Analysis of cellular function of Neurofibromatosis Type 2 tumor suppressor gene product (Merlin)

神经纤维瘤病2型抑癌基因产物（Merlin）的细胞功能分析

基本信息

批准号：
10671308
负责人：
ARAKI Norie
金额：
$ 2.43万
依托单位：
Kumamoto University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671308/
关键词：
Neurofibromatosis Type 2 NF2 Merlin Tumor suppressor gene Poly (ADP-ribose) polymerase (PARP)Ku-antigen Cellular signal transduction Nuclear export signal (NES)NF2、神経繊維腫症2型

项目摘要

The neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited disorder, and strongly associated with development of benign intracranial tumors including bilateral vestibular schwannomas and meningiomas. NF2 gene was recently cloned, and the protein it encodes (termed as merlin/schwannomin) was found to be striking similarity to the moesin-ezrin-radixin (MER) family of cytoskeleton-associated proteins. To elucidate the biological function of NF2 protein (merlin), we analyzed the mutation pattern of merlin, its cellular localization, and the cellular signals via merlin. 1) NF2 gene mutations in NF2 patients were analyzed by protein truncation test. The majority of NF2 mutations are nonsense, frame shift, or exon-missing mutations. More than 70%of the mutations are clustered in the region encoding the N-terminal half of merlin. In addition, post-transrational proteolytic cleavage of N-terminal merlin by the ubiquitous protease calpain was found in some schwannomas and meningioma … More s. These results suggest that the N-terminal region of merlin is the most important for the tumor suppressive function, 2) Cellular expression systems of mutants or wild NF2 were established, and their cellular localization was analyzed by confocal lazer scanning Microscopy (CLSM). The wild merlin showed cytoplasmic and submembranous localization that was found to be directed by its nuclear export signal (NES) dependent transport mechanism. The N-terminal mutation resulted in nuclear accumulation of merlin. 3) Cellular binding proteins of merlin were purified from bovine brain extracts and identified as poly ADP-ribose polymerase, DNA-PK subunits Ku-antigen 85 and 70 by the analysis of their internal amino-acid sequences. The N-terminal(19-339) region of merlin is essential for their interaction. The immuno-precipitation, western blotting, and CLMS study confirmed their cellular co-localization. NF2 mutations impair the merlin-related complex formation and their cellular signal transport. This loss of cellular function of merlin may link to the intracranial tumor generation. Less

2 型神经纤维瘤病 (NF2) 是一种常染色体显性遗传性疾病，与良性颅内肿瘤（包括双侧前庭神经鞘瘤和脑膜瘤）的发展密切相关，最近克隆了 NF2 基因，并发现了其编码的蛋白质（称为 merlin/schwannomin）。与细胞骨架相关蛋白的 moesin-ezrin-radixin (MER) 家族惊人的相似。 NF2 蛋白（merlin）的功能，我们分析了 merlin 的突变模式、其细胞定位以及通过 merlin 发出的细胞信号。 1）通过蛋白截短试验分析了 NF2 患者的 NF2 基因突变。超过 70% 的突变集中在编码 merlin N 末端的区域。在一些神经鞘瘤和脑膜瘤中发现了普遍存在的蛋白酶钙蛋白酶对 merlin N 端的蛋白水解裂解……更多这些结果表明 merlin 的 N 端区域对于肿瘤抑制功能是最重要的，2) 细胞表达系统建立了突变体或野生 NF2，并通过共焦激光扫描显微镜 (CLSM) 分析了它们的细胞定位。显示细胞质和膜下定位，发现这是由其核输出信号 (NES) 依赖性转运机制引导的。N 端突变导致 merlin 的核积累。 3) 从牛脑提取物中纯化并鉴定了 merlin 的细胞结合蛋白。通过分析其内部氨基酸序列，将其鉴定为聚 ADP-核糖聚合酶、DNA-PK 亚基 Ku 抗原 85 和 70。 merlin 的 N 末端 (19-339) 区域对于它们的相互作用至关重要。免疫沉淀、蛋白质印迹和 CLMS 研究证实它们的细胞共定位会损害 merlin 相关复合物的形成及其细胞信号传输。 merlin 细胞功能的丧失可能与颅内肿瘤的产生有关。