Mutation within the transmembrane domain of oxidized low-density lipoprotein receptor 1 influences oxidized low-density lipoprotein-induced signal transduction.
Zhen Ma, Ran Xu, Jing Lu, Xiong Huang, Hao Jia, Zhiwen Ding, Jie Yuan, Yunzeng Zou
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引用次数: 0
Abstract
ObjectiveTo investigate the important active sites within the NTFs to affect the in vitro interaction of oxidized low-density lipoprotein (ox-LDL) with its receptor, OLR1.MethodsSimulation analysis online was performed to generate various OLR1 chimeras, truncation mutants, and site-specific mutations. They were transfected in COS-7 cells and subjected to ox-LDL stimulations to observe the different reactions. Immunoprecipitation-mass spectrometry (IP-MS) was performed to show what proteins combined with OLR1 mutants in reaction to ox-LDL. Lipid uptake in human monocytes (THP-1) originated foam cells overexpressing somatic mutant of OLR1 were also determined. Further studies focusing on these regions were conducted using truncation mutants and site-specific mutants such as G43A, V44A, L45A, C46A, and L47A.ResultsAmino acids within the TM were highly conserved, spanning amino acids 35 to 57. The induction of intracellular p-ERK1/2 in response to ox-LDL stimulation was highly promoted in Chimera 3 possessing the TM from OLR1 like OLR1/WT (p < 0.05). Sequence alignment revealed two conserved regions within the TM of OLR1, Leu45-Cys46-Leu47 and Val55-Leu56-Gly57. Western blot showed that most of the TM changes ablated ERK1/2 activation in response to ox-LDL stimulation (p < 0.05). One human somatic mutation at L45F revealed significantly lower p-ERK1/2 levels with enhanced intake of ox-LDL in THP-1-derived foam cells than the control cells (p < 0.05). L45A and C46A molecular complexes were identified. After ox-LDL stimulation, these underlined interactions with keratins, namely KRT2 and KRT6A.ConclusionThese findings emphasize the vital role of the TM in the interactions between OLR1 and ox-LDL and point to an exciting possibility that signal transduction induced by ox-LDL through its receptor OLR1 may involve complex interactions with cytoskeletal proteins.
目的探讨氧化低密度脂蛋白（ox-LDL）与其受体OLR1在体外相互作用中的重要活性位点。方法通过在线模拟分析，生成各种OLR1嵌合体、截断突变体和位点特异性突变体。将它们转染到COS-7细胞中，并进行ox-LDL刺激，观察不同反应。采用免疫沉淀-质谱法（IP-MS）显示哪些蛋白与OLR1突变体在ox-LDL反应中结合。也测定了过表达OLR1体细胞突变体的人单核细胞（THP-1）源泡沫细胞的脂质摄取。利用截断突变体和位点特异性突变体G43A、V44A、L45A、C46A和L47A对这些区域进行了进一步的研究。结果TM内氨基酸高度保守，分布在35 ~ 57个氨基酸之间。ox-LDL刺激对细胞内p- erk1 /2的诱导作用在具有OLR1/WT (p p p p) TM的嵌合体3中被高度促进
期刊介绍：
Innate Immunity is a highly ranked, peer-reviewed scholarly journal and is the official journal of the International Endotoxin & Innate Immunity Society (IEIIS). The journal welcomes manuscripts from researchers actively working on all aspects of innate immunity including biologically active bacterial, viral, fungal, parasitic, and plant components, as well as relevant cells, their receptors, signaling pathways, and induced mediators. The aim of the Journal is to provide a single, interdisciplinary forum for the dissemination of new information on innate immunity in humans, animals, and plants to researchers. The Journal creates a vehicle for the publication of articles encompassing all areas of research, basic, applied, and clinical. The subject areas of interest include, but are not limited to, research in biochemistry, biophysics, cell biology, chemistry, clinical medicine, immunology, infectious disease, microbiology, molecular biology, and pharmacology.
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