The long-term objective of this research is to understand the biochemical and genetic mechanisms regulating human purine nucleotide synthesis. Studies of inherited enzyme defects underlying excessive purine nucleotide and uric acid production in some families with gout have contributed to concepts of the control of rates of purine synthesis de novo. One such defect is superactivity of phosphoribosylpyrophosphate (PRPP) synthetase (PRS), the enzyme catalyzing synthesis of the regulatory substrate PRPP. The proposed investigations focus on expression of two X chromosome-linked human (h) genes, hPRPS1 and hPRPS2, which encode highly homologous PRS isoforms, hPRS1 and hPRS2, respectively. The specific aims are: 1) to delineate the structural and regulatory determinants of normal hPRPS1 and hPRPS2 gene expression; and 2) to define the precise genetic defects and resulting aberrant molecular mechanisms underlying X-linked catalytic superactivity of hPRS.

In order to pursue these specific aims, molecular genetic, protein chemical, and enzyme analytical methods will be employed. The structure of hPRPS1 and hPRPS2 promoter and adjacent 5= flanking sequences will be defined by sequencing of cloned PRPS genomic DNA. hPRPS promoter function and cis-acting regulatory elements in the 5= flanking DNAs will be studied in murine and human cell lines transfected with hPRPS promoter region reporter gene plasmid constructs. Pertinent DNA sequences will be tested for nuclear protein and specific transcription factor binding by gel mobility shift assays and, where appropriate, DNA footprinting and site-directed mutation will be utilized to define and delimit key protein-binding sequences in the DNA. PRPS gene promoter activities will be correlated with PRS transcript levels, PRS isoform contents and activities, and PRPP and purine nucleotide synthesis in differentiated human cell lines representative of tissues showing differential expression of PRPS1 and/or PRPS2 transcript abundance. The influences of growth promoting agents, viral transformation, and cell cycle traversal on hPRPS promoter activities and mouse PRS transcript and isoform levels will be tested in murine cell lines. These studies are aimed at: defining models for cell-specific and gene-differential regulation of PRPS gene expression; identifying where in genetic information transfer control occurs; and ascertaining the molecular mechanisms involved.

Altered pretranslational regulation of hPRPS1 expression in PRS catalytic superactivity may reflect transcriptional dysregulation due to structural and/or functional defects in the control of the PRPS1 promoter. This hypothesis will be tested by comparing the structure of promoter regions of affected patients and normal individuals and by measuring the respective PRPS1 promoter activities in murine cells and in normal and patient fibroblasts. Finally, post-translational control of hPRS isoform expression by specific interaction with a PRS-associated 39kDa protein (PAP39) will be examined in studies assessing the specificity and functional consequences of the PAP39-PRS isoform complex and the results of selective disruption of the complex and its reconstitution from recombinant components.

R01DK028554

1980-09-01

2002-11-30