Enzymes of Skin Lipid Metabolism

描述（由申请人提供）：硫酸胆固醇和蜡单酯是皮肤中丰富的脂质，其生物学作用和生物合成途径在很大程度上仍不明确。硫酸胆固醇由SULT 2B1磺基转移酶合成，被认为是表皮脂质屏障形成中的重要调节和结构组分。产生蜡单酯的生物合成途径尚未在哺乳动物中确定，但通过类比植物和低等生物，认为涉及两种酶的顺序作用，脂肪酰基辅酶A（CoA）还原酶和蜡酯合酶。我们最初的目标将是表征缺乏胆固醇磺基转移酶的小鼠的表型。靶向基因破坏产生了SULT2B1敲除小鼠，将研究其皮肤屏障功能，脂质代谢和生殖。第二个目标是确定胆固醇磺基转移酶在体外的作用，通过表征中国仓鼠卵巢细胞系，过表达SULT 2B1和原代小鼠胚胎成纤维细胞和角质形成细胞，缺乏这种酶。将分析这些细胞的甾醇代谢、基因调控和分化。我们的第三个目标是确定哺乳动物蜡单酯的生物合成途径。生物信息学和表达克隆已经产生了编码两种脂肪酰CoA还原酶和蜡合酶的小鼠cDNA。这些酶的生化特性、亚细胞定位和组织分布将被确定。最终的目标是产生蜡合酶缺陷的小鼠。由此产生的敲除动物将用于确定蜡单酯在皮肤、眼睛和免疫系统中的功能。拟议的研究将为皮肤脂质代谢的酶以及独特的脂质在预防感染，脱水和磨损中发挥的作用提供新的见解。这项研究与皮肤病、干眼症和胆固醇代谢有关。

DESCRIPTION (provided by applicant): Cholesterol sulfate and wax monoesters are abundant lipids in the skin whose biological roles and biosynthetic pathways remain largely undefined. Cholesterol sulfate is synthesized by the SULT2B1 sulfotransferase enzyme and is postulated to be an essential regulatory and structural component in the formation of the epidermal lipid barrier. The biosynthetic pathway that produces wax monoesters has not been defined in mammals but by analogy to plants and lower organisms is thought to involve the sequential actions of two enzymes, a fatty acyl coenzyme A (CoA) reductase and a wax ester synthase. Our initial goal will be to characterize the phenotype of mice that lack cholesterol sulfotransferase. Targeted gene disruption has produced SULT2B1 knockout mice that will be studied with respect to skin barrier function, lipid metabolism and reproduction. The second goal is to determine the role of cholesterol sulfotransferase in vitro by characterizing Chinese hamster ovary cell lines that over-express SULT2B1 and primary mouse embryo fibroblasts and keratinocytes that lack this enzyme. Sterol metabolism, gene regulation, and differentiation of these cells will be analyzed. Our third goal is to define the mammalian wax monoester biosynthetic pathway. Bioinformatics and expression cloning have produced mouse cDNAs encoding two fatty acyl CoA reductases and a wax synthase. The biochemical properties, subcellular Iocalizations, and tissue distributions of these enzymes will be determined. The final objective is to produce mice that are deficient in the wax synthase enzyme. The resulting knockout animals will be used to determine the function of wax monoesters in the skin, eye, and immune system. The proposed studies will provide new insight into the enzymes of skin lipid metabolism and the roles that unique lipids play in preventing infection, dehydration, and abrasion. The research is relevant to skin disease, dry eye syndrome, and cholesterol metabolism.