Structure and Function of Concentrative Nucleoside Transporters

描述（由申请人提供）：核苷转运蛋白（NT）是设计用于将核苷转运到人体内的整合膜蛋白。核苷跨细胞膜转运在生理学上是重要的，因为它为细胞中的DNA/RNA合成提供了核苷的主要来源，并且它负责腺苷信号传导的终止。关于NT的另一个重要事实是许多核苷衍生的抗癌和抗病毒药物通过NT转运到细胞中。尽管它们在生理学和药理学上很重要，但由于缺乏转运蛋白的原子结构，NTs的分子机制在很大程度上是未知的。该领域的重大进展依赖于NTs结构的确定和运输机制的阐明。我们的长期目标是通过结构和功能的研究来了解浓缩型核苷转运蛋白（CNTs）选择性转运核苷的基本机制。CNT属于溶质载体转运蛋白超家族（SLC 28）的一个家族，并且使用离子梯度来主动地逆着它们的化学梯度转运核苷。我们选择使用来自霍乱弧菌（vcCNT）的CNT同系物作为我们的结构和功能研究的模型系统。vcCNT是研究hCNT的优良模型系统，其与hCNT具有显著的序列同源性和功能特性。我们解决了与尿苷复合的vcCNT的晶体结构。该结构揭示了转运蛋白的整体结构和核苷结合位点，提供了一般情况下vcCNT和CNT选择性核苷转运的机制见解。这些新的结果使我们提出了一个组合的结构-功能研究，具有以下三个目标：1）确定与不同核苷和核苷药物复合的vcCNT的晶体结构，2）vcCNT与hCNT的功能表征和比较，以及3）交替构象的vcCNT的结构确定。这些研究将大大促进我们对原核和真核碳纳米管转运核苷和核苷药物机制的理解，最终将促进新治疗试剂的开发。

DESCRIPTION (provided by applicant): Nucleoside transporters (NTs) are integral membrane proteins that are designed to transport nucleosides into the human body. Nucleoside transport across cell membranes is physiologically important as it provides the major source of nucleosides for DNA/RNA synthesis in cells and it is responsible for the termination of adenosine signaling. Another important fact about NTs is that many nucleoside-derived anticancer and antiviral drugs are transported into cells by way of NTs. Despite their importance in physiology and pharmacology, the molecular mechanism of NTs is largely unknown due to the lack of atomic structures of the transporters. Great advances in the field depend on the structure determination and illumination of the transport mechanism of NTs. Our long- term goal is to understand the fundamental mechanism of selective nucleoside transport by concentrative nucleoside transporters (CNTs) with structural and functional studies. CNTs belong to a family of the solute carrier transporter superfamily (SLC28) and use an ion gradient to transport nucleosides actively against their chemical gradients. We have chosen to use a CNT homolog from Vibrio cholerae (vcCNT) as a model system for our structural and functional studies. vcCNT is an excellent model system to study hCNTs that shares significant sequence homology and functional properties with hCNTs. We solved the crystal structure of vcCNT in complex with uridine. The structure reveals the overall architecture and the nucleoside-binding site of the transporter, providing mechanistic insights of selective nucleoside transport by vcCNT and CNT in general. These new results have led us to propose a combined structure-function study with the following three aims: 1) Determination of the crystal structures of vcCNT in complex with different nucleosides and nucleoside drugs, 2) Functional characterization and comparison of vcCNT with hCNTs, and 3) Structure determination of vcCNT in alternate conformations. The proposed studies will significantly advance our understanding of the mechanism of nucleoside and nucleoside drug transport by prokaryotic and eukaryotic CNTs, which eventually will facilitate the development of new therapeutic reagents.