Structural and mechanistic studies of INDY proteins

INDY 蛋白的结构和机制研究

• 批准号: 8531420
• 负责人: DANENG WANG
• 金额: $ 36.87万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8531420
• 关键词: Adipocytes; Adverse effects; Affinity; Amino Acid Sequence; Anions; Bacteria; Binding; Binding Sites; Biological Assay; Bipolar Disorder; Caloric Restriction; Cardiovascular Diseases; Cations; Cell membrane; Cells; Citrates; Coupling; Crystallization; Dependence; Diabetes Mellitus; Dimerization; Doctor of Philosophy; Drug Design; Family; Fatty Acids; Fatty acid glycerol esters; Genes; Glycolysis; Hepatocyte; Homologous Gene; Human; Insulin Resistance; Ions; Kinetics; Knockout Mice; Knowledge; LDL Cholesterol Lipoproteins; Longevity; Mammals; Measures; Membrane; Membrane Transport Proteins; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Mutagenesis; Mutation; Obesity; Paper; Pathway interactions; Principal Investigator; Proteins; Protomer; Rattus; Reporting; Role; Site; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; Succinates; Testing; Therapeutic Agents; Triglycerides; Weight Gain; base; carboxylate; citrate carrier; design; dicarboxylate; dimer; energy balance; fatty acid biosynthesis; fly; improved; inhibitor/antagonist; member; nanobodies; novel; novel strategies; oxidation; programs; protein structure; public health relevance; small molecule; sodium ion; symporter

DESCRIPTION (provided by applicant): In liver and adipose cells, cytosolic citrate is a major precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. The cytosolic citrate concentration partially depends on its direct import across the plasma membrane via the Na+-dependent citrate transporter, a member of the divalent anion/Na+ symporter (DASS) family. Mutations of the transporter gene in flies (INDY) result in reduced fat storage through calorie restriction. Knockout mice of the homologous gene are both slimmer and protected from obesity and insulin resistance. Thus, its central role in fatty acid biosynthesis makes NaCT a particularly attractive target of small-molecule therapeutic agents for obesity, diabetes and cardiovascular diseases. We have recently determined the 3.2 ¿ crystal structure of a bacterial INDY homolog in its inward-facing conformation. The crystal structure allows us to propose a detailed transport mechanism for the protein, including substrate specificity, ion specificity, ion- substrate coupling and conformational changes that the protein undergoes to accomplish substrate translocation across the membrane. In the current project, we will test this transport mechanism using mutagenesis and transport assays. We will further characterize the transport mechanism of the protein by determining the structure of the outward-facing conformation of the INDY protein from bacteria and mammals. Understanding of the transport mechanism of these transporters, particularly their substrate and ion specificity, will help in the design of drugs for obesity and diabetes.

描述（由应用提供）：在肝脏和脂肪细胞中，柠檬酸含量是合成脂肪酸，三酰基甘油，胆固醇和低密度脂蛋白的主要先驱。细胞质柠檬酸盐浓度部分取决于其通过Na+依赖性柠檬酸转运蛋白（Divalent阴离子/Na+ Semporter（DASS）家族的成员）的Na+依赖性柠檬酸转运蛋白的直接进口。转运蛋白基因在苍蝇中的突变（Indy）导致通过卡路里限制减少脂肪储存。同源基因的基因敲除小鼠既纤细，又受到保护，免受肥胖和胰岛素抵抗。这是它在脂肪酸生物合成中的核心作用，使NACT成为肥胖，糖尿病和心血管疾病的小分子治疗剂的特别有吸引力的靶标。我们最近已经确定了细菌Indy同源物在其内向构象中的3.2晶体结构。晶体结构使我们能够为蛋白质提出详细的传输机制，包括底物特异性，离子特异性，离子底物耦合和构象变化 蛋白质经历以完成整个膜的底物运输。在当前的项目中，我们将使用诱变和转运方法测试这种运输机制。我们将通过确定来自细菌和哺乳动物的Indy蛋白的向外构象的结构来进一步表征蛋白质的转运机制。了解这些转运蛋白的运输机制，尤其是其底物和离子特异性，将有助于设计肥胖和糖尿病的药物。