Klyuyeva Alla V, Goggans Kelli R, Kedishvili Natalia Y, Belyaeva Olga V
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL, United States.
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL, United States.
Methods Enzymol. 2020;637:493-512. doi: 10.1016/bs.mie.2020.02.013. Epub 2020 Mar 28.
Several human enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins exhibit catalytic oxidoreductive activity toward retinoid substrates in vitro. For some retinoid-active enzymes, their physiological significance for retinoid metabolism is supported by phenotypes linked to naturally occurring mutations in human genes or by targeted gene knockout studies of their murine homologs. However, for those enzymes that are not well conserved or display properties different from their murine counterparts, evaluation of their physiological roles can be challenging. Here, we describe the adaptation of stratified organotypic culture of human epidermis for evaluating the contribution of human putative SDR retinol dehydrogenases to biosynthesis of all-trans-retinoic acid in a three-dimensional cellular model highly sensitive to the levels of all-trans-retinol and all-trans-retinoic acid. In addition to providing a valuable readout of metabolic changes occurring in the presence or absence of the enzyme of interest, this model allows for evaluation of the effects of various retinoid and rexinoid therapeutic compounds on retinoic acid signaling, cell growth and differentiation.
蛋白质短链脱氢酶/还原酶（SDR）超家族中的几种人类酶在体外对类视黄醇底物表现出催化氧化还原活性。对于一些具有类视黄醇活性的酶，人类基因中自然发生的突变所关联的表型或其小鼠同源物的靶向基因敲除研究，支持了它们在类视黄醇代谢中的生理意义。然而，对于那些保守性不强或表现出与小鼠对应物不同特性的酶，评估它们的生理作用可能具有挑战性。在此，我们描述了人类表皮分层器官型培养的适应性，用于在对全反式视黄醇和全反式视黄酸水平高度敏感的三维细胞模型中评估人类假定的SDR视黄醇脱氢酶对全反式视黄酸生物合成的贡献。除了提供在有或没有感兴趣的酶存在时发生的代谢变化的有价值读数外，该模型还允许评估各种类视黄醇和视黄酸类治疗化合物对视黄酸信号传导、细胞生长和分化的影响。
