BTB-Kelch家族蛋白KLHL32参与炎症反应调控的初步分析  

Preliminary analysis of KLHL32,a BTB-Kelch protein,in the regulation of inflammation

作　　者：王妮[1] 陈新玉[1] 欧小利[1] 姜勇[1] 梅柱中[1] 

机构地区：[1]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,广东广州510515

出　　处：《中国病理生理杂志》2014年第5期870-875,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81070955)

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。AIM ： To identify and characterize the human KLHL32 protein, a member of BTB-Kelch family, in the regulation of inflammation. METHODS： Real-time PCR was applied to analyze the expression of KLHL32 mRNA in phorbol 12-myristate 13-acetate （PMA）-induced macrophage-like THP-1 cells stimulated hy flagellin from S. typhimurium （ ST-FLA）, an activator of Toll-like receptor 5 （ TLR5 ）. The full length of human KLHL32 coding region was amplified by RT-PCR, a.nd the amplicon was subcloned into a eukaryotic expression plasmid pcDNA3. 1. The effects of KLHL32 over- expression on TNF-α-induced activation of NF-κB was analyzed by dual-luciferase reporter assay. The protein interaction between KLHL32 and Cul-3 was determined by co-immunoprecipitation. RESULTS： The mRNA expression of KLHL32 was down-regulated in PMA-induced THP-1 cells upon 100 μg/L ST-FI,A treatment. Human KLHL32 was successfully chined anti subecloned into a eukaryotic expression plasmid to establish a recombinant plasmid peDNA3. 1-KLHL32-FLAG. Over-expression of KLHL32 in the HEK293 cells didn＇ t interfere the activity of transcription faetor NF-κB activated by TNF-α. KLH132 interacted with Cul-3 protein through its BTB domain. CONCLUSION： Human KLHL32 protein has been identified for the first time. It interacts with Cul-3 through its BTB domain and may function as a potential E3 ubiquitin ligase in the cells. The activation of TLR5 receptor leads to down-regulation of KLHL32, suggesting that KLHL32 may play a role in the cellular inflammatory response.

关 键 词：KLHL32蛋白 E3泛素连接酶 TOLL样受体5 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]