NUCLEOSIDE TRANSPORTERS--STRUCTURE/FUNCTION AND REGULATI

核苷转运蛋白——结构/功能和调控

• 批准号: 6514405
• 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 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514405
• 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.

核苷转运蛋白对于将合成核苷药物转运至靶细胞中非常重要，这些药物在靶细胞中被代谢并产生细胞毒性。 核苷类药物用于化疗（例如白血病中的 Ara C）和抗病毒剂（例如治疗艾滋病中的 AZT）。 药理学上，Na非依赖性核苷转运系统可分为硝基苄基硫代肌苷（NBMPR）敏感（ES）系统和NBMPR不敏感（EI）系统。 NBMPR 是一种核苷转运抑制剂。 最近，我们实验室克隆了人Na非依赖性平衡核苷转运蛋白（ENT1（编码ES）和ENT2（编码EI）），并开发了核苷转运蛋白缺陷细胞系PK15NTD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。 通过分子水平上核苷转运的知识，长期目标是促进用于治疗疾病的核苷类似物药物的设计。 在此应用中，我们将从功能和药理学角度表征在 PK15NTD 细胞中稳定表达的克隆人 ENT1 和 ENT2（目标 1）。 从克隆蛋白获得的结果将与人结肠癌细胞系 T84 中的内源 ES 和 EI 转运蛋白进行比较。 通过动力学研究，我们将确定 ENT1 和 ENT2 转运核苷的结构决定因素。 在目标 2 中，我们将研究 ENT1 和 ENT2 的结构/功能关系。我们将定义糖基化在 ENT1 和 ENT2 功能中的作用，因为糖基化对于核苷转运很重要。 核苷转运蛋白功能对硫醇敏感，硫醇敏感基团在 ENT2 中位于面外，但在 ENT1 中位于细胞质内。 我们建议鉴定赋予 ENT1 和 ENT2 硫醇敏感性的半胱氨酸残基。 ENT1和ENT2对NBMPR的敏感性有3000倍的差异，对鸟苷和胞苷转运的亲和力有8-10倍的差异，将构建ENT1/ENT2嵌合体来鉴定ENT1和ENT2中的结合结构域和底物识别结构域。蛋白激酶 C (PKC) 抑制 T84 细胞中克隆的 ENT1 和 ENT2 以及内源性 ES 和 EI 转运蛋白。 第三个目的是研究PKC抑制ENT1和ENT2的分子机制。 这些拟议的研究应深入了解核苷转运的分子机制和激酶调节，以及核苷转运蛋白转运的核苷/核苷药物的结构决定因素。