NEUROFIBROMATOSIS TYPE I GENE PRODUCT

I 型神经纤维瘤病基因产物

• 批准号: 3416994
• 负责人: Fuyuhiko Tamanoi
• 金额: $ 18.94万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3416994
• 关键词: Escherichia coli; antibody; arachidonate; cell membrane; chimeric proteins; gene mutation; genetic mapping; guanine nucleotide binding protein; human genetic material tag; human tissue; insect virus; intermolecular interaction; laboratory rabbit; membrane lipids; nerve /myelin protein; neural growth associated protein; neurofibromatosis; nucleic acid structure; oncoproteins; phosphatidate; phosphatidylinositols; phosphorylation; posttranslational modifications; protein purification; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture; transfection

Neurofibromatosis type I (NF1) is a genetic disease of peripheral nervous system that afflicts 1 in 3500 people worldwide, We have recently demonstrated that the NF1 gene product is a member of GAP (GTPase activating protein) protein family which down-regulate ras proteins, guanine nucleotide binding protein involved in regulation of cell growth as well as neoplasia. In this application, we propose to carry out a detailed characterization of the NF1 protein by concentrating on the following experiments. [I] Characterization of NF1 GAP activity and interaction between NF1 and ras proteins (1) The catalytic domain of NF1 will be purified from E. coli or insect cells expressing NF1 and their GAP activity as well will be investigated. (2) Interaction between NF1 and ras proteins will be studied by biochemical experiments. (3) Regions in the catalytic domain responsible for the GAP activity as well as for the interaction with ras proteins will be determined by in vitro mutagenesis of the NF1 protein. A region(s) conferring specificity of target ras proteins will be also be determined. (4) Lipids also appears to be a player in the interaction. Lipid sensitivity of NF1-GAP activity will be addressed by using various lipids and fatty acids. [II] Function of regions flanking the catalytic domain will be investigated by comparing GAP activity of the catalytic domain polypeptide and a polypeptide containing both the catalytic and the flanking regions. [III] NF1 protein in vivo will be characterized by preparing a battery of antibodies. Posttranslational modification, subcellular localization and the presence of any associated proteins will be investigated. [IV] Mutant NF1 protein will be characterized by the purification of known mutant proteins as well as by screening various tissue samples including ones derived from neurofibromatosis patients.

I 型神经纤维瘤病 (NF1) 是一种周围神经遗传性疾病 全球每 3500 人中就有 1 人受到这一系统的影响，我们最近 证明NF1基因产物是GAP（GTPase 激活蛋白）下调ras蛋白的蛋白家族， 鸟嘌呤核苷酸结合蛋白参与细胞生长调节 以及肿瘤。 在本申请中，我们建议进行详细的 通过集中于以下方面来表征 NF1 蛋白 实验。 [I] NF1 GAP 活性和相互作用的表征 NF1 和 ras 蛋白之间 (1) NF1 的催化结构域为 从表达 NF1 及其 GAP 活性的大肠杆菌或昆虫细胞中纯化 也将受到调查。 (2) NF1与ras蛋白的相互作用 将通过生化实验进行研究。 (3) 催化区域 负责 GAP 活动以及与 ras 蛋白将通过 NF1 蛋白的体外诱变来确定。 赋予靶 ras 蛋白特异性的区域也将是 决定。 (4) 脂质似乎也是相互作用的参与者。 NF1-GAP 活性的脂质敏感性将通过使用各种方法来解决 脂质和脂肪酸。 [II] 催化侧翼区域的功能 将通过比较催化的 GAP 活性来研究结构域 结构域多肽和同时含有催化和 侧翼地区。 [III] 体内NF1蛋白的特点是 准备抗体电池。 翻译后修饰， 亚细胞定位和任何相关蛋白质的存在将 被调查。 [IV] 突变型 NF1 蛋白的特征在于 纯化已知的突变蛋白以及通过筛选各种 组织样本，包括来自神经纤维瘤病患者的组织样本。