Structure and function of nucleotide sugar transporters.

核苷酸糖转运蛋白的结构和功能。

• 批准号: 10375458
• 负责人: Isabelle Rhyssa Joe Eduria Baconguis
• 金额: $ 32.34万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10375458
• 关键词: Address; Back; Binding; Binding Sites; Biochemical; Biological Assay; Cell Surface Proteins; Cell surface; Complex; Crystallization; Cytidine Monophosphate N-Acetylneuraminic Acid; Cytoplasm; Disease; Distant; Eukaryota; Foundations; Future; Gene Family; Genes; Genetic Diseases; Glycoconjugates; Goals; Golgi Apparatus; Homology Modeling; Human; Label; Lipids; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Mus; Mutation; Neoplasm Metastasis; Nucleotides; Orthologous Gene; Parasites; Pathogenesis; Pathogenicity; Pattern; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiological; Physiology; Play; Property; Protein Family; Proteins; Reaction; Regulation; Resolution; Role; Structural Models; Structure; Structure-Activity Relationship; Therapeutic; Time; Virulence; Virulence Factors; antiviral nucleoside analog; cancer cell; disease-causing mutation; drug development; experimental study; fungus; glycosylation; glycosyltransferase; insight; novel; novel therapeutic intervention; nucleoside analog; pathogen; sialic acid permease; side effect; solute; sugar nucleotide; therapeutic development; therapeutic target; tumor

Project summary Nucleotide sugar transporters (NSTs) are a family of proteins that are a critical part of the glycosylation machinery in all eukaryotes as they are responsible for transporting nucleotide sugars from the cytoplasm, where they are synthesized, into the Golgi lumen where they are then utilized by glycosyltransferases to glycosylate proteins and lipids. NSTs provide an additional essential role of transporting the nucleotide monophosphate byproduct of the glycosyltransferase reaction, which is inhibitory towards glycosyltransferases, back to the cytoplasm where it can be recycled. Since NST activity controls the concentrations of both nucleotide sugars and nucleotide monophosphates in the Golgi lumen, disruption of NST activity can have many adverse physiological effects as is seen in a number of diseases caused by either mutations in NST genes or dysregulation of NST activity. However, selective inhibition of NSTs also has the potential to be exploited for therapeutic benefit in targeting parasites that depend on particular glycoconjugates for virulence as well as in blocking certain glycosylation patterns that promote tumor metastasis. Our long-term goal is to understand the molecular details that underlie substrate recognition, substrate selectivity, and the mechanism of transport of NSTs. Although NSTs were first described nearly four decades ago, many of these questions remain largely unanswered, primarily due to a lack of structural information for NSTs as well as limited methods for biochemical characterization of this family of proteins. To overcome these obstacles, we have developed methods to express, purify, and crystallize a mammalian NST, the mouse CMP-sialic acid transporter (CST). We have also developed novel binding and transport assays that will allow us to address questions regarding substrate recognition and selectivity. These methods will allow us to determine high- resolution structures of CST in complex with its substrates CMP and CMP-sialic acid, as well as structures that represent different states of the transport cycle. These structures combined with biochemical studies will answer the fundamental questions regarding the structure-function relationship of NSTs, which will further our understanding of the role NSTs play in physiology and aid in the development of drugs to target NSTs.

项目概要 核苷酸糖转运蛋白 (NST) 是一个蛋白质家族，是糖基化的关键部分 所有真核生物中的机器，因为它们负责从细胞质运输核苷酸糖， 它们在高尔基体腔中合成，然后被糖基转移酶利用 糖基化蛋白质和脂质。 NST 提供了转运核苷酸的额外重要作用 糖基转移酶反应的单磷酸副产物，对糖基转移酶有抑制作用， 回到细胞质，在那里它可以被回收。由于 NST 活性控制着两者的浓度 高尔基体腔中的核苷酸糖和核苷酸单磷酸，破坏 NST 活性可能会产生 许多不良生理影响，如 NST 突变引起的许多疾病 基因或 NST 活性失调。然而，选择性抑制 NST 也有可能 用于靶向依赖特定糖缀合物毒力的寄生虫的治疗益处 以及阻断某些促进肿瘤转移的糖基化模式。我们的长期目标是 了解底物识别、底物选择性和机制的分子细节 NST 的运输。尽管 NST 是在近四十年前首次被描述的，但其中许多问题 很大程度上仍未得到解答，主要是由于缺乏 NST 的结构信息以及有限的 该蛋白质家族的生化表征方法。为了克服这些障碍，我们 开发了表达、纯化和结晶哺乳动物 NST（小鼠 CMP-唾液酸）的方法 运输者（CST）。我们还开发了新颖的结合和运输检测方法，使我们能够解决 有关底物识别和选择性的问题。这些方法将使我们能够确定高 CST 与其底物 CMP 和 CMP-唾液酸复合物的解析结构，以及 代表运输周期的不同状态。这些结构与生化研究相结合将 回答有关 NST 结构与功能关系的基本问题，这将进一步促进我们的研究 了解 NST 在生理学中发挥的作用，并有助于开发针对 NST 的药物。