Molecular mechanism of nucleobase/vitamin C transporters

核碱基/维生素C转运蛋白的分子机制

基本信息

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
项目类别：
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&apos 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

项目摘要

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) 基因家族的成员在哺乳动物的所有活体和维生素 C 领域运输核碱基，维生素 C（L-抗坏血酸）是一种必需的微量营养素。自由基的抗氧化剂清除剂，作为许多酶反应的辅助因子，核碱基的运输涉及 DNA 和 RNA 合成等关键过程，此外，核碱基的细胞递送在治疗应用中引起了特别的兴趣，因为核碱基类似物目前用于治疗实体瘤、淋巴增殖性疾病、肝炎和艾滋病等病毒感染以及一些炎症性疾病。，例如克罗恩病，尽管 NAT 在健康、疾病和药物治疗中很重要，但有关其转运机制的详细信息对于开发其作为高效药物靶点的潜力至关重要，在这项多项 PD/PI 提案中，我们以最近令人兴奋的 2.85 Å 分辨率的细菌 NAT 同系物 (PaaTCp) 的鉴定和结晶为基础，试图了解 NAT 家族成员之间的机制共性和差异。 C 分别以 H+ 和 Na+ 依赖的方式，该项目旨在阐明细菌和人类 NAT 家族成员的底物识别和易位的基本机制。具体目标如下： (1) 确定底物和药物结合位点目标是将 PaaTCp 与其底物（嘌呤和维生素 C）和核碱基类似物共结晶，然后使用结构作为功能验证的指导。通过突变研究结合放射性示踪剂结合来确定底物和药物结合位点；(2) 开发 PaaTCp 的转运模型，目标是定量了解 H+- 和 Na+ 依赖性底物。运输，包括识别 H+ 和 Na+ 位点以及阐明潜在离子（H+ 和 Na+）耦合机制的化学计量，并精确描述运输动力学；以及药物如何影响这些转变。以向外和向内的构象结晶 PaaTCp，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替代构象的结构时，推断构象变化；(4) 通过探索底物结合的关键要素及其偶联，确定我们在 PaaTCp 中的结构和功能发现与理解人类 SVCT 功能的相关性；离子动力来开发具有 PaaTCp 折叠的蛋白质的普遍适用的功能机制模型。