Sulfate conjugation is an important pathway in the metabolism of many drugs, other xenobiotic compounds,hormones and neurotransmitters. During the past two decades, the applicant's laboratory has systematically explored the pharmacogenetics and pharmacogenomics of enzymes that participate in sulfate conjugation in humans. In pursuit of that goal, we have applied the techniques of molecular biology and genomics to clone and characterize genes encoding sulfotransferase (SULT) enzymes in humans and - during the most recent funding period - genes encoding the two human isoforms for PAPS synthetase (PAPSS), the enzymes that catalyze the synthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor for all mammalian SULT enzymes. During that same time period, we also began to apply a """"""""genotype-to phenotype"""""""" pharmacogenetic research strategy by resequencing genes encoding SULTs and PAPSS isoforms to identify common DNA sequence variation and, subsequently, to functionally characterize variant allozymes encoded by common nonsynonymous coding single nucleotide polymorphisms (cSNPs). We observed that one of the most common causes for those functional alterations was a decrease in the quantity of enzyme protein. Cellular phenotypes resulting from those changes in encoded amino acid sequence were determined, and several of these genetic polymorphisms were then used to test medically relevant hypotheses. That pharmacogenetic research strategy will now be moved """"""""beyond the open reading frame"""""""" to include functional characterization of common genetic polymorphisms within the 5'-flanking regions of genes encoding selected SULTs and the two PAPSS isoforms, followed by genotype-phenotype correlation analysis performed with human biopsy tissue that will include both ORF- and 5'-FR genetic polymorphisms. We also propose to explore underlying mechanisms by which nonsynonymous cSNPs decrease the quantity of enzyme protein - a very common functional consequence of the presence of those polymorphisms. The proposed experiments will serve to increase our understanding of molecular mechanisms responsible for the genetic regulation of proteins that participate in sulfate conjugation, and --ultimately -- should help make it possible to predict individual variations in the sulfation and, therefore, the efficacy or toxicity of compounds metabolized by this phase II pathway for drug and xenobiotic biotransformation.
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