REGULATION OF CYSTATHIONASE EXPRESSION

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The long-term objective of this research is to understand the regulation of transsulfuration in mammalian cells. The transsulfuration pathway results in the transfer of the sulfur atom of methionine into cysteine. The early enzymes of the path are shared with the transmethylation pathway by which methyl groups are contributed to a number of important metabolic intermediates. There is almost no knowledge of the molecular mechanisms which underlie expression of enzymes in either path. Our specific objectives are to elucidate the molecular mechanisms which regulate expression of cystathionase (CSE), the terminal enzyme of transsulfuration. We shall examine CSE regulation in developing rat liver, which shows a similar temporal pattern of CSE expression to human liver. CSE protein synthesis and degradation rates will be determined at different stages of development. The concentration of functional CSE mRNA will be ascertained for each stage and for rat brain tissue, where CSE activity is very low. To obtain more detailed understanding of CSE genetic regulation, we shall isolate cDNA clones representing CSE mRNA, employing our specific antisera for library screening and/or polysome immunoselection. Using the cloned cDNA, we shall analyze transcriptional and post-transcriptional regulation of CSE gene expression as well as determine the number of gene copies, presence of introns, and methylation states of the CSE gene(s). There are numerous clinical and basic problems that relate to transsulfuration enzymes. Absence of CSE at birth may make cysteine an essential amino acid for the neonate. Genetic absence of enzyme activity results in cystathioninuria which has been associated with mental retardation. The hepatic production of cyteine by transsulfuration is crucial in maintaining glutathione levels. This thiol is of major importance in protecting cells from radiation and toxic drugs including acetaminophen and chemotherapeutic agents. Thus, an understanding of the regulation of transsulfuration could have far reaching implications for human health. (B)