The long-term goals of this project consist of characterizing the biosynthesis of all-trans-retinoic acid (RA) at the molecular, biochemical and endocrine levels. Data from the previous project period corroborate a model of RA biosynthesis that embodies interactions between retinoid-binding proteins and retinoid-specific enzymes that recognize the binding proteins as substrates and as positive or negative effectors. The work proposed will continue to evaluate the hypothesis that a major physiological pathway of RA biosynthesis has as its first and rate-limiting step conversion of retinol into retinal by all-trans-retinol dehydrogenase (RoDH) isozymes, members of the short-chain dehydrogenase/reductase family, which recognize as substrate holo-cellular retinol-binding protein.
The specific aims are to: 1) complete isolating and characterizing the cDNAs that encode RoDH isozymes; 2) establish the cellular distribution of RoDH mRNA synthesis in the embryo and throughout the lifespan of the adult; 3) express cDNAs that encode RoDH isozymes to characterize the enzymes kinetically and physically and to examine interactions with retinoid-binding proteins and retinal dehydrogenases; 4) isolate genomic clones encoding RoDH genes, and map their promoters to obtain insight into regulation of expression; 5) produce and evaluate RoDH gene knockouts by homologous recombination. Far less has been revealed about the enzymes which catalyze retinoid metabolism, than about the retinoid receptors and retinoid binding-proteins, even though retinoid function depends upon the integrated relationship among all three groups of proteins. This project provides the first detailed inroad into enzymes that specifically catalyze RA biosynthesis and represents the only all-trans-retinol dehydrogenase clones isolated. Thus, this work may provide unique insight into the mechanisms of RA-biosynthesis and its regulation. As a result, additional insight ought to be forthcoming into the physiological processes regulated by the RA signaling system, such as development, and diseases associated with its malfunctions, such as aging, cancer, birth defects and skin diseases.
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