Characterization of the Zn finger domain of the oncogenic protein MDM2

致癌蛋白 MDM2 锌指结构域的表征

• 批准号: 12670079
• 负责人: SUKEGAWA Jun
• 金额: $ 2.18万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670079/
• 关键词: GTP binding protein; Ran; MDM2; Zn finger

It has been considered that the Zn finger-like structure observed in Nup153, a component of mammalian nuclear pore complexes, would recognize and bind to some specific nucleotide sequence(s) as other common Zn finger proteins do. We have found, however, that the Zn finger-like structure binds specifically to one of the small GTP binding protein, Ran. The Zn finger-like structure of Nup153 consists of four repeats of an amino acid sequence containing regularly spacing cysteine residues. Because data base search showed several proteins possess one or two repeats of the Zn finger-like structure similar to that of Nup153, we asked if each repeat unit of the Nupl53 Zn finger-like structure is still capable of binding to Ran. Two-hybrid assay showed that peptides with two repeat units are capable of binding to Ran. However, single repeat units didn't show the activity. We asked next if MDM2, an oncogenic protein that contains a single unit of the Zn finger-like amino acid sequence, recognizes Ran. Although two-hybrid assay failed to show Ran-binding activity of MDM2, in vitro binding experiments indicated that MDM2 is indeed capable of binding to Ran. We are now pinpointing the Ran-binding sequence on MDM2.

人们认为，在哺乳动物核孔复合物的组成部分Nup 153中观察到的锌指状结构将像其他常见锌指蛋白一样识别并结合某些特定的核苷酸序列。然而，我们已经发现，锌指状结构特异性结合到一个小GTP结合蛋白，RAN。Nup153的锌指状结构由含有规则间隔的半胱氨酸残基的氨基酸序列的四个重复组成。由于数据库搜索显示几种蛋白质具有一个或两个重复的锌指状结构类似于NUP153，我们问是否NUP153锌指状结构的每个重复单元仍然能够结合到RAN。双杂交实验表明，具有两个重复单元的肽能够与Ran结合。然而，单个重复单位没有显示出活性。接下来，我们询问MDM2是否识别Ran，MDM2是一种含有单个锌指样氨基酸序列的致癌蛋白。虽然双杂交试验未能显示MDM2的Ran结合活性，但体外结合实验表明MDM2确实能够与Ran结合。我们现在正在精确定位MDM2上的RAN结合序列。