NUCLEOSIDE TRANSPORTERS--STRUCTURE/FUNCTION AND REGULATI

核苷转运蛋白——结构/功能和调节

• 批准号: 6750672
• 负责人: CHUNG-MING TSE
• 金额: $ 25.75万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6750672
• 关键词: chimeric proteins; colon neoplasms; cysteine; cytosine arabinoside; deoxycytidine; enzyme activity; gemcitabine; gene induction /repression; glycosylation; hypoxanthines; inosine; intermolecular interaction; membrane transport proteins; molecular cloning; neoplasm /cancer genetics; nucleosides; phorbols; protein kinase C; protein structure function; thiols; tissue /cell culture

Nucleoside transporters are important in transporting synthetic nucleoside drugs into their target cells where these drugs are metabolized and become cytotoxic. Nucleoside drugs are used in chemotherapy (eg. Ara C in leukemia) and as antiviral agents (eg. AZT in the treatment of AIDS). Pharmacologically, the Na independent nucleoside transport systems can be separated into the nitrobenzylthioinosine (NBMPR) sensitive (ES) and the NBMPR insensitive (EI) systems. NBMPR is an inhibitor of nucleoside transport. Recently, our laboratory cloned the human Na-independent Equilibrative Nucleoside Transporters (ENT1(encoding ES) and ENT2 (encoding EI)) and developed a Nucleoside Transporter Deficient cell line, PK15NTD cells. This will allow us to study the structure/function relationship of the nucleoside transporters and the structural determinants of nucleosides and nucleoside drugs as substrates of the nucleoside transporters. Through the knowledge of nucleoside transport at the molecular level, a long term goal is to facilitate the design of nucleoside analogue drugs for the treatment of diseases. In this application, we will characterize functionally and pharmacologically the cloned human ENT1 and ENT2 stably expressed in PK15NTD cells (Aim 1). Results obtained from the cloned proteins will be compared with the endogenous ES and EI transporters in human colonic cancer cell line, T84. Using kinetic studies, we will identify structural determinants of nucleosides for the transport by ENT1 and ENT2. In Aim 2, we will study the structure/function relationship of ENT1 and ENT2. We will define the role of glycosylation in the function of ENT1 and ENT2 since glycosylation is important for nucleoside transport. Nucleoside transporter function is thiol-sensitive and the thiol sensitive group is exofacial in ENT2 but is cytoplasmic in ENT1. We propose to identify cysteine residues that confer thiol sensitivity of ENT1 and ENT2. ENT1 and ENT2 have a 3000 fold difference in sensitivity to NBMPR and an 8-10 fold difference in the affinity for guanosine and cytidine transport, ENT1/ENT2 chimeras will be constructed to identify the binding domain and substrate recognition domain in ENT1 and ENT2. Protein kinase C (PKC) inhibits the cloned ENT1 and ENT2 and endogenous ES and EI transporters in T84 cells. Experiments are proposed in the third aim to study the molecular mechanisms of how PKC inhibits ENT1 and ENT2. These proposed studies should provide insights into the molecular mechanisms and kinase regulation of nucleoside transport, and the structural determinants of nucleosides/nucleoside drugs for transport by the nucleoside transporters.

核苷转运蛋白在将合成的核苷药物转运到其靶细胞中是重要的，在靶细胞中这些药物被代谢并变得具有细胞毒性。 核苷类药物用于化疗（例如，Ara C在白血病中）和作为抗病毒剂（例如，AZT在治疗艾滋病中的作用）。 在药理学上，钠非依赖性核苷转运系统可分为硝基苄硫代肌苷（NBMPR）敏感（ES）系统和NBMPR不敏感（EI）系统。 NBMPR是核苷转运的抑制剂。 本实验室最近克隆了人Na非依赖性平衡核苷转运蛋白（ENT 1（编码ES）和ENT 2（编码EI）），并建立了核苷转运蛋白缺陷细胞系PK 15 NTD细胞。 这将使我们能够研究核苷转运蛋白的结构/功能关系以及作为核苷转运蛋白底物的核苷和核苷药物的结构决定因素。 通过在分子水平上对核苷转运的了解，一个长期目标是促进用于治疗疾病的核苷类似物药物的设计。 在本申请中，我们将对在PK 15 NTD细胞中稳定表达的克隆的人ENT 1和ENT 2进行功能表征和鉴定（Aim 1）。 将从克隆的蛋白质获得的结果与人结肠癌细胞系T84中的内源性ES和EI转运蛋白进行比较。 使用动力学研究，我们将确定由ENT 1和ENT 2运输的核苷的结构决定因素。 在目标2中，我们将研究ENT 1和ENT 2的结构/功能关系。我们将定义糖基化在ENT 1和ENT 2功能中的作用，因为糖基化对核苷转运很重要。 核苷转运蛋白功能是硫醇敏感的，硫醇敏感基团在ENT 2中是外表面的，但在ENT 1中是细胞质的。 我们建议鉴定赋予ENT 1和ENT 2硫醇敏感性的半胱氨酸残基。 ENT 1和ENT 2对NBMPR的敏感性相差3000倍，对鸟苷和胞苷转运的亲和力相差8-10倍，将构建ENT 1/ENT 2嵌合体以鉴定ENT 1和ENT 2中的结合结构域和底物识别结构域。蛋白激酶C（PKC）抑制克隆的ENT 1和ENT 2以及T84细胞中的内源性ES和EI转运体。 第三个目标是研究PKC抑制ENT 1和ENT 2的分子机制。 这些拟议的研究应提供深入了解核苷转运的分子机制和激酶调节，以及核苷/核苷药物通过核苷转运蛋白转运的结构决定因素。

项目成果

D-Glucose upregulates adenosine transport in cultured human aortic smooth muscle cells.

D-葡萄糖上调培养的人主动脉平滑肌细胞中的腺苷转运。

• DOI: 10.1152/ajpheart.00921.2004
• 发表时间: 2005
• 期刊: American journal of physiology. Heart and circulatory physiology
• 作者: Leung,GeorgePH;Man,RickyYK;Tse,Chung-Ming
• 通讯作者: Tse,Chung-Ming

A purine-selective nucleobase/nucleoside transporter in PK15NTD cells.

PK15NTD 细胞中的嘌呤选择性核碱基/核苷转运蛋白。

• DOI: 10.1152/ajpregu.00016.2008
• 发表时间: 2008
• 期刊: American journal of physiology. Regulatory, integrative and comparative physiology
• 作者: Hoque,KaziMirajul;Chen,Linxi;Leung,GeorgePH;Tse,Chung-Ming
• 通讯作者: Tse,Chung-Ming