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Structure and properties of Max protein which regulates the function of a proto-oncogene product, c-Myc

调节原癌基因产物 c-Myc 功能的 Max 蛋白的结构和特性

基本信息

批准号：
09470492
负责人：
UESUGI Seiichi
金额：
$ 7.49万
依托单位：
Yokohama National University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470492/
关键词：
proto-oncogene DNA binding protein transcription factor c-Myc Max mutant protein DNA-porotein interaction structure 蛋白質

项目摘要

Max Protein regulates the function of a proto-oncogene product, Myc, by formation of a heterodimer with Myc or a homodimer with itself and binding to DNA. We established a procedure for preparation of Max and two types of its DNA binding domains by genetic engineering techniques and examined their structure and properties. Max proteins were in equilibrium between a monomer and a dimer in solution. The dimer content increasesd with increasing protein concentration and decreasing temperature. Increase in α-helical content of the protein simultaneously accompanied the dimer increase. Upon addition of a duplex DNA containing the specific recognition base sequence, the α-helical content increased further with increasing DNA concentration. When a DNA duplex containing a sequence different in the central two base pairs was used, the α-helical content increase was smaller. A strictly specific recognition of the cognate sequence was observed at high temperature. The biological activity of Max is modulated by phosphorylation of Ser residues in the N-terminal region. We prepared mutant proteins where one or two of the Ser residues were replaced with Asp residue(s) as a mimic of the phosophorylated Max and examined DNA binding ability of the mutant proteins. The mutant protein-DNA complexes showed lower stability and the complex of the mutant protein with double mutation showed the lowest stability. We also developed a synthetic method for obtaining phosphorylated Max by chemically joining a phosphorylated peptied synthesized chemically and the remaining part of protein prepared by genetic engineering.

MAX蛋白通过与Myc形成异源二聚体或与自身形成同源二聚体并与DNA结合来调节原癌基因产物Myc的功能。我们建立了用基因工程技术制备MAX及其两种DNA结合域的方法，并对它们的结构和性质进行了检测。MAX蛋白在溶液中处于单体和二聚体之间的平衡状态。二聚体含量随蛋白质浓度的增加和温度的降低而增加。蛋白质的α-螺旋含量增加的同时伴随着二聚体的增加。当加入含有特定识别碱基序列的双链DNA后，α-螺旋含量随着DNA浓度的增加而进一步增加。当使用含有中心两个碱基对不同序列的DNA二聚体时，α-螺旋含量增加较小。在高温下观察到同源序列的严格特异性识别。MAX的生物活性受N-末端丝氨酸残基的磷酸化调控。我们制备了突变蛋白，其中一个或两个Ser残基被天冬氨酸残基(S)取代，作为磷酸化MAX的模拟蛋白，并检测了突变蛋白的DNA结合能力。突变蛋白-DNA复合体的稳定性较差，其中双突变蛋白复合体的稳定性最差。我们还开发了一种合成方法，通过将化学合成的磷酸化多肽与通过基因工程制备的剩余部分蛋白质化学连接，获得磷酸化的MAX。