Structure and Function of Concentrative Nucleoside Transporters

说明(申请人提供)：核苷转运体(NTS)是一种完整的膜蛋白，旨在将核苷转运到人体内。核苷跨细胞膜转运具有重要的生理意义，因为它为细胞内DNA/RNA合成提供核苷的主要来源，并负责腺苷信号的终止。关于NTS的另一个重要事实是许多核苷衍生的抗癌和抗病毒药物是通过NTS转运到细胞内的。尽管NTS在生理学和药理学上很重要，但由于缺乏转运体的原子结构，NTS的分子机制在很大程度上还不清楚。这一领域的重大进展取决于NTS的结构确定和传输机制的阐明。我们的长期目标是通过结构和功能研究了解集中核苷转运体(CNTs)选择性核苷转运的基本机制。碳纳米管属于溶质载体超家族(SLC28)的一个家族，它利用离子梯度来主动运输核苷，以对抗其化学梯度。我们选择使用霍乱弧菌的CNT同源物(VcCNT)作为结构和功能研究的模型系统。VcCNT与hCNTs具有显著的序列同源性和功能特性，是研究hCNTs的良好模型系统。我们解决了尿苷络合物中vcCNT的晶体结构问题。该结构揭示了转运体的整体结构和核苷结合部位，提供了vcCNT和CNT选择性核苷转运的机制见解。这些新的结果促使我们提出了一项结构-功能相结合的研究，目的有三个：1)确定不同核苷和核苷药物的vcCNT的晶体结构；2)vcCNT的功能表征和与hCNTs的比较；以及3)vcCNT的交替构象结构测定。这些研究将极大地促进我们对原核和真核碳纳米管转运核苷和核苷药物的机制的了解，最终将促进新的治疗试剂的开发。

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.