本文選題：蛋白質(zhì)相互作用 + PDZ結(jié)構(gòu)域��； 參考：《北京協(xié)和醫(yī)學院》2012年博士論文

【摘要】：·第一部分研究了PDZ結(jié)構(gòu)域結(jié)合中間序列的結(jié)合特性 蛋白質(zhì)-蛋白質(zhì)相互作用在生命活動中廣泛存在,主要由蛋白質(zhì)結(jié)構(gòu)域來高效介導。大規(guī)模的蛋白質(zhì)相互作用可以通過研究結(jié)構(gòu)域的結(jié)合特性來實現(xiàn)。PDZ、SH3、WW等結(jié)構(gòu)域通過一個或多個識別“口袋”來識別和結(jié)合配體蛋白的一段保守的短肽序列。然而蛋白質(zhì)相互作用可能比我們現(xiàn)在認識到的復雜,還有很多的相互作用未被研究清楚。就PDZ結(jié)構(gòu)域而言,它通常結(jié)合配體蛋白C末端4-5個氨基酸殘基,近來研究發(fā)現(xiàn)其也能夠結(jié)合配體蛋白的中間序列,與自身或其他結(jié)構(gòu)域聚合,或與膜上的脂類結(jié)合。這些結(jié)合方式使蛋白質(zhì)的相互作用呈現(xiàn)出多樣性與復雜性。 首先,為了系統(tǒng)地高通量地研究PDZ結(jié)構(gòu)域結(jié)合中間序列的結(jié)合特性,我們在酵母雙雜交系統(tǒng)中構(gòu)建了中間序列隨機八肽文庫。文庫的構(gòu)建策略是將所有序列的C末端設(shè)計成相同的序列,從而保證PDZ結(jié)構(gòu)域結(jié)合的是中間序列。而后,使用文庫篩選了24個PDZ結(jié)構(gòu)域,其中包括已知文獻報道的能夠結(jié)合中間序列的PDZ結(jié)構(gòu)域；實驗室已經(jīng)構(gòu)建好的PDZ結(jié)構(gòu)域和一些能夠結(jié)合脂類的PDZ結(jié)構(gòu)域。實驗表明有14個結(jié)構(gòu)域具有結(jié)合中間序列的特性,其中6個結(jié)構(gòu)域未曾被報道。序列具有偏好性,偏好較強的具有一致序列。與C末端序列的結(jié)合特性相比,中間序列的結(jié)合特性表現(xiàn)出多樣性,只有ZO1-PDZ1結(jié)構(gòu)域的中間序列特性與其C末端特性相似。篩選結(jié)果也反映出PDZ結(jié)構(gòu)域?qū)τ谥虚g序列和C末端序列的偏好性。另外,HtrA2-PDZ的偏好性較弱,沒有一致序列,表現(xiàn)為結(jié)合疏水多肽。中間序列的篩選結(jié)果說明有更多的PDZ結(jié)構(gòu)域可以結(jié)合中間序列。此外,應用中間序列的結(jié)合特性可以來尋找已知相互作用的結(jié)合位點,也可以利用中間序列或一致序列來搜索數(shù)據(jù)庫發(fā)掘潛在的天然相互作用蛋白。 ·第二部分研究了日本血吸蟲蛋白GIPC3的分子特征及其PDZ結(jié)構(gòu)域的配體結(jié)合特性 血吸蟲病仍然是一個全球性的嚴重的公共衛(wèi)生問題,流行于77個國家和地區(qū),感染者超過2.3億。依賴于單一藥物吡喹酮大規(guī)模長期治療引起對抗藥性問題的關(guān)注。PDZ結(jié)構(gòu)域蛋白被認為是下一代藥物開發(fā)的潛在靶點。在本論文的第二部分主要研究日本血吸蟲PDZ結(jié)構(gòu)域蛋白SjGIPC3的分子特征及利用隨機多肽文庫探索其PDZ結(jié)構(gòu)域的結(jié)合特性。 SjGIPC3是含有單個PDZ結(jié)構(gòu)域的蛋白。多重序列比對分析顯示SjGIPC3與其同源物在GH1與PDZ結(jié)構(gòu)域相對保守,但PDZ結(jié)構(gòu)域中的C末端結(jié)合環(huán)處并不保守,其中的經(jīng)典的GLGF模體被SFGL替代。進化分析表明日本血吸蟲SjGIPC3與吸蟲同源物形成一分支,而與其他物種相距較遠。分期轉(zhuǎn)錄分析顯示,SjGIPC3基因在侵入宿主后轉(zhuǎn)錄水平顯著升高；在雄性成蟲的轉(zhuǎn)錄水平為最高。 在酵母雙雜交系統(tǒng)中,我們初步探索了SjGIPC3-PDZ結(jié)合C末端序列的結(jié)合特性。用SjGIPC3全長蛋白或其PDZ結(jié)構(gòu)域為誘餌蛋白,篩選了本室利用人基因組構(gòu)建的新型隨機多肽文庫,實驗表明SjGIPC3PDZ結(jié)構(gòu)域主要結(jié)合Ⅰ類和Ⅱ類配體,但以結(jié)合Ⅰ類配體為主；用SjGIPC3全長蛋白為誘餌篩選文庫得到的C末端序列比用PDZ結(jié)構(gòu)域為誘餌得到的序列的規(guī)律性更強,提示SjGIPC3蛋白的N端和C端區(qū)域可能對PDZ結(jié)構(gòu)域的配體結(jié)合特性產(chǎn)生影響。根據(jù)PDZ結(jié)構(gòu)域的結(jié)合規(guī)律,利用本室開發(fā)的Tailfit軟件預測并在酵母雙雜交系統(tǒng)中驗證了4個潛在的SjGIPC3的天然配體,其中之一為NMDA受體。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363

【參考文獻】

相關(guān)期刊論文 前4條

1 李秋霞;羅茂林;李茹柳;陳蔚文;;緊密連接蛋白ZO-1研究概述[J];廣州中醫(yī)藥大學學報;2007年06期

2 郭佳巖,宋婀莉,黃海明,馬素參,高友鶴;利用隨機多肽文庫研究ZO-1中PDZ3結(jié)構(gòu)域的配體結(jié)合特點[J];中國生物化學與分子生物學報;2004年03期

3 田瑞;李英娜;宋婀莉;高詩娟;黃海明;張玲;馬素參;高友鶴;;通過篩選隨機多肽文庫研究ZO-1中PDZ1結(jié)構(gòu)域的配體結(jié)合特點[J];中國生物化學與分子生物學報;2006年06期

4 吳志豪;王建;賀福初;;大規(guī)模酵母雙雜交技術(shù)研究蛋白質(zhì)相互作用的應用[J];遺傳;2006年12期

，

本文編號：1977966