Structure and function of nucleotide sugar transporters.

项目摘要 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，其对糖基转移酶具有抑制作用， 回到细胞质中循环利用。由于NST活性控制着两者的浓度， 核苷酸糖和核苷酸单磷酸，NST活性的破坏可以有 许多不利的生理作用，如在由NST中的任一突变引起的许多疾病中所见 基因或NST活性失调。然而，NST的选择性抑制也有可能被 在靶向依赖于特定糖缀合物的毒力的寄生虫中用于治疗益处 以及阻断促进肿瘤转移的某些糖基化模式。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 运输NST。虽然NST在近四十年前首次被描述，但许多问题 主要是由于缺乏NST的结构信息以及有限的 该蛋白家族的生物化学表征方法。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 转运蛋白（CST）。我们还开发了新的结合和运输试验，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-唾液酸复合的拆分结构，以及 代表运输周期的不同状态。这些结构与生物化学研究相结合， 回答关于NST结构-功能关系的基本问题，这将进一步我们 了解NST在生理学中的作用，并帮助开发针对NST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.