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: 9127471
• 负责人: Matthias Quick
• 金额: $ 43.55万
• 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/9127471
• 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; 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(申请人提供)：核酸基/抗坏血酸转运体(NAT)基因家族的成员在哺乳动物中运输所有物种的核酸基和维生素C。在人体内，维生素C(L-抗坏血酸)是一种基本的微量营养素，它作为自由基的抗氧化剂清除，并在许多酶反应中作为辅助因子。碱基的运输涉及DNA和RNA合成、细胞信号传递和代谢调节等关键过程。此外，由于核酸基类似物目前被用于治疗实体肿瘤、淋巴增生性疾病、肝炎和艾滋病等病毒感染以及一些炎症性疾病(如克罗恩病)，因此核酸基的细胞递送在治疗应用中获得了特别的兴趣。尽管NAT在健康、疾病和药物治疗中非常重要，但有关其转运机制的详细信息仍然有限，这对于开发其作为高效药物靶点的潜力至关重要。在这个包含多个PD/PI的提案中，我们试图了解NAT家族成员之间的机械共性和差异。在我们最近令人兴奋的鉴定和结晶细菌NAT同源物(PaaTCp)的基础上，2.85ä分辨率分别以H+和Na+依赖的方式运输碱基和维生素C，该项目旨在阐明细菌和人类NAT家族成员底物识别和转位的基本机制。我们提出了以下具体目标：(1)确定底物和药物结合部位(S)。目标是使PaaTCp与其底物(嘌呤和维生素C)和碱基类似物共结晶，然后以这些结构为指南，通过结合放射性示踪剂结合的突变研究从功能上验证底物和药物结合部位；(2)开发PaaTCp的运输模型。其目的是对依赖于H+和Na+的底物转运有一个定量的了解，包括H+和Na+位置的鉴定和潜在离子(H+和Na+)偶联机制的化学计量学的阐明，并准确地描述运输的动力学；(3)说明与(Co)底物转运相关的构象变化以及药物如何影响这些转变。我们的目标是使PaaTCp以外向和向内的构象结晶，并使用交联和半胱氨酸可及性分析来验证结构，或者当无法获得替代构象的结构时，推断构象变化；(4)通过探索底物结合的关键元素及其与离子推动力的耦合，建立具有PaaTCp折叠的蛋白质的通用适用机制模型，从而建立我们在PaaTCp中的结构和功能发现与理解人类SVCT功能的相关性。