Substrate Specificity Determinants in Nutrient Solute Carrier Transporters

总结 在人类中，有超过460种溶质载体（SLC）转运蛋白介导溶质的输入和流出， 包括神经递质营养物质和离子SLC正在成为各种疾病的关键治疗靶点。 疾病和失调的根源例如，营养SLC转运蛋白在重编程代谢中起主要作用。 通过为细胞提供用于治疗癌症、自身免疫性疾病和心脏缺血的营养物质， 构建生物量或作为信号分子，增强细胞增殖和分化，或调节细胞 死亡我们的主要目标是描述营养物质中的底物和抑制剂特异性决定因素， 转运蛋白，并开发新的策略来调节其功能。我们采取综合方法 包括计算生物学和有机化学，再加上生物化学和生物物理学， 方法和体外疾病模型，来表征丙氨酸-丝氨酸-半胱氨酸转运蛋白2（ASCT 2， SLC1A5）。 在本项目的目标1中，我们将在多个基因组中生成和完善人SLC 1家族的同源模型。 构象我们将描述模型的底物结合位点的结构特征和最近发现的 决定底物和抑制剂特异性的变构位点。关键残基将采用定点 诱变和电生理学方法。最后，我们将开发一个可公开访问的数据库， 包括所有可建模的人类SLC转运蛋白的同源模型，并将疾病相关突变映射到 模型，并提供其预测的功能效果。 目标二：合理设计调节营养素功能的小分子工具化合物 通过不同的机制进行运输。这将扩大转运蛋白抑制剂的化学空间， 对目标1中开发的模型进行测试。我们将重点关注以下方法：（一）优化和 表征新型构象特异性抑制剂，如可光活化的和荧光的化合物， 与ASCT 2的底物结合位点相互作用;（B）设计靶向结构域-结构域的变构抑制剂 ASCT 2中的界面，以测试该转运蛋白的推定的升降机机制;以及（C）开发具有 对ASCT 2和L型氨基酸转运蛋白1（LAT 1，SLC 7A 5）具有双重特异性，我们假设 由于它们在重编程代谢中的协同性， 网络. 如果成功的话，这个项目将提高我们对新兴的结构-功能关系的理解。 治疗目标该项目还将提供新的化学探针，以进一步表征 这些蛋白质及其在疾病中的作用，以及一个有用的公开在线资源。

SUMMARY In humans, there are over 460 Solute Carrier (SLC) transporters that mediate the import and efflux of solutes, including neurotransmitters, nutrients, and ions. The SLCs are emerging as key therapeutic targets for a variety of diseases and disorders. For example, nutrient SLC transporters play a major role in reprogrammed metabolic networks in cancer, autoimmune disease, and heart ischemia, by supplying cells with nutrients that are used to build biomass or serve as signaling molecules that enhance cell proliferation and differentiation, or regulate cell death. Our broad goal is to describe the substrate and inhibitor specificity determinants in nutrient transporters and develop novel strategies to modulate their functions. We take an integrative approach that includes computational biology and organic chemistry, coupled with biochemical and biophysical approaches, and in vitro models of disease, to characterize the Alanine-Serine-Cysteine Transporter 2 (ASCT2, SLC1A5). In Aim 1 of this project, we will generate and refine homology models for the human SLC1 family in multiple conformations. We will describe structural features of the models’ substrate binding site and recently discovered allosteric site(s), that determine substrate and inhibitor specificity. Key residues will be tested with site-directed mutagenesis and electrophysiological approaches. Finally, we will develop a publicly-accessible database that includes homology models for all modelable human SLC transporters, and maps disease-related mutations onto the models and provides their predicted functional effect. In Aim 2, we will rationally design small molecule tool compounds that modulate the function of nutrient transporters via distinct mechanisms. This will expand the chemical space of transporter inhibitors and provide a test for the models developed in Aim 1. We will focus on the following approaches: (A) optimizing and characterizing novel conformation-specific inhibitors, such as photoactivatable and fluorescent compounds that interact with the substrate binding site of ASCT2; (B) designing allosteric inhibitors targeting the domain-domain interface in ASCT2, to test the putative elevator mechanism of this transporter; and (C) developing inhibitors with dual specificity for ASCT2 and the L-type Amino Acid Transporter 1 (LAT1, SLC7A5), which we hypothesize to have a pronounced inhibitory effect on cell proliferation due to their cooperativity in reprogrammed metabolic networks. If successful, this project will improve our understanding of structure-function relationships in emerging therapeutic targets. This project will also provide novel chemical probes to further characterize the structure of these proteins and their role in disease, as well as a useful publicly available online resource.