Molecular mechanism of nucleobase/vitamin C transporters

核碱基/维生素C转运蛋白的分子机制

• 批准号: 9900837
• 负责人: Matthias Quick
• 金额: $ 35.32万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900837
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Anemia; Anions; Antioxidants; Archaea; Ascorbic Acid; Ascorbic Acid Deficiency; Automobile Driving; Bacteria; Bicarbonates; Binding; Biochemical; Biochemical Reaction; Biological Assay; Carrier Proteins; Cells; Cessation of life; Collaborations; Coupling; Crohn' s disease; Crystallization; Crystallography; Cysteine; DNA biosynthesis; Dependence; Disease; Drug Binding Site; Drug Delivery Systems; Drug Targeting; Electrophysiology (science); Elements; Exhibits; Family; Family member; Fatigue; Free Radical Scavengers; Gene Family; Goals; Health; Hemorrhage; Hepatitis; Homologous Gene; Human; Impaired wound healing; Individual; Inflammatory; Intestines; Ions; Kidney; Kinetics; Life; Lymphoproliferative Disorders; Mammals; Measurement; Mediating; Membrane; Mental Depression; Metabolic; Methods; Micronutrients; Modeling; Molecular; Molecular Conformation; Mutation; Organism; Pathway interactions; Petechiae; Pharmaceutical Preparations; Pharmacotherapy; Physiology; Process; Protein Conformation; Proteins; Protista; Purines; RNA chemical synthesis; Rattus; Regulation; Resolution; Resources; Role; Scurvy; Signal Transduction; Site; Solid Neoplasm; Structure; Substrate Specificity; Therapeutic; Time; Ursidae Family; Virus Diseases; Vitamins; absorption; ascorbate; cofactor; crosslink; design; fungus; interest; member; multidisciplinary; nucleobase; nucleobase analog; nutrition; protein function; public health relevance; radiotracer; sodium DEPENDENDENT vitamin C transporter 1; sodium-dependent vitamin C transporter 2; solute; stoichiometry; uptake

 DESCRIPTION (provided by applicant): Members of the nucleobase/ascorbate transporter (NAT) gene family transport nucleobases in all kingdoms of live and vitamin C in mammals. In humans, vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant scavenger of free radicals and as a cofactor in many enzymatic reactions. Transport of nucleobases is implicated in crucial processes such as DNA and RNA synthesis, cell signaling, and metabolic regulation. In addition, the cellular delivery of nucleobases has gained special interest in therapeutic applications as nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases, e.g., Crohn's disease. Despite the importance of NATs in health, disease, and pharmacotherapy, detailed information about their transport mechanism, which is crucial to exploit their potential as target for drugs with high efficacy, is limited. In this multple PD/PI proposal we seek to understand mechanistic commonalities and differences among members of the NAT family. Building on our recent exciting identification and crystallization of a bacterial NAT homolog (PaaTCp) at 2.85 Å resolution that transports nucleobases and vitamin C in H+ and Na+-dependent fashion, respectively, this project is designed to elucidate basic mechanisms of substrate recognition and translocation in both bacterial and human NAT family members. We propose the following Specific Aims: (1) to identify the substrate and drug binding site(s). The goal is to co-crystallize PaaTCp with its substrates (purines and vitamin C) and nucleobase analogs and then use the structures as a guide to functionally validate the substrate and drug binding sites by mutational studies in conjunction with radiotracer binding; (2) to develop a model of transport for PaaTCp. The goal is to obtain a quantitative understanding of H+- and Na+-dependent substrate transport, including the identification of the H+ and Na+ sites and the elucidation of the stoichiometry of the potential ion (H+ and Na+) coupling mechanism, and to describe precisely the kinetics of transport; (3) to illustrate conformational changes associated with (co)substrate translocation and how drugs affect these transitions. The goal is to crystallize PaaTCp in outward- and inward-facing conformations, and to use crosslinking and cysteine accessibility assays to validate the structures, or when structures with alternate conformations are not attainable, to deduce conformational changes; (4) to establish the relevance of our structural and functional findings in PaaTCp to understanding the function of the human SVCTs by exploring the key elements of substrate binding, and its coupling to the ion motive force to develop a general applicable mechanistic model of function for proteins with PaaTCp fold.

 描述（由适用提供）：在哺乳动物中所有活的王国和维生素C的核心酶/抗坏血酸转运蛋白转运蛋白（NAT）基因家族转运核基础上。在人类中，维生素C（L-抗坏血酸）是必不可少的微量营养素，可作为自由基的抗氧化剂清除剂，在许多酶促反应中用作辅助因子。核基底的运输在关键过程中隐含，例如DNA和RNA合成，细胞信号传导和代谢调节。此外，由于目前使用核类似物在治疗实体瘤，淋巴细胞增生性疾病，肝炎和艾滋病等炎性疾病等核类似物中，核核酶的细胞递送已在治疗应用中获得了特殊兴趣。尽管NAT在健康，疾病和药物疗法中的重要性，但有关其运输机制的详细信息，但在这项多重PD/PI建议中，我们试图了解NAT家族成员之间的机械共享和差异。基于我们最近以2.85Å分辨率对细菌NAT同源物（PAATCP）的令人兴奋的鉴定和结晶的基础，该分辨率分别以H+​​和Na+依赖性的方式运输核仁蛋白C和维生素C，该项目旨在阐明细菌和人类NAT家族成员的基本识别和转化的基本机制。我们提出以下特定目的：（1）识别底物和药物结合位点。目的是将PAATCP与其底物（嘌呤和维生素C）和核碱酶类似物共结合，然后使用结构作为指南，通过突变研究与放射性臂结合结合进行突变研究，以便在功能上验证底物和药物结合位点； （2）为PAATCP开发运输模型。目的是获得对H+和Na+依赖性底物转运的定量理解，包括鉴定H+和Na+位点以及对电势离子（H+和Na+）偶联机制的化学计量的阐明，并确切地描述转运的动力学； （3）说明与（CO）底物翻译相关的构象变化以及药物如何影响这些过渡。目标是在向外和向内的会议上结晶PAATCP，并使用交联和半胱氨酸可访问性测定法来验证结构，或者当无法获得替代会议的结构时，以推导构象变化； （4）通过探索底物结合的关键要素及其与离子动力的耦合，以开发一种与PAATCP折叠一起开发蛋白质功能的一般机械模型，以建立PAATCP中结构和功能性发现的相关性，以了解人类SVCT的功能。