Supplement to Structure and function of nucleotide sugar transporters.

项目总结(来自原始申请) 核苷酸糖转运体(NSTs)是糖基化过程中的重要组成部分。 所有真核生物中的机械，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，在那里它们被糖基转移酶利用来 糖基化蛋白质和脂类。NST为核苷酸的运输提供了额外的基本作用 糖基转移酶反应的副产物单磷酸，对 糖基转移酶，回到细胞质中，在那里它可以被循环使用。由于NST活动控制着 高尔基体腔内核苷酸糖和核苷酸单磷酸盐的浓度，干扰 NST活动可能有许多不利的生理影响，这在由以下原因引起的许多疾病中都可以看到 要么是NST基因突变，要么是NST活性失调。然而，选择性抑制NSTs也 在针对依赖于特定疾病的寄生虫方面具有潜在的治疗效益 用于毒力和阻断某些促进肿瘤的糖基化模式的糖偶联物 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，以及NSTs的运输机制。尽管NST最先被描述为 40年前，其中许多问题在很大程度上仍然没有得到回答，主要是因为缺乏结构性 NSTs的信息以及对这一蛋白质家族进行生物化学表征的有限方法。至 克服这些障碍，我们已经开发出表达、纯化和结晶哺乳动物的方法 NST，小鼠cMP-唾液酸转运蛋白(CST)。我们还开发了新的绑定和运输 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CST与其底物CMP和CMP的络合物的高分辨率结构。 唾液酸，以及代表运输周期不同状态的结构。这些结构 结合生化研究将回答有关结构-功能的基本问题 NSTs的关系，这将进一步加深我们对NSTs在生理学中的作用和帮助 开发针对NST的药物。

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.