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: 9346958
• 负责人: Matthias Quick
• 金额: $ 2.89万
• 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 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9346958
• 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; Dependency; 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; Organism; Pathway interactions; Petechiae; Pharmaceutical Preparations; Pharmacotherapy; Physiology; Process; Proteins; Protista; Purines; RNA chemical synthesis; Rattus; Regulation; Resolution; Resources; Role; Scurvy; Signal Transduction; Site; Solid Neoplasm; Structural Genes; 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; 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（由申请人提供）：核碱基/抗坏血酸转运蛋白（NAT）基因家族成员在哺乳动物的所有生物界和维生素C中转运核碱基。维生素C（L-抗坏血酸）是人体必需的微量营养素，可作为自由基的抗氧化剂清除剂和许多酶促反应中的辅助因子。核碱基的转运与DNA和RNA合成、细胞信号传导和代谢调节等关键过程有关。此外，核碱基的细胞递送在治疗应用中获得了特别的兴趣，因为核碱基类似物目前用于治疗实体瘤、淋巴增生性疾病、病毒感染如肝炎和AIDS以及一些炎性疾病，例如，克罗恩氏病。尽管NAT在健康，疾病和药物治疗中的重要性，但关于其转运机制的详细信息是有限的，这对于开发其作为高效药物靶点的潜力至关重要。在这个多PD/PI提案中，我们试图了解NAT家族成员之间的机制共性和差异。我们最近令人兴奋的鉴定和结晶的细菌NAT同源物（PaaTCp）在2.85 μ m的分辨率，运输核碱基和维生素C在H+和Na+依赖的方式，分别，该项目旨在阐明底物识别和易位的基本机制，在细菌和人类NAT家族成员。我们提出了以下具体目标：（1）确定底物和药物结合位点。目标是使PaaTCp与其底物（嘌呤和维生素C）和核碱基类似物共结晶，然后使用结构作为指导，通过突变研究结合放射性示踪剂结合来功能性地验证底物和药物结合位点;（2）开发PaaTCp的转运模型。目的是获得对H+和Na+依赖的底物转运的定量理解，包括H+和Na+位点的鉴定和潜在离子（H+和Na+）耦合机制的化学计量的阐明，并精确地描述转运的动力学;（3）说明与（共）底物转运相关的构象变化以及药物如何影响这些转变。目标是使PaaTCp以面向外和面向内的构象结晶，并使用交联和半胱氨酸可及性测定来验证结构，或者当无法获得具有交替构象的结构时，推断构象变化;（4）通过探索底物结合的关键元件，建立我们在PaaTCp中的结构和功能发现与理解人类SVCT功能的相关性，以及它与离子动力的耦合，以开发具有PaaTCp折叠的蛋白质的功能的通用机制模型。