The primary focus of the present proposal is on how the machinery for sulfation, a common posttranslational modification of proteins, lipids and carbohydrates (especially proteoglycans) is organized and controlled in higher organisms. The integrated pathway for sulfate uptake, activation and utilization encompasses multiple components and multiple intracellular compartments. Central to this process is the bifunctional PAPS synthetase which synthesizes phosphoadenosyiphosphosulfate (PAPS) from ATP and sulfate- in a two-step reaction. Recent work, including the discovery of the PAPS synthetase gene family, the identification of mutations in PAPS synthetase that lead to both human and animal chondrodystrophies and elucidation of unique enzymatic properties, underlines the significance of this enzyme in the overall sulfation process. Two specific questions will be addressed in this continuation proposal. 1) What features of the fused bifunctional PAPS synthetase and individual monofunctional sulfurylases and kinases account for the significant mechanistic differences between them, especially with respect to the channeling phenomenon? Mutagenic analysis and structural elucidation approaches will be employed. 2) What is the role of the multiple PAPS synthetase family members with respect to tissue- and developmental-specific expression patterns? A multi-faceted approach will be used to quantitate each isoform, elucidate mechanisms of transcriptional regulation and determine the consequences of manipulation of isoform expression. These studies are aided by the availability of a mutant model system with a defect in sulfation that results in altered proteoglycan production and abnormal skeletal growth and development. The range of methodology necessary for accomplishing these goals includes: cDNA cloning and sequencing, enzyme assay and kinetic analysis, cell and tissue culture and propagation of the mutant mouse strain. These studies have the long-term goal of providing a model of how this critically important pathway is regulated and then to correlate defects in the overall pathway with abnormal growth and development.
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