研究领域

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The vitamin folic acid is essential to the production of purines and thymidylate required for the synthesis of DNA and RNA. Of special importance is the role of folate in providing sufficient amounts of adenosylmethionine required for more than a hundred different methylation reactions fundamental to a wide range of metabolic functions. All these processes are particularly important to rapidly dividing cells in any tissue. Recently, folate status and its metabolic functions have been shown to relate to the incidence of atherosclerosis in adults and to neural tube defects in children. It has been shown that folate supplements to women, begun prior to conception, decrease the incidence of neural tube defects in babies by up to 66%, but the way in which this is achieved metabolically is unclear; does it relate to nucleotides, methylation, gene(s) expression, polymorphism in a gene(s) or some other process? My laboratory has been investigating several aspects of folate-mediated processes: both the mitochondria and the cytoplasm of eukaryotes have folate-dependent functions, but their roles are not clearly understood, especially as regards the mitochondrial compartment. We demonstrated over-expression of a bifunctional folate enzyme in the mitochondria of tumour cells and showed that it is highly expressed in fetal but not in normal adult tissues. We postulate that it may have a role in fetal development and/or mitochondrial biogenesis and perhaps relates to neural tube defects. Gene disruption experiments in mammalian cells in culture have been performed to understand the role of this enzyme and others in the two cellular compartments. Using these cells we have recently obtained mice with one of the nuclear alleles encoding the mitochondrial protein inactivated. A breeding program with these mice demonstrated that the null mutant mice die in utero at about day 13 of development, apparently due to a defect in the ability of the fetal liver to establish hematopoesis. We are currently working with cell lines from mutants and wild type to delineate possible metabolic consequences of the gene deletion. Many of the folate-dependent enzymes are multifunctional, having more than one catalytic activity per polypeptide. One of our goals has been to understand the benefits of this type of structure to its function in mammalian cells. My laboratory has cloned and expressed several such enzymes and have X-ray crystallographic structure of the bifunctional enzyme that uses NADP. The objectives are to understand the mechanism of the enzyme(s) using site-directed mutagenesis, assisted by 3-D structure, to assign functions to key amino acid residues. The specific goals in this portion of the program are: 1.to extend crystallography studies to the mitochondrial version of the bifunctional enzyme; 2.identify residues involved in catalysis by each activity; 3.to create monofunctional versions of the enzymes; 4.to understand the unique replacement of NADP by NAD, Mg and phosphate by the enzyme expressed in fetal mitochondria and tumour cells. Re-introducing the cDNA that encodes these mutant proteins having altered properties into the gene-disrupted mammalian cells then provides a vehicle to test hypotheses of metabolic function.