NUCLEOSIDE TRANSPORTERS--STRUCTURE/FUNCTION AND REGULATI

核苷转运蛋白——结构/功能和调节

• 批准号: 6377611
• 负责人: CHUNG-MING TSE
• 金额: $ 25.76万
• 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/6377611
• 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核苷药物转运到其靶细胞中很重要，这些药物被代谢并变为细胞毒性。 核苷药物用于化学疗法（例如白血病中的ARA C）和抗病毒剂（例如，在AIDS治疗中AZT）。 从药理上讲，NA独立的核苷转运系统可以分离为硝基苯基噻吩氨酸（NBMPR）敏感（ES）和NBMPR不敏感（EI）系统。 NBMPR是核苷转运的抑制剂。 最近，我们的实验室将人NA非依赖性的平衡核苷转运蛋白（ENT1（编码ES）和ENT2（编码EI））克隆，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，以及核苷和核苷药物的结构决定因素作为核苷转运蛋白的底物。 通过了解分子水平的核苷转运，一个长期目标是促进核苷类似物药物的设计用于治疗疾病。 在此应用中，我们将在功能和药理上表征在PK15NTD细胞中稳定表达的克隆的人ENT1和ENT2（AIM 1）。 从克隆的蛋白质获得的结果将与人类结肠癌细胞系中的内源性ES和EI转运蛋白进行比较，T84。 使用动力学研究，我们将确定核苷的结构决定因素，以通过ENT1和ENT2运输。 在AIM 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。 核苷转运蛋白的功能是硫醇敏感的，硫醇敏感的基团在ENT2中是外生的，但在ENT1中是胞质的。 我们建议确定赋予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，并且将构建对鸟嘌呤和胞苷转运的亲和力的8-10倍差异，ENT1/ENT2 Chimeras将构建以识别ENT1和ENT2中的结合域和底物识别域。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。 在第三个目标中提出了实验，以研究PKC如何抑制ENT1和ENT2的分子机制。 这些提出的研究应提供有关核苷转运的分子机理和激酶调节的见解，以及核苷/核苷药物的结构决定因素，以通过核苷转运蛋白转运。