【作者】 刘迎；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 本论文工作的重点分为两部分:第一部分是人类Brd4中两个bromodomain的克隆、表达和纯化,研究了它们在溶液中的存在状态,并用核磁共振的方法解析了溶液中Brd4 BD2的结构,研究了两个bromodomain与乙酰化的组蛋白尾巴的相互作用,测定了它们的解离常数,分析了Brd4 BD2分别在游离状态下和配基结合状态下主链的动力学特性,并用分子动力学模拟的方法计算了由一个双乙酰化的组蛋白小肽和两个BD2组成的三元复合物模型;第二部分是关于人类Mogl蛋白的表达纯化以及溶液结构的解析,并初步研究了它与Ran·GDP的相互作用。论文的第一章阐述了第一部分的内容。第一章的第一部分详细介绍了Brd4在生物体内的重要作用。Brd4是一个多功能蛋白,它与不同的蛋白结合以发挥不同的功能。通过结合P-TEFb和mediator,调控转录的延长过程;通过结合RFC,调控DNA复制过程,并调节细胞G1/S期的转变;通过与SPA-1结合,参与细胞信号传导途径,并调控细胞G2/M期的转变;Brd4还参与到某些病毒的生命周期中去,或者调控病毒基因的转录及复制,或者辅助病毒在细胞有丝分裂时均匀的分布到子代细胞中。所有的这些功能都是与它结合乙酰化的染色质相联系的。Brd4是BET家族的成员,解读属于它自己的组蛋白密码,并将其翻译成不同的细胞调控过程。第一章的第二部分详尽的描述了各种蛋白质的克隆、表达、纯化过程以及论文中所涉及到的其它实验过程。第一章的第三部分是实验的结果和讨论。Brd4 BD2的溶液结构表明,它是由保守的左手四螺旋束构成,长的ZA loop上一些独特的二级结构的修饰使得BD2呈现出一个独特的疏水口袋,以识别某些乙酰化的组蛋白尾巴。Brd4的BD1和BD2都能够结合H4-AcK5和H4-AcK12,只是亲和性有一些微小的差别,而且它们与单乙酰化的小肽结合都很弱,解离常数都在毫摩尔数量级。我们鉴定了BD1和BD2在溶液中都主要以单体存在,而且当它们以等摩尔比例混合时,也没有异二聚体形成,这一结果与之前其它研究小组的预测是完全不同的。Brd2BD1和TAFⅡ250通过形成同二聚体或者异二聚体而极大程度的增强了与乙酰化组蛋白尾巴的亲和性。而Brd4不能以类似的方式增强与组蛋白尾巴的结合,因此,我们推测应该有其它的机制来辅助这一过程。HPV E2蛋白建立了一个很好的模型,很可能在正常细胞中,一个尚未被发现的类似E2的蛋白辅助Brd4与染色质的结合,当然这个蛋白也可能就是mediator中的某一组分。主链动力学分析表明,Brd4 BD2在结合了配基之后,微秒-毫秒数量级的交换过程在一定程度上受到抑制,而皮秒-纳秒数量级的快速运动则变化的不规律。论文的第二章阐述了第二部分的内容。到目前为止Mogl这个蛋白研究的并不多,而且主要都是来自于酵母的信息。论文中列举了已有文献报道的一些已知的功能,其中最主要的就是它能够与Ran结合,参与调控生物分子的核质转运过程。Ran在这一转运体系中扮演着重要的角色,Mogl与Ran·GTP和Ran·GDP都能够紧密结合,并促使Ran将核苷酸释放出去,空载状态下的Ran仍然能够与Mogl紧密结合。但这种结合在核质转运系统中确切的分子机制还不清楚。Mogl参与SLN1-SKN7信号传导途径,能够直接与途径中的某些蛋白相互作用,而且从某种意义上说,这一功能应该还是与核质转运相联系的。此外,Mogl可能还参与RNA代谢和脂类代谢过程。我们表达纯化了人类Mogl和Ran蛋白,并用核磁共振方法解析了hMogl的溶液结构,研究了它们相互作用的特性,希望能够找到它们的结合界面,或者得到复合物的晶体结构,这部分的工作还在进行中。这部分工作中,hMogl和hRan的表达质粒构建是由孙建平博士完成的,hMogl的表达纯化以及所有蛋白样品的制备是我完成的,hMogl溶液结构的解析是胡琦博士完成的,其它的实验包括长晶体的尝试是由我和胡琦共同完成的。更多还原

【Abstract】 This PhD thesis focuses on two parts: The first part is the cloning, expression and purification of the two bromodomains in human Brd4. Their properties in solution were studied, and the solution structure of Brd4 BD2 was determined. The interactions between BDs of Brd4 and acetylated histone tails have also been studied, and their dissociation constants were measured. Backbone dynamic properties of both apo- and ligand bound- BD2 were analyzed. A ternary complex model is presented, which is composed of one molecule of diacetylated histone peptide and two molecules of Brd4 BD2. It was calculated by molecular dynamics simulation. The second part is about the expression, purification and structure determination of human Mogl. Interactions between hMogl and hRanGDP were investigated primarily. Chapter I describes the first part of our work.In the first part of Chapter I, there is a particular description of the importance of Brd4 in organisms. It is a multifunctional protein, and plays different roles through interaction with different binding partners. Brd4 regulates transcription elongation process through interaction with P-TEFb and mediator. It regulates DNA replication process and G1/S transition through interaction with RFC. It is involved in signal transduction pathway, and regulates G2/M transition by association with SPA-1. It is also involved in the life cycles of some viruses. It regulates the transcription or replication of viral genes, or facilitates their partition during mitosis. All these functions are linked to its association with acetylated chromatin. Brd4 belongs to the BET family. It reads its own histone code, and then translates it into its own regulatory processes.The protein cloning, expression, purification and other experimental processes were described in detail in the second part of Chapter I.The third part of Chapter I describes the results and discussion of the corresponding experiments. Solution structure of Brd4 BD2 demonstrates a conserved left-handed four-helix bundle. Special decorations on the long ZA loop present a unique surface on the functional hydrophobic pocket, which determines the recognition for acetylated histone tails. Both BD1 and BD2 bind to H4-AcK5 and H4-AcK12, but with subtle difference. Their bindings are rather weak. The dissociation constants were estimated at milimolar scale. BD1 and BD2 of Brd4 were both identified to be mainly monomeric in solution. No heterodimers were observed when they were mixed at equal molar. These results are different from the predictions of another group. While Brd2 BD1 and TAFII250 enhance their affinities with acetylated histone tails through formation of a homodimer or a heterodimer, and Brd4 fails to function similarly. We predict that this protein should have its own mechanism to hold onto chromosomes during mitosis and regulation of associated cellular processes. HPV E2 sets up a good model. It is possible that, in normal living cells, an unknown E2 like protein facilitates the reinforcement of Brd4 and acetyl-chromatin association. It is also possible that this unknown protein comes from the mediator complex. Analysis of backbone dynamics demonstrated that, ligand binding depressed the microsecond-millisecond conformational exchanges in some degree, while changes in picosecond-nanosecond time scale fast motions were irregular.Chapter II describes the second part of our work.Mog1 has not been investigated well up to now. The primary information comes from the yeast. We list some of its functions that have been published. One of the most important is that, Mog1 binds to Ran, and participates in the regulation of nucleocytoplasm transport. Ran plays important roles in this system. Mog1 interacts with both Ran-GTP and Ran-GDP. The interaction makes Ran release the nucleotide. The unloaded Ran still binds to Mog1. But the mechanism of this interaction in the transport system is not known. Mog1 is also involved in the SLN1-SKN7 pathway. It directly binds to some components of the pathway. In some sense, this function still links to nuclear transport. Besides, Mog1 may participate in RNA and lipid metabolism.We expressed and purified the Mog1 and Ran protein, determined the solution structure of hMog1, and investigated some properties of their interaction. We hope to find the binding interface or obtain the crystal structure of their complex. This part of work is still in process.This part of work was done cooperatively with Jianping Sun and QiHu. Plasmids of hMog1 and hRan were constructed by Dr. Jianping Sun. Solution structure of hMog1 was determined by Dr. QiHu. Expression, purification, and all the sample preparation of hMog1 were of my work. All other experiments, including protein complex crystallization, were carried out by QiHu and me.更多还原