突变蛋白K488X-menin通过Hsp70和CHIP构成的泛素-蛋白酶体系统快速降解  

Rapid degradation of protein mutant K488X-menin by ubiquitin-proteasome system composed of Hsp70 and CHIP

作　　者：曾珍 张倩倩 张景 杨敬业 卢清香 田洪霞 周忠雪 周定安 ZENG Zhen;ZHANG Qianqian;ZHANG Jing;YANG Jingye;LU Qingxiang;TIAN Hongxia;ZHOU Zhongxue;ZHOU Ding'an(Clinical Laboratory Center,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Clinical Research Center,Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Gastroenterology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学附属医院临床检验中心,贵州贵阳550004 [2]贵州医科大学临床研究中心,贵州贵阳550004 [3]贵州医科大学附属医院消化内科,贵州贵阳550004

出　　处：《贵州医科大学学报》2025年第3期336-345,共10页Journal of Guizhou Medical University

基　　金：国家自然科学基金地区项目(32060165)。

摘　　要：目的探究突变蛋白K488X-menin在多发性内分泌腺瘤1型(multiple endocrine neoplasia type 1,MEN1)降解的机制。方法以中国贵州黔南平塘1个具有MEN1的3代大家庭为研究对象,收集先证者临床相关的病史和临床资料,全外显子(whole exome sequencing,WES)、一代测序检测致病基因;用生物信息学对野生型menin(WT-menin)和突变体menin(K488X-menin)蛋白结构进行分析;基因克隆构建野生型质粒、定点突变技术构建突变型质粒,转染到293T细胞中,并用放线菌酮(cycloheximide,CHX)和蛋白酶体抑制剂(MG132)单独或先后作用于转染了质粒的293T细胞,Western blot分析WT-menin和K488X-menin蛋白表达水平;免疫共沉淀(Co-immunoprecipitation,Co-IP)分析K488X-menin与热休克蛋白70(heat shock protein 70,Hsp70)、热休克蛋白70羧基末端反应蛋白(carboxyl terminus of hsp70-interacting professional,CHIP)相互作用情况;体外泛素化分析menin、Hsp70、CHIP与泛素之间的相关性。结果MEN1先证者影像学结果支持MEN1的临床诊断,并在MEN1家族中仅发现先证者和其儿子存在MEN1 exon 10 c.1462A>T突变;Western blot实验表明K488X-menin蛋白出现截断和表达下调(P<0.05);K488X-menin可以被CHX阻止蛋白质合成,而蛋白酶体抑制剂MG132可以逆转CHX作用的蛋白下调(P<0.05);Co-IP证实WT-menin和K488X-menin与Hsp70均可发生蛋白相互作用,Hsp70或(和)CHIP使K488X-menin蛋白表达出现下调,并且Hsp70和CHIP共同作用使K488X-menin蛋白表达显著下调和K488X-menin泛素化(P<0.05)。结论突变体K488X-menin蛋白表达不稳定,通过Hsp70和CHIP参与的泛素-蛋白酶体系统的泛素化降解,导致抑癌作用丧失,诱导MEN1疾病的发生。Objective To explore the degradation mechanism of protein mutant K488X-menin in multiple endocrine neoplasia type 1(MEN1).Methods A three-generation family with MEN1 was selected as a research subject from Pingtang,Qiannan,Guizhou in China.Proband-related clinical history and data were collected.Pathogenic genes were detected by whole exome sequencing(WES)and the first generation sequencing.Bioinformatics was used to analyze the protein structures of wild-type menin(WT menin)and mutant menin(K488X menin).Gene cloning was applied to construct wild-type plasmids,and site directed mutagenesis was used to construct mutant plasmids.These plasmids were transfected into 293T cells.Plasmids-transfected 293T cells were separately or sequentially treated with CHX and MG132.Western blot was used to detect the expression levels of WT menin and K488X menin proteins.Co-immunoprecipitation(Co-IP)was applied to examine the interaction between K488X menin and heat shock protein 70(Hsp70),K488X,and carboxyl terminus of hsp70-interacting protein(CHIP).In vitro ubiquitination assay was performed to analyze menin,Hsp70 and CHIP in relation to ubiquitin.Results The imaging results of the proband with MEN1 supported the clinical diagnosis of MEN1,and only the proband and his son in the MEN1 family were found to have a exon 10 c.1462A>T mutation in exon 10 in MEN1.Western blot result showed that K488X-menin protein exhibited a truncated form and downregulated expression(P<0.05).CHX inhibited K488X-menin protein synthesis.Proteasome inhibitor MG132 reversed the protein downregulation-induced by CHX.Co-IP verified that both WT-menin and K488X-menin interacted with Hsp70.Hsp70 or(and)CHIP downregulated K488X-menin protein expression,and the combined action of Hsp70 and CHIP significantly downregulated K488X-menin protein expression and caused the ubiquitination of K488X-menin.Conclusion The expression of the misfolded K488X-menin protein is unstable and undergoes ubiquitination degradation through the ubiquitin-proteasome system(UPS)involv

关 键 词：多发性内分泌腺瘤1型 热休克蛋白70 热休克蛋白70羧基末端反应蛋白 泛素化 K488X-menin 

分 类 号：R394.3[医药卫生—医学遗传学]