Structural and mechanistic studies of INDY proteins

描述(申请人提供)：在肝脏和脂肪细胞中，胞浆柠檬酸盐是合成脂肪酸、三酰甘油、胆固醇和低密度脂蛋白的主要前体。胞质中柠檬酸的浓度部分依赖于它通过钠离子依赖的柠檬酸转运蛋白通过质膜的直接输入，柠檬酸转运蛋白是二价阴离子/钠离子转运蛋白家族的成员。果蝇转运蛋白基因的突变通过限制卡路里导致脂肪储存减少。同源基因敲除的小鼠更苗条，并免受肥胖和胰岛素抵抗的影响。因此，它在脂肪酸生物合成中的核心作用使NACT成为治疗肥胖症、糖尿病和心血管疾病的小分子药物的特别吸引人的靶点。我们最近测定了细菌Indy同系物的内向构象的3.2°晶体结构。晶体结构使我们能够提出蛋白质的详细运输机制，包括底物专一性、离子专一性、离子-底物耦合和构象变化 蛋白质通过膜完成底物的转运。在目前的项目中，我们将使用诱变和运输试验来测试这种运输机制。我们将通过确定细菌和哺乳动物的Indy蛋白向外构象的结构来进一步表征该蛋白的运输机制。了解这些转运蛋白的转运机制，特别是它们的底物和离子特异性，将有助于设计治疗肥胖和糖尿病的药物。

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.