Structural and mechanistic studies of INDY proteins

描述（由申请方提供）：在肝脏和脂肪细胞中，胞质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。胞质柠檬酸盐浓度部分取决于其通过Na+依赖性柠檬酸盐转运蛋白（二价阴离子/Na+同向转运蛋白（DASS）家族的成员）跨质膜的直接输入。果蝇中转运蛋白基因的突变（INDY）通过热量限制导致脂肪储存减少。同源基因的敲除小鼠既更苗条，又不会肥胖和胰岛素抵抗。因此，其在脂肪酸生物合成中的核心作用使得NaCT成为肥胖症、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。我们最近确定了细菌INDY同系物的3.2晶体结构，其向内的构象。晶体结构使我们能够提出一个详细的运输机制的蛋白质，包括底物特异性，离子特异性，离子-底物耦合和构象变化， 蛋白质经历以完成底物跨膜转运。在目前的项目中，我们将使用诱变和转运试验来测试这种转运机制。我们将通过确定来自细菌和哺乳动物的INDY蛋白的外向构象的结构来进一步表征该蛋白的转运机制。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.