Structure and mechanism of mammalian stearoyl-CoA desaturases

哺乳动物硬脂酰辅酶A去饱和酶的结构和机制

• 批准号: 10202589
• 负责人: AH-LIM TSAI
• 金额: $ 63.29万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202589
• 关键词: Acyl Coenzyme A; Address; Anabolism; Binding; Biochemical Reaction; Biological Assay; Cancer Cell Growth; Catalysis; Cell Proliferation; Cells; Coenzyme A; Communication; Complex; Crystallization; Cytochromes b5; Detergents; Diabetes Mellitus; Drug Targeting; Electron Transport; Endoplasmic Reticulum; Enzymes; Family; Fatty acid glycerol esters; Foundations; Freezing; Geometry; Goals; High Fat Diet; Histidine; Homeostasis; Hydrogen; In Vitro; Insecta; Ions; Iron; Kinetics; Knowledge; Length; Life; Ligands; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Mediating; Membrane; Membrane Proteins; Modeling; Molecular Conformation; Movement; Mus; Mutation; NADH; Nitric Oxide; Obesity; Oxidation-Reduction; Oxidoreductase; Oxygen; Palmitoyl Coenzyme A; Pathway interactions; Phospholipids; Positioning Attribute; Problem Solving; Proteins; Reaction; Resolution; Role; Saturated Fatty Acids; Specificity; Spin Trapping; Stearoyl-CoA Desaturase; Structure; System; Testing; Visualization; Zinc; base; cancer cell; cytochrome b5 reductase; diabetic; drug development; fatty acid oxidation; in vitro Assay; insight; lipid biosynthesis; member; nanodisk; novel; obesity treatment; oxidation; pi bond; preference; proteoliposomes; reconstitution; screening; stearoyl-coenzyme A

Mammalian stearoyl-CoA desaturase (SCD) is a member of a super family of redox enzymes that have four transmembrane helices and a diiron center composed of nine conserved histidine residues. This superfamily has over 20,000 members from all kingdoms of life and yet very little is known about the function and mechanism of these proteins. The overall goal of the proposal is to understand the mechanism of SCD at the atomic resolution. SCD resides on endoplasmic reticulum membrane and catalyzes the formation of a double bond on saturated fatty acyl-CoAs. Mice with reduced SCD activity do not become obese or diabetic when fed with a high-fat diet, illustrating the significance of SCD activity in energy homeostasis. SCD expression level is upregulated in cancer cells because of a higher demand for membrane biosynthesis, and inhibition of SCD activity thwarts cancer cell growth. These results led to intense efforts on targeting SCDs for the treatment of obesity, diabetes, and cancer, but the efforts are hampered by a lack of 3-dimenional structures of SCD and incomplete understanding of the catalytic mechanism. It remains unclear how SCDs recognize their substrates and achieve specificity in double bond formation. The all-histidine coordinated diiron center is different from other diiron centers of known structures and its mechanism of catalysis has not been examined. In addition, sustained SCD activity requires two additional membrane proteins, cytochrome b5 (b5) and cytochrome b5 reductase (b5R), which mediate electron transfer from NADH to the diiron center. Yet how the diiron center is redox-cycled through dynamic interactions with b5 and b5R remains a mystery. We expressed and purified functional mouse SCD1 and solved its crystal structure to 2.6 Å resolution. We also expressed and purified full length b5 and b5R, and assembled the enzymatic reactions in vitro. In addition, we produced stable binary complexes between b5R and b5 and between b5 and SCD1, and we established assays to measure electron transfer between the binding partners. These preliminary results allowed us to propose the following three Specific Aims: Aim 1: To characterize the structure and function of SCD1. Aim 2: To understand the oxidative and reductive pathways of the SCD1 diiron center. Aim 3: To understand the structural basis of electron transfer between b5 and SCD1 and between b5R and b5.

哺乳动物硬脂酰辅酶A去饱和酶（SCD）是氧化还原酶超家族的成员， 四个跨膜螺旋和由九个保守的组氨酸残基组成的二铁中心。这 超家族有来自所有生命王国的超过20，000个成员，然而对它的功能知之甚少。 和这些蛋白质的作用机制。建议书的整体目标是让市民了解社署的运作机制， 原子分辨率。SCD存在于内质网膜上，并催化 饱和脂肪酰辅酶A上的双键。SCD活性降低的小鼠不会变得肥胖或糖尿病 当喂食高脂饮食时，说明SCD活性在能量稳态中的重要性。SCD 由于对膜生物合成的更高需求，表达水平在癌细胞中上调， 抑制SCD活性阻碍癌细胞生长。这些结果导致了针对SCD的密集努力， 肥胖症、糖尿病和癌症的治疗，但由于缺乏三维模型， SCD的结构和催化机理的不完全理解。 SCD如何识别底物并实现双键的特异性仍不清楚 阵全组氨酸配位的双铁中心不同于其他已知结构的双铁中心 其催化机理尚未研究。此外，持续的SCD活动需要两个 其他膜蛋白，细胞色素b5（b5）和细胞色素b5还原酶（b5R），介导电子 从NADH转移到二铁中心。然而，二铁中心如何通过动态相互作用进行氧化还原循环， 与b5和b5R的关系仍然是个谜。 我们表达并纯化了功能性小鼠SCD 1，并将其晶体结构解析到2.6 nm分辨率。 我们还表达和纯化了全长b5和b5R，并在体外组装了酶促反应。在 此外，我们在b5 R和b5之间以及b5和SCD 1之间产生了稳定的二元复合物， 建立了测定结合配偶体之间电子转移的测定法。这些初步结果 我们提出了以下三个具体目标： 目的1：研究SCD 1的结构和功能。 目的2：了解SCD 1二铁中心的氧化和还原途径。 目的3：了解b5和SCD 1之间以及b5 R之间电子转移的结构基础 和B5。