Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington (G.Z., C.J.S., H.X., N.I.); Department of Chemistry, Washington State University, Pullman, Washington (E.M.P., J.P.J.); and Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, Washington (K.-L.H., N.P.K.).
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington (G.Z., C.J.S., H.X., N.I.); Department of Chemistry, Washington State University, Pullman, Washington (E.M.P., J.P.J.); and Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, Washington (K.-L.H., N.P.K.)
Drug Metab Dispos. 2021 Mar;49(3):202-211. doi: 10.1124/dmd.120.000296. Epub 2020 Dec 18.
All--retinoic acid (RA) is a critical endogenous signaling molecule. RA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to RA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to RA formation in human liver. Human recombinant AOX formed RA from retinaldehyde (K ∼1.5 ± 0.4 µM; k ∼3.6 ± 2.0 minute). In human liver S9 fractions (HLS9), RA formation was observed in the absence of NAD, suggesting AOX contribution to RA formation. In the presence of NAD, Eadie-Hofstee plots of RA formation in HLS9 indicated that two enzymes contributed to RA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of RA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of RA in the presence of NAD correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to RA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter RA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to RA and contributes to hepatic RA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic RA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic RA concentrations and signaling and alter vitamin A homeostasis.
全反式视黄酸（RA）是一种重要的内源性信号分子。RA 主要由视黄醛通过醛脱氢酶 1A1（ALDH1A1）合成，但醛氧化酶（AOX）也可能有助于 RA 的生物合成。本研究的目的是检验 AOX 对人肝中 RA 形成有重要贡献的假设。人重组 AOX 可从视黄醛中形成 RA（K∼1.5±0.4µM；k∼3.6±2.0 分钟）。在人肝 S9 级分（HLS9）中，在没有 NAD 的情况下观察到 RA 的形成，表明 AOX 有助于 RA 的形成。在 NAD 存在的情况下，HLS9 中 RA 形成的 Eadie-Hofstee 图表明有两种酶参与 RA 的形成。这两种酶基于 AOX 抑制剂肼屈嗪（20%-50%抑制）和 ALDH1A1 抑制剂 WIN18,446（50%-80%抑制）对 RA 形成的抑制作用，被鉴定为 AOX 和 ALDH1A1。通过液相色谱-串联质谱（LC-MS/MS）定量鉴定特征肽，在 HLS9 中 AOX 的表达为 9.4-24 pmol mg S9 蛋白，而 ALDH1A1 的表达为 156-285 pmol mg S9 蛋白。在 NAD 存在下 RA 形成的速度与 ALDH1A1 和 AOX 蛋白的表达显著相关。总之，这些数据表明 AOX 和 ALDH1A1 均有助于人肝中 RA 的生物合成，其中 ALDH1A1 是高亲和力、低容量酶，而 AOX 是低亲和力、高容量酶。结果表明，在 ALDH1A 功能障碍或过量维生素 A 的情况下，AOX 可能在调节肝脏维生素 A 稳态方面发挥重要作用，而 AOX 的抑制可能改变 RA 的生物合成和信号转导。意义声明：本研究提供了直接证据，表明人 AOX 将视黄醛转化为 RA，并有助于肝 RA 的生物合成。发现 AOX 可能负责整体肝 RA 形成的 20%-50%表明，通过药物相互作用、遗传多态性或疾病状态改变 AOX 活性可能会影响肝 RA 浓度和信号转导，并改变维生素 A 稳态。
