This is an application for a Mentored Research Scientist Development Award. The long term objective of the research is to identify and characterize human alcohol dehydrogenases that are responsible for retinoic acid synthesis in human tissues. Retinoic acid regulates the expression of multiple genes during fetal-development and in adulthood. Abnormal retinoic acid concentrations in developing tissues result in malformations similar to those seen in Fetal Alcohol Syndrome. Ethanol inhibits retinoic acid synthesis in several tissues. However, the mechanism of this effect is not known. It was suggested that ethanol inhibits the rate-limiting step in retinoic acid synthesis, oxidation of retinol to retinal. Two types of alcohol dehydrogenases are capable of oxidizing retinol - cytosolic and microsomal dehydrogenases. Only cytosolic retinol dehydrogenases are known to be inhibited by ethanol. It is not clear yet which enzymes oxidize retinol in vivo. Retinol is present in tissues in two forms: free and bound to cellular retinol binding proteins. It is not known which form of retinol is oxidized to retinoic acid in vivo. The working hypothesis of this research proposal is that the overall level of cellular retinoic acid production from the free and bound retinol is determined by the individual catalytic properties of both cytosolic and microsomal retinol dehydrogenases and their content in specific tissues. Ethanol inhibition of retinoic acid synthesis will be stronger in tissues where retinoic acid is produced mainly by alcohol-sensitive retinol dehydrogenases.
The specific aims of this application are to: 1) clone the cDNAs for the new human and cytosolic retinol dehydrogenases by screening human cDNA libraries with specific cDNA probes and by RT-PCR amplification of the mRNA from different human tissues with degenerate oligonucleotide primers; 2) express the cDNAs in E. Coli and Baculovirus systems and purify the recombinant enzymes and binding proteins; 3) determine the content of individual retinol dehydrogenases in human tissues that synthesize retinol acid by Western and Northern blot analysis; 4) determine the retinol-oxidizing capacity of recombinant retinol dehydrogenases with the free and bound retinol by kinetic analysis; 5) determine the kinetics of inhibition of retinol dehydrogenases by ethanol, 4-methylpyrazole and citral; 6) determine the effect of ethanol and retinoic acid on the activity; and expression level of retinol dehydrogenases in human cell cultures. Career development objectives are to gain experience in kinetic characterization of alcohol dehydrogenases, methods of identification of the products of retinal metabolism by HPLC, in situ RNA hybridizations, immunohistochemistry, and expression of recombinant proteins in eukaryotic cells. The long-term objective is to establish an independent research program in this field of alcohol research.
| Kedishvili, N Y; Gough, W H; Davis, W I et al. (1998) Effect of cellular retinol-binding protein on retinol oxidation by human class IV retinol/alcohol dehydrogenase and inhibition by ethanol. Biochem Biophys Res Commun 249:191-6 |
