the DNA base T), it is a common target for antibiotic and anticancer drugs. The proposed catalytic mechanism for TSase comprises a series of bond cleavages and formations including activation of two C-H bonds: a rate-limiting C-H→C hydride transfer and a faster C-H→O proton transfer. This provides an excellent model system to examine the structural and dynamic effects of the enzyme on different C-H cleavage steps in the same catalyzed reaction. Our experiments found that the KIE on the hydride transfer is temperature independent while the KIE on the proton transfer is temperature dependent, implying the protein environment is better organized for H-tunneling in the former. Our QM/MM calculations revealed that the hydride transfer has a transition state (TS) that is invariable with temperature while the proton transfer has multiple subsets of TS structures, which corroborates with our experimental results. The calculations also suggest that collective protein motions rearrange the network of H-bonds to accompany structural changes in the ligands during and between chemical transformations. These computational results not only illustrate functionalities of specific protein residues that reconcile many previous experimental observations, but also provide guidance for future experiments to verify the proposed mechanisms. In addition, we conducted experiments to examine the importance of long-range interactions in TSase-catalyzed reaction, using both kinetic and structural analysis. Those experiments found that a remote mutation affects the hydride transfer by disrupting concerted protein motions, and Mg2+ binds to the surface of TSase and affects the hydride transfer at the interior active site. Both our experiments and computations have exposed interesting features of ecTSase that can potentially provide new targets for antibiotic drugs targeting DNA biosynthesis. The relationship between protein structure, motions, and catalytic activity learned from this project may have general implications to the question of how enzymes work.

La conversion par les thymidylate synthases du désoxyuridine 5'-monophosphate (dUMP) en désoxythymidine 5'-monophosphate (dTMP) est une des étapes clés de la biosynthèse des désoxyribonucléotides pyrimidiques. Chez les procaryotes (bactéries et archées), il existe deux familles de thymidylate synthases non homologues : ThyA et ThyX. La distribution phylogénétique quasi-exclusive des deux familles est complexe et ne suit pas les relations phylogéniques entre les organismes. Les protéines de ces deux familles ne présentent aucune similarité de séquence, de structure et leurs mécanismes et efficacités catalytiques diffèrent. Les travaux présentés dans cette thèse ont permis de montrer que l'activité de ThyX est essentielle à la survie de la bactérie Rhodobacter capsulatus en absence de thymidine exogène. A partir des résultats de tests de complémentation génétique et de la modélisation mathématique du métabolisme des folates nous avons pu mettre en évidence que seulement une faible activité dihydrofolate réductase était nécessaire à la survie des organismes utilisant ThyX. D'autre part, j'ai pu montrer par des approches génétiques et par l'étude statistique de plus de 400 génomes procaryotes que les organismes utilisant ThyX ont une réplication et un taux de croissance lents, et possèdent majoritairement un petit génome. Je propose un modèle dans lequel la faible activité catalytique de ThyX limite la réplication de l'ADN e! t par conséquent l'expansion du génome. Enfin, j'ai étudié le métabolisme des pyrimidines chez la bactérie pathogène humaine, Helicobacter pylori. J'ai pu en particulier mettre en évidence qu'en absence de voie de récupération de l'uracile, cette bactérie est capable de métaboliser un analogue toxique le 5-fluorouracile (5FU), utilisé comme anticancéreux. J'ai par ailleurs montré que l'orotate phosphoribosyltransférase de H. pylori était capable de restaurer l'auxotrophie pour l'uracile d'une souche d'Escherichia coli indiquant que cette enzyme pourrait être responsable de la sensibilité de H. pylori au 5FU. Les travaux présentés dans cette thèse ont permis une meilleure compréhension des conséquences physiologiques de l'utilisation de la thymidylate synthase ThyX. Ils ont notamment permis d'expliquer la distribution apparemment sporadique des deux familles de thymidylate synthases dans le monde procaryote.

The therapeutic potential of a novel multi-targeted small-interfering RNA (siRNA) was investigated in human breast cancer cells. Previous studies had identified an siRNA that specifically and potently inhibited expression of thymidylate synthase (TS) by directly targeting human TS mRNA. TS is a folate-dependent enzyme that catalyzes the key reaction involved in synthesizing nucleotide precursors for DNA biosynthesis, and as such, it plays a critical role in maintaining cell growth. The goal of this thesis was to design and develop a novel siRNA molecule that targeted TS mRNA as well as a cellular mRNA that encodes a different cellular protein involved in cancer cell growth and proliferation, such as a member of the ErbB family. Gene sequence analysis was performed and identified an overlapping sequence between TS and ErbB2 mRNAs. An siRNA duplex was then designed to simultaneously target human TS and ErbB2 mRNA. Transfection of the multi-targeted siRNA (TS1M17) revealed that both ErbB2 and TS proteins were significantly suppressed in a time and dose-dependent manner in ErbB2-overexpressing human breast cancer SKBR3 cells. The corresponding mRNA levels, as determined by RT-PCR, were also decreased. Protein levels of other ErbB family members, including ErbB1 and ErbB3, remained unchanged with siRNA treatment. An ErbB2-specific siRNA (B2450) inhibited ErbB2, but had no effect on TS expression demonstrating the specificity of the multi-targeted siRNA against both TS and ErbB2. Mismatched (TS1-Mismatch) and control (GL2) siRNAs had no inhibitory effects on expression of the two target proteins. Suppression of activated ErbB2, as determined by expression of phosphorylated ErbB2 protein, was observed with transfection of TS1M17 siRNA. In addition, the expression of downstream signaling proteins, such as phosphorylated mitogen activated protein kinase (p-MAPK), p27Kip1, p21Cip1, cyclin D1, and survivin were significantly changed. In contrast, control siRNAs did not exert any inhibitory effects on downstream signaling. Taken together, these findings suggest that TS1M17 siRNA inhibits signaling of the ErbB2 pathway. The effect of TS1M17 siRNA on cytotoxicity was analyzed by WST-1 assay. Upon transfection into SKBR3 cells, the TS1M17 siRNA significantly suppressed cell proliferation with an IC50 value of 0.65 nM, which is 154-fold more potent than ErbB2- and TS-specific siRNAs. This study suggests that targeting expression of ErbB2 and TS, two key proteins involved in distinct and critical pathways for cancer growth and proliferation, with a single siRNA molecule may provide a novel approach for cancer chemotherapy.

Thymidylate synthase (TS), encoded by the ThyA gene, is essential for the growth and survival of Mycobacterium tuberculosis and therefore is a potential drug target. Thymidylate synthase binds both a substrate, 2'-deoxyuridine-5'monophosphate (dUMP) as well as a cofactor, (6R,S)-5,10-methylenetetrahydrofolate (mTHF), providing the ability to inhibit a single target by two separate classes of molecules. 5'-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is a very tight binding mechanism based inhibitor, shown to have a Ki of 2nM for Mtb TS. Pemetrexed and Raltitrexed are both anti-folates, targeting the cofactor binding site of thymidylate synthase. The x-ray crystal structures of Mycobacterium tuberculosis thymidylate synthase were solved in the binary complexes ThyA-dUMP and ThyA-FdUMP at 2.5 A and 2.4 A resolutions, respectively. The ternary complex, ThyA-dUMP-Pemetrexed was solved to a resolution of 1.7 A. The enzyme is comprised of 8 alpha-helices as well as 23% of the protein formed by beta-sheets, including the dimer interface which is a beta-sandwich. Examination of the dUMP binding site allowed the identification of key conserved residues that play a role in ligand binding and catalysis. Comparison of the dUMP-Pemetrexed ternary complex with that of the human crystal structure shows two fewer interactions in the Mtb enzyme. One is due to the replacement of a Met with a Val which doesn't allow hydrophobic interactions with the ring system of Pemetrexed, and the other is the replacement of an Asn with a Trp, depriving the Mtb protein of a hydrogen bond at the N7 of the pyrrolo ring. A spectrophotometric assay that monitored DHF formation was used to determine the inhibition of Pemetrexed and Raltitrexed on Mtb TS. Both were verified as noncompetitive inhibitors, and Pemetrexed was found to have an IC50 of 17muM and a Ki of 16.8muM, while Raltitrexed had an IC50 of 3.5muM and a Ki of 3.2muM.