Identification of host proteins interacting with the integrin-like A domain of Toxoplasma gondii micronemal protein MIC2 by yeast-two-hybrid screening.
Identification of host proteins interacting with the integrin-like A domain of Toxoplasma gondii micronemal protein MIC2 by yeast-two-hybrid screening.
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通过酵母双杂交筛选鉴定与弓形虫微线体蛋白 MIC2 的类整合素 A 结构域相互作用的宿主蛋白
DOI：
10.1186/s13071-014-0543-1
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发表时间：
2014-11-26
影响因子：
3.2
通讯作者：
Zhao J
中科院分区：
文献类型：
--
作者：
Wang Y;Fang R;Yuan Y;Hu M;Zhou Y;Zhao J
背景：弓形虫是一种专性胞内原虫，是引起重要的人畜共患病弓形虫病的病原体。这种寄生虫利用一种称为滑翔运动的独特运动形式来寻找和入侵宿主细胞。微线粘附素MIC 2通过其胞外粘附结构域与底物和宿主细胞受体结合，在这些过程中发挥关键作用。虽然已知MIC 2在入侵期间介导寄生虫和宿主细胞之间的重要相互作用，但与MIC 2相互作用的特异性宿主蛋白尚未明确鉴定。本研究利用酵母双杂交系统寻找与MIC 2相互作用的宿主蛋白。方法：将MIC 2的不同粘附结构域克隆到pGBKT 7载体中，并与GAL 4 DNA结合结构域融合表达。通过免疫印迹分析诱饵蛋白在酵母细胞中的表达，并通过与仅表达GAL 4 DNA结合结构域的pGBKT 7空载体的比较来测试它们的自激活。为了鉴定与MIC 2相互作用的宿主蛋白，将小鼠cDNA文库克隆到GAL 4激活结构域表达载体中，使用MIC 2的整合素样A结构域（残基74-270）作为诱饵，通过酵母双杂交筛选。在初步筛选和排除假阳性命中后，对阳性猎物进行测序并使用BLAST分析和Gene Ontology Classifications进行分析。鉴定了弓形虫MIC 2基因，分别为LAMTOR 1（late endosomal/lysosomal adaptor，MAPK and mTOR activator 1）和RNaseH 2 B（ribonuclease H2 subunit B）。结论：本研究首次报道了弓形虫MIC 2与宿主蛋白LAMTOR 1和RNaseH 2B的相互作用。这些数据将有助于我们更好地了解MIC 2的功能，并表明MIC 2除了与宿主细胞结合外，还可能在调节宿主信号转导和其他生物学过程中发挥作用。
Background:Toxoplasma gondii is an obligate intracellular protozoan, causing the important zoonosis toxoplasmosis. This parasite utilizes a unique form of locomotion called gliding motility to find and invade host cells. The micronemal adhesin MIC2 plays critical roles in these processes by binding to substrates and host cell receptors using its extracellular adhesive domains. Although MIC2 is known to mediate important interactions between parasites and host cells during invasion, the specific host proteins interacting with MIC2 have not been clearly identified. In this study, we used a yeast-two-hybrid system to search for host proteins that interact with MIC2.Methods:Different adhesive domains of MIC2 were cloned into the pGBKT7 vector and expressed in fusion with the GAL4 DNA-binding domain as baits. Expression of bait proteins in yeast cells was analyzed by immuno-blotting and their autoactivation was tested via comparison with the pGBKT7 empty vector, which expressed the GAL4 DNA binding-domain only. To identify host proteins interacting with MIC2, a mouse cDNA library cloned into a GAL4 activation-domain expressing vector was screened by yeast-two-hybrid using the integrin-like A domain of MIC2 (residues 74-270) as bait. After initial screening and exclusion of false positive hits, positive preys were sequenced and analyzed using BLAST analysis and Gene Ontology Classifications.Results:Two host proteins that had not previously been reported to interact with T. gondii MIC2 were identified: they are LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) and RNaseH2B (ribonuclease H2 subunit B). Gene Ontology analysis indicated that these two proteins are associated with many cellular processes, such as lysosome maturation, signaling transduction, and RNA catabolism.Conclusion:This study is the first one to report interactions between Toxoplasma gondii MIC2 and two host proteins, LAMTOR1 and RNaseH2B. The data will help us to gain a better understanding of the function of MIC2 and suggest that MIC2 may play roles in modulating host signal transduction and other biological processes in addition to binding host cells.
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DOI：
10.1074/jbc.m110.177394
发表时间：
2011-03-25
期刊：
The Journal of biological chemistry
影响因子：
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作者：
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影响因子：
64.5
作者：
Reijns MA;Rabe B;Rigby RE;Mill P;Astell KR;Lettice LA;Boyle S;Leitch A;Keighren M;Kilanowski F;Devenney PS;Sexton D;Grimes G;Holt IJ;Hill RE;Taylor MS;Lawson KA;Dorin JR;Jackson AP
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影响因子：
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影响因子：
3.2
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