The metabolic roles of human cytosolic and mitochondrial serine hydroxymethyltransferase (SHMT) and the factors that contribute to their gene expression and catalytic activity will be determined using molecular biology techniques. The role of the SHMT catalyzed formation of 5- formyltetrahydrofolate, and the effects of -5-formyltetrahydrofolate accumulation on cellular metabolism and homeostasis will be investigated. The following approaches will be taken: l. The human SHMT genes will be cloned, sequenced and analyzed for consensus DNA regulatory elements. Consensus or novel cis-acting sequence elements that contribute to SHMT expression will be identified using chloramphenicol acetyltransferase assays and in vivo footprint analysis. The associated trans-acting factors will be identified and characterized by gel-mobility shift assays and southwestern analysis. 2 The mechanisms through which external stimuli regulate SHMT expression will be elucidated. Cultured cells will be treated with stimuli that are known to influence SHMT activity. The effects of these stimuli on SHMT mRNA levels and synthesis rates will be determined using a reverse transcriptase-polymerase chain reaction assay and nuclear runoff assays. Changes in mRNA will be compared to SHMT enzyme activity. The effects of external stimuli on folate one-carbon pools, intracellular serine/glycine concentrations and on the 5-formyltetrahydrofolate futile cycle will also be determined by HPLC analysis. 3. The role of the 5-formyltetrahydrofolate futile cycle in mammalian cellular homeostasis will be studied. Cell culture models will be developed where the futile cycle is manipulated by methenyltetrahydrofolate synthetase inhibitors, varying intracellular serine/glycine ratios and by disrupting and/or over expressing SHMT and methenyltetrahydrofolate synthetase. The effects of 5- formyltetrahydrofolate accumulation on cellular glycine concentrations, one-carbon metabolism, global protein synthesis, and gene transcription in mammalian cells will be investigated. The long term goal of these studies is to (l) differentiate the metabolic roles of mitochondrial and cytosolic SHMT and determine if their regulation influences the supply of folate-activated one-carbon units, (2) determine how 5-formyltetrahydrofolate concentrations are regulated in mammalian cells, (3) define the role of the SHMT catalyzed formation of 5- formyltetrahydrofolate in influencing cell metabolism and proliferation.
