Characterization of the Zn finger domain of the oncogenic protein MDM2

研究认为，哺乳动物核孔复合体Nup153中观察到的类锌指结构与其他常见的锌指蛋白一样，能够识别并结合特定的核苷酸序列(S)。然而，我们发现锌指状结构与一种小的GTP结合蛋白RAN特异结合。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与Nup153相似的一个或两个重复的锌指状结构，我们询问Nupl53锌指状结构的每个重复单元是否仍然能够与RAN结合。双杂交实验表明，含有两个重复单位的多肽能够与RAN结合。然而，单一重复单位没有显示出这种活性。接下来，我们询问MDM2是否识别RAN。MDM2是一种致癌蛋白，含有单个单位的锌指状氨基酸序列。虽然双杂交实验未能显示MDM2与RAN结合的活性，但体外结合实验表明MDM2确实能够与RAN结合。我们现在正在定位MDM2上的Ran结合序列。

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.