Date of Award

2010

Document Type

Campus Access Dissertation

Department

College of Pharmacy

Sub-Department

Pharmaceutical Science

First Advisor

Sondra Berger

Abstract

Part I:

Vitamins such as folic acid and nicotinamide (NAm) play an essential role in genome stability. Folic acid is a precursor of 5,10-methylenetetrahydrofolic acid (MTHF) that is partitioned into pathways leading to TTP or methionine synthesis. Deficiency of MTHF or imbalance in the pathways utilizing MTHF may cause uracil incorporation into DNA and/or a decrease in one-carbon groups required for DNA methylation. The default pathway for excision of uracil in DNA is the base excision repair (BER) pathway. NAm is a precursor of NAD+, the substrate of two families of enzymes, poly(ADP-ribose) polymerases (PARPs) and sirtuins (SIRTs). PARP-1 is thought to mediate the repair of BER intermediates and to suppress homologous recombination (HR); SIRT1 is a histone deacetylase. Thus, the balance between folic acid and NAm may regulate the repair of BER intermediates by alternative pathways such as HR and alter the accessibility of transcriptional, replication, and repair complexes to DNA. The hypothesis under examination is that a deficiency of or imbalance between folic acid and NAm decreases genome stability and increases carcinogenesis. To test it, studies were designed and conducted on two levels: in a cell culture model and in samples from breast cancer patients.

In the cell model HCT116, EC 50 of folate and Nam, precursor of NAD, were determined. Customized RPMI 1640 depletion medium was used in the study offering an advantage relative to standard approaches. Various ratios of two nutrients were tested and preliminary results showed cell growth rate changed as the ratio of the two nutrients changed even though concentrations of both nutrients were above their EC100. It indicated a role of imbalance of folic acid and Nam in cancer cell growth. In the study of breast cancer patients, blood samples were collected from breast cancer patients to analyze the polymorphism of TYMS and MTHFR, that encode key enzymes in folate metabolism. Folate and NAD status were also examined. MTHFR 677T allele frequency was significantly lower in the studied population compared to the healthy population suggesting a protective role of the T allele in breast cancer. However, due to insufficient sample size, this observation was not statistically significant.

Part II:

Thymidylate synthase (TS) catalyzes the reaction that forms dTMP from dUMP. Previous data suggested that human thymidylate synthase (hTS) exists in two major conformations, active and inactive. Two hTS mutants were created to mimic the active and inactive forms. The mutant, designated R163K, has an arginine to lysine substitution at residue 163 and crystallizes in an active conformation in the native state. The other mutant, A191K, has an alanine to lysine substitution at residue 191 and is stabilized in the inactive form. We postulated that hTS exists in two conformations in solution. To test it, circular dichroism was chosen to detect solution structures and the effects of ligand binding. Relative to hTS, the CD spectra of both R163K and A191K showed a decreased ellipticity. Upon ligation with dUMP, A191K exhibited a further decrease in ellipticity and showed no apparent response to phosphate. In contrast, the ellipticity of R163K was increased by phosphate and dUMP produced a further increase in ellipticity. In addition, a study of temperature effects on TS catalysis showed that hTS has a 2.2-fold higher activation energy compared to R163K, consistent with the hypothesis that hTS exists in an inactive conformation in solution. Crystal structures of hTS indicated that enzyme conformation is related to the structure of an eukaryotic-specific region, insert 1. Insert 1 contains a serine residue that is phosphorylated by the protein kinase, casein kinase 2 (CK2). Phosphorylation of the enzymes by CK2 was analyzed by isoelectrofocusing. A191K showed multiple phosphorylation sites while hTS and R163K only showed one site, indicating that phosphorylation by CK2 is conformation-selective.

After validating that the mutants are models for the two major conformations, an investigation aimed at understanding the physiological relevance of conformational switching of TS was conducted. Phylogenetic evidence supported an evolutionary selection pressure towards conformational switching of TS. Crystal structures suggested that the catalytic thiol of R163K is more susceptible to modification by oxidation compared to hTS. Thus, we postulated that the inactive conformer is more resistant to oxidation than the active conformer. To test the hypothesis, we conducted a series of studies. Utilizing the thiol-reactive agent, dithionitrobenzoic acid (DTNB), detailed modification rates and levels of different thiols including Cys 195, the critical cysteine for TS catalysis, were collected. After modification by DTNB, Cys 195 of R163K was resistant to oxidation, relative to hTS and A191K; however, R163K exhibited the most rapid rate of modification by DTNB. In contrast, A191K exhibited the slowest rate of DTNB modification yet results from a study of TS catalysis after modification by DTNB showed that Cys 195 in A191K is more susceptible to modification by DTNB in vitro. While the overall rates of modification are consistent with our hypothesis, based on crystallographic data, the susceptibility of Cys 195 to modification is opposite to our expectation. To further study the conformers' response to oxidation, in vivo studies followed. Chinese hamster lung cells were manipulated to overexpress hTS and R163K. Cells were exposed to tert-butyl hydroperoxide (TBHP)-induced oxidative stress. hTS-expressing cells showed protection from oxidation compared to R163K-expressing cells. Studies of protection from TBHP-mediated cytotoxicity by either thymidine or N-acetylcysteine suggested that the structure of the conformer per se rather than its role in catalysis plays a role in the response. This is the first study to investigate the physiological role of TS enzyme. The data indicate that conformational switching of hTS has a protective role under oxidizing conditions.

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