Biosynthesis of 9-cis-retinoic acid in vivo. The roles of different retinol dehydrogenases and a structure-activity analysis of microsomal retinol dehydrogenases.
Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion protein-based luciferase reporter assay, the formation of 9-cis-retinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cis-retinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.
Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden.
K
K
Tryggvason
A
A
Romert
U
U
Eriksson
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D000077556:Alitretinoin; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017830:Octoxynol / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D051381:Rats; D050697:Retinal Dehydrogenase; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism
D003062:Codon; D003902:Detergents; D014801:Vitamin A; D000077556:Alitretinoin; D014212:Tretinoin; D017830:Octoxynol; D000429:Alcohol Oxidoreductases; C109795:cis-retinol - androgen dehydrogenase; D000426:Alcohol Dehydrogenase; D008156:Luciferases; D000445:Aldehyde Oxidoreductases; D050697:Retinal Dehydrogenase; D019286:Endopeptidase K