张柯,刘清波,胡永林,王大成.巴尔通氏体效应蛋白BepE干扰人源Csk蛋白信号通路的结构与功能分析[J].生物化学与生物物理进展(待发表)
巴尔通氏体效应蛋白BepE干扰人源Csk蛋白信号通路的结构与功能分析
Structural and functional analysis of disruption of the human Csk signaling pathways by effector protein BepE from Bartonella henselae
投稿时间:2017-07-14  修订日期:2017-09-29
中文关键词:  巴尔通氏体,BepE,EPIYA,Csk,晶体结构
英文关键词:Bartonella  henselae,BepE,EPIYA,Csk,Crystal  Structure
基金项目:中国科学院战略性先导科技专项(XDB08020202),国家生物大分子重点实验室资助项目
作者单位E-mail
张柯 (1)中国科学院生物物理研究所生物大分子国家重点实验室(2)中国科学院大学 adxyz1990@163.com 
刘清波 中国科学院生物物理研究所生物大分子国家重点实验室  
胡永林 中国科学院生物物理研究所生物大分子国家重点实验室 yonglin@ibp.ac.cn 
王大成 中国科学院生物物理研究所生物大分子国家重点实验室 dcwang@sun5.ibp.ac.cn 
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中文摘要:
      巴尔通氏体作为多种人类疾病的病原菌,可分泌多种毒力效应蛋白。其中的7个毒力效应蛋白BepA-BepG由一个IV型分泌系统VirB注入宿主细胞内,干扰宿主细胞的多种信号传递通路。在这7个效应蛋白中,BepD-F的N端含有特征的EPIYA基序。注入宿主细胞后,这些EPIYA基序上的酪氨酸残基被宿主细胞中的SFK激酶磷酸化,磷酸化后的EPIYA基序可与宿主细胞中含SH2结构域的蛋白结合并干扰宿主细胞的SH2蛋白相关信号通路。在此之前,已经有多种病原菌的效应蛋白被发现含有EPIYA基序,并且通过磷酸化的EPIYA干扰哺乳动物宿主的SH2信号通路。这些毒性效应蛋白除EPIYA基序之外并没有明显的序列同源性。与此相应,在人类蛋白质组中目前只发现了6种含有EPIYA基序的蛋白,这个基序出现的频率显著低于基于随机预测的频率,这可能是在人类进化过程中含有EPIYA基序的蛋白会干扰正常信号通路传导而被淘汰。JAM-A是已报道的6个人体内EPIYA蛋白之一,它存在于人血小板中的,可通过招募Csk至血小板来避免血栓的形成。已有的报道证明了BepE可与人源Csk相互作用,通过影响Csk的功能进而干扰一系列信号通路。人源Csk蛋白作为一个可以同时被来自于病原菌毒力蛋白和内源蛋白中磷酸化EPIYA基序识别并结合的分子,为我们研究并对比这两种含EPIYA基序的蛋白质对人类细胞中SH2信号通路的作用提供了一个理想的靶标。本文报道了Csk与BepE及JAM-A两种磷酸化多肽复合物高分辨率晶体结构并分别测定其亲和力。Csk与多肽复合物结构显示,Csk与两种多肽结合方式相似,都是通过SH2结构域与磷酸化酪氨酸结合,多肽链垂直于SH2结构域中的β片层。SPR实验结果显示,来源于巴尔通氏体的BepE比人源JAM-A与Csk亲和力高,这暗示毒力蛋白通过磷酸化的EPIYA基序以高亲和力结合人体内含SH2结构域的蛋白进而干扰SH2蛋白所涉及的信号通路。
英文摘要:
      ABSTRACT: The vasculotropic pathogenic bacteria Bartonella henselae secretes 7 Bep effector proteins (BepA-G) and injects these proteins into host cells using a type IV secretion system. Among these effector proteins, BepD-F contain multiple copies of Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in their N-terminal regions. After injection into host cells, the tyrosine residues in these EPIYA motifs will be phosphorylated by SFK, and the tyrosine-phosphorylated EPIYA motifs can subsequently interact with various SH2 domain-containing proteins from host cell and interfere with the SH2 signaling pathways of the host cells. In addition to B. henselae Bep effector proteins, numerous pathogenic bacteria effector proteins had been identified previously to contain EPIYA motifs. They were also found to utilize their EPIYA motifs to disrupt the SH2-signaling pathways of respective host cells, although they share no obvious overall sequence similarities. In contrast, only 6 proteins containing EPIYA motifs were found in human proteome, much lower than random occurrences, probably due to its toxicity in human cells. JAM-A is one of EPIYA motif containing proteins occurring in blood platelet, which can recruit Csk to avoid thrombus formation. BepE was reported to interact with Csk and disturb Csk-related signaling pathways. Here we reported the crystal structures of complexes between Csk and the phosphorylated EPIYA motifs from BepE and JAM-A, and biophysical measurements of their binding affinities. The complex structures show that Csk binds with two phosphopeptides in a similar way. The Csk SH2 domain binds with phosphotyrosine. Phosphopeptides are perpendicular to β-sheets in SH2 domain. Results of SPR show that the affinity of BepE with Csk is much higher than that of JAM-A. These results suggest that the EPIYA motif of toxic proteins binds with the SH2 domain of human Csk by a higher affinity, then disturb the SH2 domain proteins involved signaling pathways. The results reported here laid a solid foundation for our understanding of the structural basis of how toxic EPIYA motifs interrupt the SH2 signaling pathways of host cells.
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