BTB-Kelch家族蛋白KLHL32参与炎症反应调控的初步分析

发布时间：2018-04-25 23:11

本文选题：KLHL蛋白 + E泛素连接酶　；参考：《中国病理生理杂志》2014年05期

【摘要】：目的:克隆人BTB-Kelch家族蛋白KLHL32编码基因并初步鉴定其在细胞中的功能。方法:采用实时荧光定量PCR分析Toll样受体5(Toll-like receptor 5,TLR5)特异性激活剂鼠伤寒沙门菌鞭毛蛋白(flagllin from S.typhimurium,ST-FLA)刺激经佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导分化的THP-1巨噬细胞中KLHL32 mRNA表达水平的变化,应用RT-PCR克隆人KLHL32编码区的cDNA序列并将其克隆至真核表达载体pcDNA3.1,采用萤光素酶双报告基因表达系统分析KLHL32过表达对转录因子核因子κB(NF-κB)活性的影响,并采用免疫共沉淀实验分析了KLHL32与Cul-3蛋白在细胞内的相互结合。结果:ST-FLA(100μg/L)刺激经PMA诱导分化的THP-1巨噬细胞4 h引起KLHL32 mRNA的表达水平下调;成功克隆了人KLHL32基因并将其克隆至真核表达载体中获得重组质粒pcDNA3.1-KLHL32-FLAG,在HEK293细胞中过表达KLHL32蛋白对TNF-α诱导的转录因子NF-κB激活没有明显影响,但KLHL32可通过其氨基端的BTB结构域与Cul-3相互结合。结论:成功鉴定了人KLHL32蛋白,其通过BTB结构域与Cul-3蛋白相互结合,可能是一种潜在的E3泛素连接酶,证实了TLR5受体激活可诱导其表达水平的下调,提示KLHL32蛋白在细胞中可能参与炎症细胞信号转导的调控。
[Abstract]:Aim: to clone the gene encoding human BTB-Kelch family protein KLHL32 and to identify its function in cells. Methods: the expression of KLHL32 mRNA in THP-1 macrophages induced by phorbol 12-myristate 13-acetatePMA-induced by flagellin from S.typhimurium ST-FLAwas analyzed by real-time fluorescence quantitative PCR (PCR), a specific activator of Toll receptor 5(Toll-like receptor 5 (TLR5), in Salmonella typhimurium. The cDNA sequence of human KLHL32 coding region was cloned by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1. the effect of KLHL32 overexpression on the activity of transcription factor nuclear factor 魏 B (NF- 魏 B) NF- 魏 B was analyzed by using fluorescent enzyme double reporter gene expression system. The binding of KLHL32 and Cul-3 protein in cells was analyzed by immunoprecipitation assay. Results the expression of KLHL32 mRNA in THP-1 macrophages induced by PMA was down-regulated by 1: ST-FLA-100 渭 g / L stimulation for 4 h. Human KLHL32 gene was successfully cloned and cloned into eukaryotic expression vector to obtain recombinant plasmid pcDNA3.1-KLHL32-FLAG. Overexpression of KLHL32 protein in HEK293 cells had no significant effect on the activation of transcription factor NF- 魏 B induced by TNF- 伪. However, KLHL32 can bind to Cul-3 through the BTB domain of its amino terminal. Conclusion: human KLHL32 protein has been successfully identified. It may be a potential E3 ubiquitin ligase through the interaction of BTB domain with Cul-3 protein, which indicates that the activation of TLR5 receptor can induce the down-regulation of its expression level. These results suggest that KLHL32 protein may be involved in the regulation of signal transduction in inflammatory cells.
【作者单位】：南方医科大学病理生理教研室广东省功能蛋白质组学重点实验室;
【基金】：国家自然科学基金资助项目(No.81070955)
【分类号】：R363

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