Structural and mechanistic studies of INDY proteins

描述（由申请人提供）：在肝脏和脂肪细胞中，胞质柠檬酸盐是合成脂肪酸、甘油三酯、胆固醇和低密度脂蛋白的主要前体。胞内柠檬酸盐浓度部分取决于其通过钠离子依赖的柠檬酸转运体（二价阴离子/钠离子同向转运体（DASS）家族的一员）穿过质膜的直接进口。果蝇的转运体基因突变（INDY）通过限制卡路里导致脂肪储存减少。同源基因的敲除小鼠既苗条，又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成为肥胖、糖尿病和心血管疾病小分子治疗剂的一个特别有吸引力的靶点。我们最近确定了细菌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提出蛋白质的详细运输机制，包括底物特异性，离子特异性，离子-底物偶联和构象变化

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.