Structure and function of nucleotide sugar transporters.

项目总结 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，它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利用。由于NST活动控制着两者的浓度 高尔基体腔中的核苷酸糖和核苷酸单磷酸，NST活性的中断可以有 许多不利的生理影响，如在许多疾病中看到的由NST突变引起的 基因或NST活性失调。然而，选择性抑制NSTs也有可能是 在针对依赖于特定糖结合物的毒力的寄生虫方面具有治疗益处 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、底物选择性和作用机制的分子细节 NST的运输。尽管NST在近40年前就被首次描述，但其中许多问题 在很大程度上仍然没有得到答复，主要是因为缺乏关于国家技术人员的结构信息，以及有限的 这一蛋白质家族的生化特征的方法。为了克服这些障碍，我们有 开发了一种表达、纯化和结晶哺乳动物NST的方法，即小鼠cMP-唾液酸 运输商(CST)。我们还开发了新的结合和运输分析，将使我们能够解决 关于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-唾液酸络合物的拆分结构，以及 表示运输周期的不同状态。这些结构与生物化学研究相结合将 回答关于NST结构-功能关系的基本问题，这将进一步推动我们的 了解NSTs在生理学中的作用，并帮助开发针对NSTs的药物。

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.