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stearoyl-COA去饱和酶（SCD）是超级氧化还原酶家族的成员 四个跨膜螺旋和一个由九个保守的组氨酸保留的迪隆中心。这 超家族拥有超过20,000名生活王国的成员，但对此功能知之甚少 和这些蛋白质的机制。该提案的总体目标是了解SCD的机制 原子分辨率。 SCD位于内质网膜上，并催化A的形成 饱和脂肪酰基烟灰的双键。 SCD活性降低的小鼠不会肥胖或糖尿病 当喂养高脂饮食时，说明了SCD活性在能量稳态中的重要性。 scd 由于对膜生物合成的需求更高，并且在癌细胞中表达水平上调，并且 抑制SCD活性阻碍了癌细胞的生长。这些结果导致了针对SCD的巨大努力 肥胖，糖尿病和癌症的治疗 SCD的结构和对催化机制的不完全理解。 尚不清楚SCD如何识别其底物并在双键中获得特异性 形成。全吉斯汀协调的二龙中心与其他已知结构的其他二龙中心不同 尚未检查其催化机制。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（B5R），介导电子 从纳德转移到迪隆中心。然而，如何通过动态相互作用将氧化还原循环循环循环 有B5和B5R仍然是一个谜。 我们表达和纯化的功能小鼠SCD1，并将其晶体结构求解至2.6Å分辨率。 我们还表达并纯化了全长B5和B5R，并在体外组装了酶促反应。在 此外，我们在B5R和B5之间以及B5和SCD1之间产生了稳定的二进制复合物，我们 已建立的阿萨斯测量结合伙伴之间的电子转移。这些初步结果 允许我们提出以下三个特定目标： 目标1：表征SCD1的结构和功能。 目标2：了解SCD1 Diron中心的氧化和减少路径。 目标3：了解B5和SCD1之间的电子传输的结构基础以及B5R之间 和B5。