NUCLEOCYTOPLASMIC TRANSPORT--NEW FACTORS AND PATHWAYS

核质转运--新因素和途径

• 批准号: 2910425
• 负责人: AURELIAN RADU
• 金额: $ 18.09万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910425
• 关键词: cell nucleus; gene expression; genetic library; immunoprecipitation; intracellular transport; northern blottings; nuclear membrane; nucleic acid sequence; protein localization; protein structure function; transport proteins; western blottings; yeast two hybrid system

Various classes of proteins and nucleic acids are imported into and exported out of the cell nucleus through separate pathways, defined by independent sets of carrier molecules and docking sites ad the nuclear pore complexes. For some transported species the components of their transport machineries are partially or totally unknown. Our aim is to identify and characterize new nucleocytoplasmic transport factors, based on two complementary strategies. The first approach involves obtaining the full DNA sequences of potential transport factors whose existence is inferred from the presence in the GenBank EST database of shot, partial cDNA sequences, showing significant homology with known transport factors. The second strategy is based on the fact that all known transport factors dock temporarily, during passage across the nuclear pore, to proteins at the nuclear pore complexes. One of these docking site proteins, Nup98, which we isolated and shown to be implicated in multiple transport pathways, will be used for retention and isolation of new transport factors from the nucleoplasm. The proteins isolated by these two approaches will be characterized in terms of full cDNA sequence, subcellular localization, and level of expression in various tissues and developmental stages. Most importantly, the proteins interacting with these transport factors will be identified by co-immunoprecipitation, screening of expression libraries, and two-hybrid assays. The ensemble of these data, and especially the set of interacting molecules, will shed light on the specific functions of these proteins as transport factors. The relevance of these studies for human health is linked to the fact that a vast number of proteins and nucleic acids cannot exert their function unless they are imported in or exported out of the nucleus in a precise manner, and in fact there are known cases when tumors arise due to defects in nucleocytoplasmic transport pathways are used by viruses, knowing in detail these mechanisms could also offer opportunities for new anti-viral therapies and for improved gene therapy vectors.

各类蛋白质和核酸被输入并 通过不同的途径从细胞核输出，定义为 独立的载体分子组和核对接位点 孔隙复合体。对于一些运输物种来说，其组成部分 运输机械部分或完全未知。我们的目标是 识别和表征新的核细胞质转运因子，基于 两种互补的策略。第一种方法涉及获取 潜在转运因子的完整 DNA 序列，其存在是 根据 GenBank EST 数据库中存在的镜头推断，部分 cDNA 序列，与已知的转运因子显示出显着的同源性。 第二个策略基于以下事实：所有已知的运输因素 在穿过核孔的过程中，暂时停靠在蛋白质处 核孔复合体。这些对接位点蛋白之一，Nup98， 我们将其分离并证明与多种运输有关 路径，将用于保留和隔离新的运输 来自核质的因子。这两个分离出的蛋白质 方法将根据完整的 cDNA 序列进行表征， 亚细胞定位以及在各种组织中的表达水平 发展阶段。最重要的是，蛋白质相互作用 这些运输因子将通过免疫共沉淀来鉴定， 表达文库的筛选和双杂交测定。乐团的 这些数据，尤其是相互作用的分子集，将摆脱 阐明这些蛋白质作为运输因子的特定功能。 这些研究与人类健康的相关性与以下事实有关： 大量蛋白质和核酸无法发挥其功能 除非它们以精确的方式输入或输出出细胞核 事实上，有一些已知的案例是由于缺陷而产生肿瘤的 核细胞质转运途径被病毒使用，知道 详细说明这些机制也可能为新的抗病毒药物提供机会 疗法和改进的基因治疗载体。