Joompa Pattamaporn, Ponnikorn Saranyoo, Roytrakul Sittiruk, Tungpradabkul Sumalee
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Chulabhorn International College of Medicine, Thammasat University, Rangsit Campus, Pathum Thani, Thailand.
Cell Biosci. 2017 Aug 23;7:45. doi: 10.1186/s13578-017-0172-4. eCollection 2017.
is an intracellular bacteria causing Melioidosis, the disease widely disseminates in Southeast Asia and Northern Australia. has ability to invade various types of host cell and to interfere with host defense mechanisms, such as nitric oxide (NO). Due to the cross-talk among alternative killing mechanisms in host immune response against invading microbes, autophagy is the molecular mechanism belonging to intracellular elimination of eukaryotic cells that has been widely discussed. However, bacterial evasion strategy of and host-bacterial protein-protein interaction within autophagic machinery remain unknown.
Here, we demonstrated the protein-protein interaction study between different strains of , including wild type PP844 and mutant, with autophagy-related protein LC3 that has been constructed, using the modified immunoaffinity hydrophobic chromatography based-technique. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was utilized for identifying the eluted proteins obtained from the established column. In addition, the expression level of gene encoding candidate protein was predicted prior to verification using real-time quantitative reverse transcription PCR assay (RT-qPCR).
LC3 recombinant proteins could be entrapped inside the column before encountering their bacterial interacting partners. Based on affinity interaction, the binding capacity of LC3 with antibody displayed over 50% readily for hydrophobically binding with bacterial proteins. Following protein identification, bacterial ATP-binding cassette (ABC) transporter periplasmic substrate-binding protein (BPSL2203) was identified as a candidate LC3-interacting protein, which was found only in wild type. Gene expression analysis and bioinformatics of BPSL2203 were validated the proteomic result which are suggesting the role of RpoS-dependent gene regulation.
Remarkably, utilization of the modified immunoaffinity hydrophobic chromatography with LC-MS/MS is a convenient and reliable approach to a study in -LC3 protein-protein interaction.
是一种引起类鼻疽病的细胞内细菌，该疾病在东南亚和澳大利亚北部广泛传播。具有侵入各种类型宿主细胞并干扰宿主防御机制的能力，如一氧化氮（NO）。由于宿主针对入侵微生物的免疫反应中多种杀伤机制之间存在相互作用，自噬是一种属于真核细胞内清除的分子机制，已被广泛讨论。然而，的细菌逃避策略以及自噬机制内的宿主 - 细菌蛋白质 - 蛋白质相互作用仍不清楚。
在此，我们使用基于改良免疫亲和疏水色谱的技术，展示了不同菌株（包括野生型PP844和突变体）与已构建的自噬相关蛋白LC3之间的蛋白质 - 蛋白质相互作用研究。利用液相色谱串联质谱（LC - MS/MS）分析来鉴定从已建立的柱子中洗脱的蛋白质。此外，在使用实时定量逆转录PCR测定法（RT - qPCR）进行验证之前，预测编码候选蛋白的基因表达水平。
LC3重组蛋白在遇到其细菌相互作用伙伴之前可被困在柱子内。基于亲和相互作用，LC3与抗体的结合能力显示超过50%易于与细菌蛋白进行疏水结合。在蛋白质鉴定后，细菌ATP结合盒（ABC）转运蛋白周质底物结合蛋白（BPSL2203）被鉴定为候选的LC3相互作用蛋白，仅在野生型中发现。BPSL2203的基因表达分析和生物信息学验证了蛋白质组学结果，提示了RpoS依赖性基因调控的作用。
值得注意的是，将改良免疫亲和疏水色谱与LC - MS/MS结合使用是一种研究-LC3蛋白质 - 蛋白质相互作用的便捷可靠方法。
Zotero 插件
