Biomedical subjects

Patrick J Stover

Publications and source records attributed to Patrick J Stover.

14 recordsLinked to original sources

PubMed2006

Regulation of folate-mediated one-carbon metabolism by 10-formyltetrahydrofolate dehydrogenase.

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.

Carbon↗

PubMed2006

Influence of human genetic variation on nutritional requirements.

Genetic variation is known to affect food tolerances among human subpopulations and may also influence dietary requirements, giving rise to the new field of nutritional genomics and raising the possibility of individualizing nutritional intake for optimal health and disease prevention on the basis of an individual's genome. However, because gene-diet interactions are complex and poorly understood, the use of genomic knowledge to adjust population-based dietary recommendations is not without risk. Whereas current recommendations target most of the population to prevent nutritional deficiencies, inclusion of genomic criteria may indicate subpopulations that may incur differential benefit or risk from generalized recommendations and fortification policies. Current efforts to identify gene alleles that affect nutrient utilization have been enhanced by the identification of genetic variations that have expanded as a consequence of selection under extreme conditions. Identification of genetic variation that arose as a consequence of diet as a selective pressure helps to identify gene alleles that affect nutrient utilization. Understanding the molecular mechanisms underlying gene-nutrient interactions and their modification by genetic variation is expected to result in dietary recommendations and nutritional interventions that optimize individual health.

Chronic Disease↗

PubMed2005

Regulation of de novo purine biosynthesis by methenyltetrahydrofolate synthetase in neuroblastoma.

5-Formyltetrahydrofolate (5-formylTHF) is the only folate derivative that does not serve as a cofactor in folate-dependent one-carbon metabolism. Two metabolic roles have been ascribed to this folate derivative. It has been proposed to 1) serve as a storage form of folate because it is chemically stable and accumulates in seeds and spores and 2) regulate folate-dependent one-carbon metabolism by inhibiting folate-dependent enzymes, specifically targeting folate-dependent de novo purine biosynthesis. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme that metabolizes 5-formylTHF and catalyzes its ATP-dependent conversion to 5,10-methenylTHF. This reaction determines intracellular 5-formylTHF concentrations and converts 5-formylTHF into an enzyme cofactor. The regulation and metabolic role of MTHFS in one-carbon metabolism was investigated in vitro and in human neuroblastoma cells. Steady-state kinetic studies revealed that 10-formylTHF, which exists in chemical equilibrium with 5,10-methenylTHF, acts as a tight binding inhibitor of mouse MTHFS. [6R]-10-formylTHF inhibited MTHFS with a K(i) of 150 nM, and [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a K(i) of 30 nm. MTHFS is the first identified 10-formylTHF tight-binding protein. Isotope tracer studies in neuroblastoma demonstrate that MTHFS enhances de novo purine biosynthesis, indicating that MTHFS-bound 10-formylTHF facilitates de novo purine biosynthesis. Feedback metabolic regulation of MTHFS by 10-formylTHF indicates that 5-formylTHF can only accumulate in the presence of 10-formylTHF, providing the first evidence that 5-formylTHF is a storage form of excess formylated folates in mammalian cells. The sequestration of 10-formylTHF by MTHFS may explain why de novo purine biosynthesis is protected from common disruptions in the folate-dependent one-carbon network.

Animals↗

PubMed2005

Polymorphisms in cytoplasmic serine hydroxymethyltransferase and methylenetetrahydrofolate reductase affect the risk of cardiovascular disease in men.

Genetic variation in folate-regulating enzymes contributes to the risk of cardiovascular disease (CVD). The cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme is proposed to regulate a key metabolic intersection in folate metabolism. We hypothesized that a variant in cSHMT (cSHMT 1420C-->T) affects CVD risk, and that the effect depends on a linked step in the metabolic pathway catalyzed by methylenetetrahydrofolate reductase (MTHFR). A nested case-control study of incident CVD was conducted within the all-male Normative Aging Study cohort. Of the incident CVD cases, 507 had DNA samples; 2 controls/case were selected by risk set sampling (matched on age and birth year). A significant gene-gene interaction (P-values 0.0013, 0.0064) was found between MTHFR and cSHMT, and there was little or no change in the coefficients in covariate-adjusted models. The effect of MTHFR 677C-->T genotype on CVD risk varied by cSHMT 1420C-->T genotype. Among men with cSHMT 1420C-->T TT genotype, the odds ratios (OR) for CVD risk for MTHFR 677C-->T CT and TT genotypes compared with the MTHFR 677C-->T CC genotype were 3.6 (95% CI, 1.7-7.8) and 10.6 (95% CI, 2.5-46.0), respectively. Among men with the cSHMT 1420C-->T CC/CT genotype, the corresponding ORs were 1.0 (95% CI, 0.8-1.2) and 1.3 (95% CI, 0.9-1.8). Plasma total homocysteine concentrations were highest in the subgroup of men with both polymorphisms, MTHFR 677C-->T TT and cSHMT 1420C-->T TT, consistent with a higher risk of CVD in this subgroup. A more complete understanding of the molecular mechanism awaits identification of the functional effect of the polymorphism.

Adult↗

PubMed2004

Mimosine attenuates serine hydroxymethyltransferase transcription by chelating zinc. Implications for inhibition of DNA replication.

L-mimosine is a naturally occurring plant amino acid and iron chelator that arrests the cell cycle in the late G(1) phase, although its mechanism of action is not known. Some studies indicate that mimosine prevents the initiation of DNA replication, whereas other studies indicate that mimosine disrupts elongation of the replication fork by impairing deoxyribonucleotide synthesis by inhibiting the activity of the iron-dependent enzyme ribonucleotide reductase and the transcription of the cytoplasmic serine hydroxymethyltransferase gene (SHMT1). In this study, the mechanism for mimosine-induced inhibition of SHMT1 transcription was elucidated. A mimosine-responsive transcriptional element was localized within the first 50 base pairs of the human SHMT1 promoter by deletion analyses and gel mobility shift assays. The 50-base-pair sequence contains a consensus zinc-sensing metal regulatory element (MRE) at position -44 to -38, and mutation of the MRE attenuated mimosine-induced transcription repression. Mimosine treatment eliminated MRE- and Sp1-binding activity in nuclear extracts from MCF-7 cells but not in nuclear extracts from a mimosine-resistant cell line, MCF-7/2a. MCF-7 cells cultured in zinc-depleted medium for more than 16 days were viable and lacked cytoplasmic serine hydroxymethyltransferase protein, confirming that mimosine inhibits SHMT1 transcription by chelating zinc. The disruption of DNA-protein interactions by zinc chelation provides a general mechanism for the inhibitory effects of mimosine on nuclear processes, including replication and transcription. Furthermore, this study establishes that SHMT1 is a zinc-inducible gene, which provides the first mechanism for the regulation of folate-mediated one-carbon metabolism by zinc.

Chelating Agents↗

PubMed2004

Nutritional genomics.

The integration of genomics into nutritional sciences has illuminated the complexity of genome responses to nutritional exposures while offering opportunities to increase the effectiveness of nutritional interventions, both clinical and population based. Nutrients elicit multiple physiological responses that affect genome stability, imprinting, expression, and viability. These effects confer both health benefits and risks, some of which may not become apparent until later in life. Nutritional genomics challenges us to understand the reciprocal and complex interactions among the human genome and dietary components in normal physiology and pathophysiology. Understanding these interactions will refine current definitions of benefit and risk and lead to the establishment of dietary recommendations that have a high predictive value, minimize the risk of unintended consequences, and account for the modifying effects of human genetic variation. Furthermore, nutritional genomics will enable the design of effective dietary regimens for the prevention and management of complex chronic disease. This review focuses on new perspectives that have been presented to the nutritional sciences by the advent of genomics, and new challenges that demand attention because of their potential impact on, and immediate translation into, current public health nutrition recommendations and interventions.

Animals↗

PubMed2004

Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus.

Folate metabolism is necessary for the biosyntheses of purine nucleotides and thymidylate and for the synthesis of S-adenosylmethionine, a cofactor required for cellular methylation reactions and a precursor of spermidine and spermine syntheses. Disruption of folate metabolism is associated with several pathologies and developmental anomalies including cancer and neural tube defects. The enzyme 5,10-methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2) catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, and has been shown to affect intracellular folate concentrations by accelerating folate degradation. Mammalian MTHFS proteins described to date are not stable and no recombinant mammalian MTHFS protein has been successfully expressed in Escherichia coli. The three-dimensional structure of MTHFS has not been solved. The cDNA coding for Mus musculus MTHFS was isolated and expressed in E. coli with a hexa-histidine tag. Milligram quantities of recombinant mouse MTHFS were purified using metal affinity chromatography and the protein was stabilized with Tween 20. Mouse MTHFS has a molecular mass of 23kDa and is 84% identical in amino acid sequence to the human enzyme. Activity assays confirmed the functionality of the recombinant protein, with Km =5 microM for (6S)-5-formyltetrahydrofolate and Km=769 microM for Mg-ATP. This is the first example of a mammalian form of MTHFS expressed in E. coli that yielded sufficient quantities of stable purified protein to allow for detailed characterization of its three-dimensional structure and kinetic properties.

Amino Acid Sequence↗

PubMed2004

Physiology of folate and vitamin B12 in health and disease.

Folate is a water-soluble B-vitamin and enzymatic cofactor that is necessary for the synthesis of purine and thymidine nucleotides and for the synthesis of methionine from homocysteine. Impairment of folate-mediated one-carbon metabolic pathways can result from B-vitamin deficiencies and/or single nucleotide polymorphisms, and increases risk for pathologies, including cancer and cardiovascular disease, and developmental anomalies including neural tube defects. Although several well validated metabolic and genomic biomarkers for folate deficiency exist, our understanding of the biochemical and genetic mechanisms whereby impaired folate metabolism increases risk for developmental anomalies and disease is limited, as are the mechanisms whereby elevated folate intake protects against these pathologies. Therefore, current initiatives to increase folate intakes in human populations to ameliorate developmental anomalies and prevent disease, while effective, lack predictive value with respect to unintended adverse outcomes.

Folic Acid↗

PubMed2003

Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation.

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.

Biological Transport↗

PubMed2003

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers.

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.

Binding Sites↗

PubMed2003

New paradigms for nutrient control of genome translation.

Organisms respond adaptively to changing environmental conditions by altering genome expression. Translational control of gene expression by nutrients and metabolites is one of the most robust biologic mechanisms that enable rapid alterations in the cellular proteome in response to changes in the local environment. Recently, investigators have identified new mechanisms for nutrient control of translation in mammals; evidence supports the conservation of translational control mechanisms in eukaryotes that heretofore had only been demonstrated in prokaryotes.

Eukaryotic Cells↗

PubMed2002

Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses.

Folate-dependent one-carbon metabolism is required for the synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Methionine is subsequently adenylated to S-adenosylmethionine (SAM), a cofactor that methylates DNA, RNA, proteins, and many metabolites. Previous experimental and theoretical modeling studies have indicated that folate cofactors are limiting for cytoplasmic folate-dependent reactions and that the synthesis of DNA precursors competes with SAM synthesis. Each of these studies concluded that SAM synthesis has a higher metabolic priority than dTMP synthesis. The influence of cytoplasmic serine hydroxymethyltransferase (cSHMT) on this competition was examined in MCF-7 cells. Increases in cSHMT expression inhibit SAM concentrations by two proposed mechanisms: (1) cSHMT-catalyzed serine synthesis competes with the enzyme methylenetetrahydrofolate reductase for methylenetetrahydrofolate in a glycine-dependent manner, and (2) cSHMT, a high affinity 5-methyltetrahydrofolate-binding protein, sequesters this cofactor and inhibits methionine synthesis in a glycine-independent manner. Stable isotope tracer studies indicate that cSHMT plays an important role in mediating the flux of one-carbon units between dTMP and SAM syntheses. We conclude that cSHMT has three important functions in the cytoplasm: (1) it preferentially supplies one-carbon units for thymidylate biosynthesis, (2) it depletes methylenetetrahydrofolate pools for SAM synthesis by synthesizing serine, and (3) it sequesters 5-methyltetrahydrofolate and inhibits SAM synthesis. These results indicate that cSHMT is a metabolic switch that, when activated, gives dTMP synthesis higher metabolic priority than SAM synthesis.

Animals↗

PubMed2002

Securing the future of nutritional sciences through integrative graduate education.

The new millennium has ushered in unprecedented opportunities for research and discovery in the nutritional sciences. These have arisen from major advancements in the social and biological sciences, notably genetics, genomic technologies, computational statistics, and behavioral sciences. The most exciting academic challenges and employment opportunities for nutritional sciences graduates interface with other disciplines, and doctoral training programs in the nutritional sciences must respond to the wealth of these emerging opportunities. The American Society for Nutritional Sciences (ASNS) Graduate Nutrition Education Committee suggests that three major challenges face the nutritional sciences: (a) to train doctoral students to be full and active participants in interdisciplinary research and discovery, and (b) to achieve this goal while maintaining nutrition's unique academic identity and (c) fostering a cohesive academic community. The committee proposes that these objectives are best engaged at the level of graduate education and that the future of ASNS will be secured by strengthening graduate nutrition programs. This manuscript reviews educational strategies that address these challenges and advocates that ASNS actively engage graduate doctoral programs in nutritional sciences.

Education, Graduate↗

PubMed2002

Bringing individuality to public health recommendations.

The data generated from the human genome project offers unprecedented opportunities to elucidate the etiology of chronic diseases and developmental anomalies that arise from deleterious genome-diet interactions. Folate metabolism is an attractive system to explore such relationships. Folate is necessary for the synthesis of purine and thymidine deoxyribonucleotides and S-adenosylmethionine, a cofactor required for DNA methylation. Impaired folate metabolism results from primary folate deficiency, alcohol, gastrointestinal disorders that result in malabsorption, single nucleotide polymorphisms, increased folate catabolism and secondary nutrient deficiencies in vitamin B-6, vitamin B-12 and iron arising from a variety of pathologies. Any of these conditions singly or in combination influence DNA synthesis, DNA integrity, allelic-specific gene expression, chromatin structure and DNA mutation rates. Biochemical manifestations of impaired folate metabolism include increased uracil uptake into DNA, altered DNA methylation status and elevated homocysteine and S-adenosylhomocysteine in serum and tissues. These biochemical changes are associated with risk for cancer, cardiovascular disease, neural tube defects and some neuropathies and anemia, although direct causative mechanisms have not been established in all cases. Interactions between folate and the genome are reciprocal; polymorphisms in key genes influence folate nutritional requirements, indicating that dietary folate adequacy likely exerts selective pressure and thereby influences genetic variation. Other studies indicate that exposure to excess folate, perhaps at levels that occur at the upper end of the intake distribution curve, may have unintended consequences in promoting embryo viability. Therefore individualizing folic acid dietary recommendations necessitates a detailed understanding of all genetic and physiological variables that influence the interaction of folate with the genome and their relationship to the disease process.

Folic Acid↗