Mechanistic studies of prokaryotic and eukaryotic nitrate/nitrite transport

原核和真核硝酸盐/亚硝酸盐转运机制研究

• 批准号: 10302305
• 负责人: Hongjin Zheng
• 金额: $ 30.47万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302305
• 关键词: 3-Dimensional; Address; Affect; Affinity; Anti-Inflammatory Agents; Binding; Biological; Biology; Blood Circulation; Blood Glucose; Blood flow; CD69 antigen; Cancerous; Cardiovascular system; Cellular Membrane; Chemistry; Communities; Crystallization; Defect; Dyslipidemias; Equilibrium; Escherichia coli; Event; Genetic Diseases; Goals; Health; Homeostasis; Homo sapiens; Human; Human body; Hypoxia; Ions; Knowledge; Life; Light; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Molecular; Molecular Conformation; Movement; Mutagenesis; Nitrates; Nitric Oxide; Nitrites; Nitrogen; Nitrogen Dioxide; Nutrient; Oral cavity; Outcome; Pathogenesis; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Play; Process; Protein Biochemistry; Protein Conformation; Proteins; Resolution; Role; Salivary Glands; Structure; Structure-Activity Relationship; Time; antiporter; commensal bacteria; dietary; drug development; flexibility; high dimensionality; improved; inhibitor; malignant stomach neoplasm; nitrate transporter; potassium nitrate; protective effect; structural biology; translational impact; uptake

Project Summary Recent studies suggest that nitrate and nitrite molecules have profound beneficial effects to human health, though they were thought to be cancerous since 1970s. To maximize their pharmaceutical potential, we need to first understand how nitrate and nitrite circulate in humans. Such circulation depends on two critical events: active accumulation of nitrate mediated by sialin transporters in salivary glands, as well as nitrate uptake and nitrite secretion by commensal bacteria in the mouth. Thus, nitrate/nitrite molecules have to cross different cellular membranes multiple times, before being used by humans. These translocation processes are mediated by a group of membrane proteins called nitrate transporters. To understand how these transporters function, we solved high-resolution crystal structures of NarK from E. coli, and further demonstrated that, surprisingly, NarK is a nitrate/nitrite exchanger. Despite the progress we made, the detailed molecular mechanisms of nitrate/nitrite translocation are still largely unknown. In this proposal, we aim to fill the knowledge gap by: 1) understand substrate selectivity and conformational flexibility using directed-mutagenesis of NarK, so to better understand the function of NarK at the molecular level; 2) obtain high-resolution structures of NarK in previously unobserved conformation, so we can reconstruct the complete transport cycle of NarK; 3) explore the structure-function relationship of human nitrate transporter sialin, so we will understand the similarities and differences among nitrate transporters from diverse species. Overall, upon completion of the proposal, we expect to expand our general understanding of the nitrate/nitrite transport, and shed light on the crucial roles that nitrate transporters (both eukaryotic and prokaryotic) play in nitrate/nitrite circulation. The knowledge gained here will facilitate potential drug development related to bacterial nitrate transporters and human sialin.

项目概要 最近的研究表明硝酸盐和亚硝酸盐分子对人类健康具有深远的有益影响， 尽管自 20 世纪 70 年代以来，它们就被认为是癌症。为了最大限度地发挥其制药潜力，我们需要 首先了解硝酸盐和亚硝酸盐如何在人体中循环。这种循环取决于两个关键事件： 唾液腺中唾液酸转运蛋白介导的硝酸盐主动积累，以及硝酸盐吸收和 口腔中的共生细菌分泌亚硝酸盐。因此，硝酸盐/亚硝酸盐分子必须穿过不同的 在被人类使用之前，细胞膜会被多次破坏。这些易位过程是介导的 由一组称为硝酸盐转运蛋白的膜蛋白实现。为了了解这些转运蛋白如何发挥作用， 我们从大肠杆菌中解析出 NarK 的高分辨率晶体结构，并进一步证明，令人惊讶的是， NarK 是一种硝酸盐/亚硝酸盐交换剂。尽管我们取得了进展，但详细的分子机制 硝酸盐/亚硝酸盐易位仍然很大程度上未知。在本提案中，我们的目标是通过以下方式填补知识空白：1) 使用 NarK 的定向诱变了解底物选择性和构象灵活性，以便更好地 从分子水平了解NarK的功能； 2) 获得NarK的高分辨率结构 以前未观察到的构象，因此我们可以重建 NarK 的完整运输周期； 3）探索 人硝酸盐转运蛋白唾液酸蛋白的结构-功能关系，因此我们将了解其相似之处和 不同物种的硝酸盐转运蛋白之间存在差异。总体而言，在提案完成后，我们 期望扩大我们对硝酸盐/亚硝酸盐运输的一般理解，并阐明其中的关键作用 硝酸盐转运蛋白（真核和原核）在硝酸盐/亚硝酸盐循环中发挥作用。知识点 这里获得的成果将促进与细菌硝酸盐转运蛋白和人类唾液酸蛋白相关的潜在药物开发。