NEUROFIBROMATOSIS TYPE I GENE PRODUCT

I型神经纤维瘤病（NF 1）是一种遗传性周围神经系统疾病， 世界上每3500人中就有1人受到这种疾病的折磨，我们最近 证明NF-1基因产物是GAP（GTfamily）的成员， 激活蛋白）蛋白家族，其下调ras蛋白， 参与细胞生长调节的鸟嘌呤核苷酸结合蛋白， 以及肿瘤形成。 在本申请中，我们建议进行详细的 NF 1蛋白的表征集中在以下方面 实验 [I]NF 1 GAP活性和相互作用的表征 NF 1和ras蛋白之间的关系（1）NF 1的催化结构域将是 从E.表达NF 1的大肠杆菌或昆虫细胞及其GAP活性 也将被调查。 (2)NF 1与ras蛋白的相互作用 将通过生物化学实验进行研究。 (3)催化剂区域 负责差距活动以及与 ras蛋白将通过NF 1蛋白的体外诱变来确定。 赋予靶ras蛋白的特异性的区域也将被标记。 测定 (4)脂质似乎也是相互作用的参与者。 NF 1-GAP活性的脂质敏感性将通过使用各种 脂类和脂肪酸。 [II]催化蛋白质侧翼区域的功能 将通过比较催化剂的GAP活性来研究结构域 结构域多肽和含有催化结构域和催化结构域两者的多肽。 侧翼区域。[III]体内NF 1蛋白的特征在于： 准备一组抗体 翻译后修饰， 亚细胞定位和任何相关蛋白的存在将 追究 [IV]突变NF 1蛋白的特征在于： 纯化已知的突变蛋白以及通过筛选各种突变蛋白， 组织样品，包括来自神经纤维瘤病患者的组织样品。

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.