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Supplement to Structure and function of nucleotide sugar transporters.

补充核苷酸糖转运蛋白的结构和功能。

基本信息

批准号：
10798026
负责人：
Isabelle Rhyssa Joe Eduria Baconguis
金额：
$ 3.05万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-03-31
项目状态：
未结题

项目摘要

Project summary (from original application) 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.

项目摘要（来自原始申请）核苷酸糖转运蛋白（NSTs）是一类在糖基化过程中起重要作用的蛋白质家族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，在那里它们被合成，进入高尔基体腔，然后在那里它们被糖基转移酶利用，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，其抑制糖基转移酶反应。糖基转移酶，回到细胞质，在那里它可以回收。由于NST活动控制着浓度的核苷酸糖和核苷酸单磷酸在高尔基体腔，破坏 NST活性可具有许多不利的生理作用，如在由NST引起的许多疾病中所见。 NST基因突变或NST活性失调。然而，选择性抑制NST也有可能被开发用于靶向依赖于特定的寄生虫的治疗益处，糖缀合物的毒性以及阻断某些促进肿瘤的糖基化模式转移我们的长期目标是了解底物识别的分子细节，底物选择性和NST的转运机制。尽管NST最初被描述为 40年前，这些问题中的许多问题在很大程度上仍然没有答案，主要是由于缺乏结构性的 NST的信息以及用于该蛋白质家族的生物化学表征的有限方法。到克服这些障碍，我们已经开发出表达、纯化和结晶哺乳动物细胞的方法， NST，小鼠CMP-唾液酸转运蛋白（CST）。我们还开发了新的绑定和传输这将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分辨率结构的CST在复杂的与其基板CMP和CMP- 唾液酸，以及代表运输循环不同状态的结构。这些结构结合生物化学研究将回答有关结构-功能的基本问题， NST的关系，这将进一步加深我们对NST在生理学中的作用的理解，并有助于开发针对NST的药物。