PDZ结构域结合中间序列结合特性的研究

发布时间：2018-06-04 15:57

本文选题：蛋白质相互作用 + PDZ结构域　；参考：《北京协和医学院》2012年博士论文

【摘要】：·第一部分研究了PDZ结构域结合中间序列的结合特性蛋白质-蛋白质相互作用在生命活动中广泛存在,主要由蛋白质结构域来高效介导。大规模的蛋白质相互作用可以通过研究结构域的结合特性来实现。PDZ、SH3、WW等结构域通过一个或多个识别“口袋”来识别和结合配体蛋白的一段保守的短肽序列。然而蛋白质相互作用可能比我们现在认识到的复杂,还有很多的相互作用未被研究清楚。就PDZ结构域而言,它通常结合配体蛋白C末端4-5个氨基酸残基,近来研究发现其也能够结合配体蛋白的中间序列,与自身或其他结构域聚合,或与膜上的脂类结合。这些结合方式使蛋白质的相互作用呈现出多样性与复杂性。首先,为了系统地高通量地研究PDZ结构域结合中间序列的结合特性,我们在酵母双杂交系统中构建了中间序列随机八肽文库。文库的构建策略是将所有序列的C末端设计成相同的序列,从而保证PDZ结构域结合的是中间序列。而后,使用文库筛选了24个PDZ结构域,其中包括已知文献报道的能够结合中间序列的PDZ结构域；实验室已经构建好的PDZ结构域和一些能够结合脂类的PDZ结构域。实验表明有14个结构域具有结合中间序列的特性,其中6个结构域未曾被报道。序列具有偏好性,偏好较强的具有一致序列。与C末端序列的结合特性相比,中间序列的结合特性表现出多样性,只有ZO1-PDZ1结构域的中间序列特性与其C末端特性相似。筛选结果也反映出PDZ结构域对于中间序列和C末端序列的偏好性。另外,HtrA2-PDZ的偏好性较弱,没有一致序列,表现为结合疏水多肽。中间序列的筛选结果说明有更多的PDZ结构域可以结合中间序列。此外,应用中间序列的结合特性可以来寻找已知相互作用的结合位点,也可以利用中间序列或一致序列来搜索数据库发掘潜在的天然相互作用蛋白。 ·第二部分研究了日本血吸虫蛋白GIPC3的分子特征及其PDZ结构域的配体结合特性血吸虫病仍然是一个全球性的严重的公共卫生问题,流行于77个国家和地区,感染者超过2.3亿。依赖于单一药物吡喹酮大规模长期治疗引起对抗药性问题的关注。PDZ结构域蛋白被认为是下一代药物开发的潜在靶点。在本论文的第二部分主要研究日本血吸虫PDZ结构域蛋白SjGIPC3的分子特征及利用随机多肽文库探索其PDZ结构域的结合特性。 SjGIPC3是含有单个PDZ结构域的蛋白。多重序列比对分析显示SjGIPC3与其同源物在GH1与PDZ结构域相对保守,但PDZ结构域中的C末端结合环处并不保守,其中的经典的GLGF模体被SFGL替代。进化分析表明日本血吸虫SjGIPC3与吸虫同源物形成一分支,而与其他物种相距较远。分期转录分析显示,SjGIPC3基因在侵入宿主后转录水平显著升高；在雄性成虫的转录水平为最高。在酵母双杂交系统中,我们初步探索了SjGIPC3-PDZ结合C末端序列的结合特性。用SjGIPC3全长蛋白或其PDZ结构域为诱饵蛋白,筛选了本室利用人基因组构建的新型随机多肽文库,实验表明SjGIPC3PDZ结构域主要结合Ⅰ类和Ⅱ类配体,但以结合Ⅰ类配体为主；用SjGIPC3全长蛋白为诱饵筛选文库得到的C末端序列比用PDZ结构域为诱饵得到的序列的规律性更强,提示SjGIPC3蛋白的N端和C端区域可能对PDZ结构域的配体结合特性产生影响。根据PDZ结构域的结合规律,利用本室开发的Tailfit软件预测并在酵母双杂交系统中验证了4个潜在的SjGIPC3的天然配体,其中之一为NMDA受体。
[Abstract]:The first part studies the binding properties of PDZ domains combined with intermediate sequences.
Protein protein interaction is widely existed in life activities, mainly mediated by the protein domain. Large-scale protein interactions can be used to identify and combine ligand proteins by one or more "pockets" by studying the binding properties of the domains in the.PDZ, SH3, and WW domains. Short peptide sequences. However, protein interaction may be more complex than we have known now, and many interactions have not been studied. In terms of the PDZ domain, it usually combines the 4-5 amino acid residues of the ligand protein C terminal. Recent studies have found that it can also combine with the ligand of the ligand egg white, with its own or other domains. They combine with or bind to lipids on the membrane. These binding modes make protein interactions present diversity and complexity.
First, in order to systematically study the combination of PDZ domain and intermediate sequence in high flux, we construct an intermediate sequence random eight peptide library in the yeast two hybrid system. The construction strategy of the library is to design the C end of all the sequences into the same sequence, so as to ensure that the PDZ domain is combined with the intermediate sequence. The library has screened 24 PDZ domains, including the known PDZ domains that can combine with the intermediate sequence; the laboratory has constructed a good PDZ domain and some PDZ domains that can be combined with lipids. The experiment shows that 14 domains have the characteristics of combining with the intermediate sequences, of which 6 domains have not been reported. Sequences have Preference and strong preference have a consistent sequence. Compared with the binding characteristics of the C terminal sequence, the binding characteristics of the intermediate sequences are diverse. Only the intermediate sequence characteristics of the ZO1-PDZ1 domain are similar to that of the C terminal. The screening results also reflect the preference of the PDZ domain for the intermediate sequence and the C terminal sequence. In addition, HtrA2-PDZ The preference is weak and there is no consistent sequence, showing the binding of hydrophobic peptides. The screening results of the intermediate sequences show that more PDZ domains can be combined with the intermediate sequences. In addition, the binding properties of the intermediate sequences can be used to find the known binding sites, and the middle sequence or the uniform sequence can be used to search the database. Discover potential natural interaction proteins.
In the second part, we studied the molecular characteristics of Schistosoma japonicum protein GIPC3 and the ligand binding properties of PDZ domain.
Schistosomiasis is still a global and serious public health problem, which is prevalent in 77 countries and regions, with more than 230 million infected people. The attention to the problem of drug resistance by the large-scale long-term treatment of single drug praziquantel.PDZ domain protein is considered to be a potential target for the development of the next generation of drugs. In the second part of this paper The molecular characteristics of Schistosoma japonicum PDZ domain protein SjGIPC3 were studied, and the binding properties of PDZ domain were explored by random peptide library.
SjGIPC3 is a protein containing a single PDZ domain. Multiple sequence alignment analysis shows that SjGIPC3 and its homology are relatively conservative in the GH1 and PDZ domains, but the C end binding ring in the PDZ domain is not conservative, and the classical GLGF modules are replaced by SFGL. Evolutionary analysis shows that Schistosoma japonicum SjGIPC3 and the homologous of the fluke form a branch, The transcriptional analysis showed that the transcriptional level of the SjGIPC3 gene increased significantly after the invasion of the host, and the transcriptional level of the male adult was the highest.
In the yeast two hybrid system, we preliminarily explored the binding properties of SjGIPC3-PDZ binding C terminal sequence. Using SjGIPC3 full length protein or its PDZ domain as bait protein, we screened a new random peptide library constructed by human genome in this room. The experiment showed that the SjGIPC3PDZ domain was mainly combined with class I and class II ligands, but combined with I. The class of ligands is dominant; the C terminal sequence obtained by the SjGIPC3 full-length protein as the bait library is more regular than the sequence used by the PDZ domain as the bait. It is suggested that the N and C end regions of the SjGIPC3 protein may affect the ligand binding properties of the PDZ domain. According to the binding law of the PDZ domain, the Tailfi developed in this room is used. T software predicted and identified 4 potential SjGIPC3 natural ligands in yeast two hybrid system, one of which is NMDA receptor.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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