SUMO-1修饰ataxin-3对其亚细胞定位及转录抑制活性的影响

发布时间：2018-03-27 06:23

本文选题：ataxin-3　切入点：SUMO-1　出处：《中南大学》2008年博士论文

【摘要】： 背景: 遗传性脊髓小脑型共济失调(Spinocerebellar ataxia,SCA)是一类常见的神经系统遗传病,分子遗传学研究已至少定位了30种基因型,克隆了17个疾病基因,其中脊髓小脑型共济失调3型/马查多-约瑟夫病(Spinocerebellar ataxia type 3/Machado-Joesph Disease,SCA3/MJD)基因型最常见。其发病是由于致病基因MJD1编码区内3'端的CAG重复序列异常扩增导致其编码产物ataxin-3蛋白的羧基端多聚谷氨酰胺(polyglutamine,polyQ)肽链异常扩展引起。ataxin-3蛋白的生理功能目前还不明确,其羧基端polyQ肽链异常扩展导致疾病发生的具体机制还不清楚。研究发现,蛋白质翻译后修饰在其生理功能的发挥及致病机制中具有重要的意义。小泛素相关修饰物(small ubiquitin-related modifier,SUMO)是近10余年来发现的一类重要的蛋白质翻译后修饰因子。与泛素化通路相似,SUMO修饰其底物sumoylation过程:从SUMO前体合成、水解活化、共价结合底物蛋白到SUMO解离等涉及一系列酶的级联反应。sumoylation参与调节细胞内许多重要而基础的功能活动,如蛋白质的亚细胞核定位、蛋白质核转运、转录调控、DNA修复、维持基因组完整性以及信号转导等。目前研究发现SUMO存在于阿尔茨海默病、多系统萎缩、多聚谷氨酸疾病(SCA1、SBMA、DRPLA、Huntingtin病)、帕金森病等多种神经系统变性疾病的包涵体中。同时,很多与神经系统变性疾病相关的蛋白被发现是SUMO的底物蛋白,sumoylation并参与其生理及发病过程。在前期工作中,我们应用酵母双杂交技术,筛选成人脑cDNA文库发现并证实ataxin-3以其N端与SUMO-1相互作用。随后应用免疫荧光-激光共聚焦及免疫共沉淀技术在真核细胞水平证实ataxin-3与SUMO-1相互作用。应用在线SUMOplot分析程序发现ataxin-3在K166位点具有sumoylation模块(165VKGD168)以及其他可能被SUMO-1修饰的K8、K206位点。目的: 确定ataxin-3蛋白被SUMO-1修饰的具体赖氨酸位点以及SUMO-1修饰ataxin-3蛋白对其亚细胞定位及转录抑制活性的影响。方法: 1、应用overlap法和长引物法扩增ataxin-3基因定点突变,应用重组基因技术构建野生型及polyQ扩展突变型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R的真核表达载体; 2、应用免疫荧光及Western-blot检测所构建的真核表达载体的表达; 3、应用Ni-NTA沉淀及Western-blot确定ataxin-3蛋白被SUMO-1修饰的具体赖氨酸位点; 4、应用免疫荧光-激光共聚焦技术观察SUMO-1修饰ataxin-3蛋白对其亚细胞定位的影响; 5、应用Stratagene公司的哺乳动物双杂交系统、Promega公司的双萤光素酶报告基因检测系统分析ataxin-3蛋白的转录调控活性以及SUMO-1修饰对其转录调控活性的影响。结果: 1、DNA测序证实所构建的野生型及polyQ异常扩展型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R的真核表达载体的目的基因除了设计的突变位点发生了改变、野生型ataxin-3其CAG三核苷酸重复次数为20次、polyQ扩展突变型CAG三核苷酸重复次数为68次外,其余位点均与ataxin-3在GenBank中的标准序列(S75313)完全匹配。核对各载体的阅读框在插入目的基因序列后均无移码,说明各真核表达载体构建成功。 2、Western-blot证实野生型及polyQ扩展突变型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R真核表达载体均能在HEK293T细胞中正常表达;免疫荧光结果显示野生型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R在细胞中呈弥散分布,polyQ异常扩展型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R能在核内形成蛋白聚集物。 3、Ni-NTA沉淀及Western-blot实验结果显示:在ataxin-3-20Q-K166R、ataxin-3-68Q-K166R与SUMO-1共转组中,均缺少与SUMO-1相结合的条带;而野生型及polyQ扩展突变型ataxin-3-K8R、ataxin-3-K206R组则存在与SUMO-1相结合的条带。说明ataxin-3的第166位赖氨酸是SUMO-1修饰的关键氨基酸位点,第8位赖氨酸以及第206位赖氨酸不与SUMO-1结合。 4、免疫荧光-激光共聚焦结果显示ataxin-3-20Q蛋白、ataxin-3-20Q-K166R蛋白在HEK293T细胞及PC12细胞中弥散表达,两者胞浆胞核分布无明显差异,PC12细胞在NGF诱导下可见轴突生长;ataxin-3-68Q蛋白、ataxin-3-68Q-K166R蛋白在HEK293T细胞及PC12细胞的胞浆胞核均有表达,均能在核内形成蛋白聚集物;说明ataxin-3-20Q及ataxin-3-68Q在有/无SUMO-1修饰时亚细胞定位无明显变化。 5、萤光素酶活性分析结果显示:与pCMV-BD组基本酶活性比较,pCMV-BD-ataxin-3-20Q组及pCMV-BD-ataxin-3-68Q组酶活性均明显降低(P＜0.05),以后者尤为明显;与pCMV-BD-ataxin-3-20Q组比较,pCMV-BD-ataxin-3-20Q-K166组酶活性明显降低(P＜0.05);与pCMV-BD-ataxin-3-68Q组比较,pCMV-BD-ataxin-3-68Q-K166组酶活性降低,但差异无明显统计学意义(P＞0.05)。结论: 1、首次发现SUMO-1修饰野生型、polyQ扩展突变型ataxin-3的关键氨基酸位点为第166位赖氨酸,第8位赖氨酸以及第206位赖氨酸不是SUMO-1修饰ataxin-3的氨基酸位点; 2、首次发现SUMO-1修饰野生型、polyQ扩展突变型ataxin-3不能改变其亚细胞定位; 3、证实野生型、polyQ扩展突变型ataxin-3具有转录抑制活性,尤以polyQ异常扩展型ataxin-3为甚; 4、首次发现SUMO-1修饰能抑制野生型、polyQ扩展突变型ataxin-3的转录抑制活性。
[Abstract]:Background :

There are at least 30 genotypes in hereditary spinocerebrobasilar ataxia ( SCA ) , and 17 disease genes have been cloned and 17 disease genes have been cloned .

Sumoylation has been found to be the substrate protein of SUMO , sumoylation and its involvement in the physiological and pathogenesis of SUMO .

In the previous work , we used yeast two - hybrid technique to screen the cDNA library of human brain , and confirmed that atran - 3 interacted with SUMO - 1 at its N - terminus . At the same time , the interaction between atran - 3 and SUMO - 1 was confirmed by immunofluorescence - laser cofocus and immune co - precipitation technique . At the K166 locus , the strain of atran - 3 was found to have sumoylation module ( 165VKGD168 ) , and other k8 , K206 sites which may be modified by SUMO - 1 .

Purpose :

The specific lysine sites modified by SUMO - 1 and the effect of SUMO - 1 on its subcell localization and transcriptional repression activity were determined .

Method :

1 , using the overlap method and the long primer method to amplify the site - directed mutagenesis of the at - 3 gene , and constructing a eukaryotic expression vector of the wild - type and polyQ - extended mutant atran - 3 - K8r , atran - 3 - K166R , atopharyng3 - K206R by using the recombinant gene technology ;

2 , using immunofluorescence and Western - blot to detect the expression of eukaryotic expression vector ;

3 . The specific lysine site modified by SUMO - 1 was determined by the precipitation and Western - blot .

4 . The effect of SUMO - 1 on the subcell localization was observed by immunofluorescence - laser co - focusing technique .

5 . Using the mammalian double - hybrid system of Stratford Company , the transcriptional regulation activity of atras - 3 protein and the effect of SUMO - 1 modification on its transcriptional regulatory activity were analyzed by using the reporter gene of double luciferase reporter gene of the company .

Results :

1 . DNA sequencing confirmed that the target gene of the eukaryotic expression vector of the wild type and polyQ abnormal expansion type atran - 3 - K66R , atran - 3 - K206R was changed in addition to the designed mutation site . The repeat times of the wild - type at. - 3 - K206R were 20 times , the number of repeats of polyQ - extended mutant CAG was 68 times , and the remaining sites were all matched with the standard sequence in GenBank ( S75313 ) . The reading frame of each carrier was not shifted after insertion of the target gene sequence , indicating that each eukaryotic expression vector was constructed successfully .

2 . Western - blot showed that the wild - type and polyQ - extended mutant atran - 3 - K6r , atran - 3 - K166R , atran - 3 - K206R eukaryotic expression vector were able to express normal expression of the expression vector . The results showed that the wild - type ataxil - 3 - K6r , atran - 3 - K166R , atran - 3 - K206R were dispersed in the cells .

3 . The results showed that there was a lack of band with SUMO - 1 , while wild type and polyQ extended mutant atran - 3 - K66R , atran - 3 - 68Q - K166R and SUMO - 1 were absent , while wild - type and polyQ - extended mutant atran - 3 - K66R had a band which was combined with SUMO - 1 . The 166 - lysine at atran - 3 was the key amino acid site modified by SUMO - 1 , and the 8th lysine and 206 lysine were not bound to SUMO - 1 .

4 . The results of immunofluorescence - laser confocal microscopy showed that at the time of NGF induction , the expression of atran - 3 - 20Q - K166R protein was not significantly different in the cytoplasm of PC12 cells . The expression of atopharyng3 - 68Q protein and atopharyng3 - 68Q - K166R protein in both the cytoplasm and nucleus of PC12 cells showed no significant changes in the localization of the subcells in the presence / absence of SUMO - 1 modification .

5 . The luciferase activity of pCMV - BD - atran - 3 - 20Q group and pCMV - BD - atran - 3 - 68Q - K166 group were significantly lower than that of pCMV - BD - atran - 3 - 68Q group ( P < 0.05 ) .

Conclusion :

1 . It was first found that SUMO - 1 modified wild type , the key amino acid site of polyQ - extended mutant atran - 3 was 166 - lysine , the 8th lysine and the 6th lysine were not the amino acid sites of SUMO - 1 .

2 . For the first time , SUMO - 1 modified wild type , polyQ extended mutant atran - 3 could not change its subcell localization ;

3 . It was confirmed that the wild - type , polyQ - extended mutant atran - 3 had the transcriptional inhibitory activity , especially the polyQ - extended atran - 3 .

4 . For the first time , it was found that the modification of SUMO - 1 could inhibit the transcription - inhibitory activity of wild - type and polyQ - extended mutant atons - 3 .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

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