本文選題：ataxin-3　切入點：SUMO-1　出處：《中南大學》2008年博士論文

【摘要】： 背景: 遺傳性脊髓小腦型共濟失調(diào)(Spinocerebellar ataxia,SCA)是一類常見的神經(jīng)系統(tǒng)遺傳病,分子遺傳學研究已至少定位了30種基因型,克隆了17個疾病基因,其中脊髓小腦型共濟失調(diào)3型/馬查多-約瑟夫病(Spinocerebellar ataxia type 3/Machado-Joesph Disease,SCA3/MJD)基因型最常見。其發(fā)病是由于致病基因MJD1編碼區(qū)內(nèi)3'端的CAG重復序列異常擴增導致其編碼產(chǎn)物ataxin-3蛋白的羧基端多聚谷氨酰胺(polyglutamine,polyQ)肽鏈異常擴展引起。ataxin-3蛋白的生理功能目前還不明確,其羧基端polyQ肽鏈異常擴展導致疾病發(fā)生的具體機制還不清楚。研究發(fā)現(xiàn),蛋白質(zhì)翻譯后修飾在其生理功能的發(fā)揮及致病機制中具有重要的意義。 小泛素相關(guān)修飾物(small ubiquitin-related modifier,SUMO)是近10余年來發(fā)現(xiàn)的一類重要的蛋白質(zhì)翻譯后修飾因子。與泛素化通路相似,SUMO修飾其底物sumoylation過程:從SUMO前體合成、水解活化、共價結(jié)合底物蛋白到SUMO解離等涉及一系列酶的級聯(lián)反應。sumoylation參與調(diào)節(jié)細胞內(nèi)許多重要而基礎(chǔ)的功能活動,如蛋白質(zhì)的亞細胞核定位、蛋白質(zhì)核轉(zhuǎn)運、轉(zhuǎn)錄調(diào)控、DNA修復、維持基因組完整性以及信號轉(zhuǎn)導等。目前研究發(fā)現(xiàn)SUMO存在于阿爾茨海默病、多系統(tǒng)萎縮、多聚谷氨酸疾病(SCA1、SBMA、DRPLA、Huntingtin病)、帕金森病等多種神經(jīng)系統(tǒng)變性疾病的包涵體中。同時,很多與神經(jīng)系統(tǒng)變性疾病相關(guān)的蛋白被發(fā)現(xiàn)是SUMO的底物蛋白,sumoylation并參與其生理及發(fā)病過程。 在前期工作中,我們應用酵母雙雜交技術(shù),篩選成人腦cDNA文庫發(fā)現(xiàn)并證實ataxin-3以其N端與SUMO-1相互作用。隨后應用免疫熒光-激光共聚焦及免疫共沉淀技術(shù)在真核細胞水平證實ataxin-3與SUMO-1相互作用。應用在線SUMOplot分析程序發(fā)現(xiàn)ataxin-3在K166位點具有sumoylation模塊(165VKGD168)以及其他可能被SUMO-1修飾的K8、K206位點。 目的: 確定ataxin-3蛋白被SUMO-1修飾的具體賴氨酸位點以及SUMO-1修飾ataxin-3蛋白對其亞細胞定位及轉(zhuǎn)錄抑制活性的影響。 方法: 1、應用overlap法和長引物法擴增ataxin-3基因定點突變,應用重組基因技術(shù)構(gòu)建野生型及polyQ擴展突變型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R的真核表達載體; 2、應用免疫熒光及Western-blot檢測所構(gòu)建的真核表達載體的表達; 3、應用Ni-NTA沉淀及Western-blot確定ataxin-3蛋白被SUMO-1修飾的具體賴氨酸位點; 4、應用免疫熒光-激光共聚焦技術(shù)觀察SUMO-1修飾ataxin-3蛋白對其亞細胞定位的影響; 5、應用Stratagene公司的哺乳動物雙雜交系統(tǒng)、Promega公司的雙螢光素酶報告基因檢測系統(tǒng)分析ataxin-3蛋白的轉(zhuǎn)錄調(diào)控活性以及SUMO-1修飾對其轉(zhuǎn)錄調(diào)控活性的影響。 結(jié)果: 1、DNA測序證實所構(gòu)建的野生型及polyQ異常擴展型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R的真核表達載體的目的基因除了設(shè)計的突變位點發(fā)生了改變、野生型ataxin-3其CAG三核苷酸重復次數(shù)為20次、polyQ擴展突變型CAG三核苷酸重復次數(shù)為68次外,其余位點均與ataxin-3在GenBank中的標準序列(S75313)完全匹配。核對各載體的閱讀框在插入目的基因序列后均無移碼,說明各真核表達載體構(gòu)建成功。 2、Western-blot證實野生型及polyQ擴展突變型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R真核表達載體均能在HEK293T細胞中正常表達;免疫熒光結(jié)果顯示野生型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R在細胞中呈彌散分布,polyQ異常擴展型ataxin-3-K8R、ataxin-3-K166R、ataxin-3-K206R能在核內(nèi)形成蛋白聚集物。 3、Ni-NTA沉淀及Western-blot實驗結(jié)果顯示:在ataxin-3-20Q-K166R、ataxin-3-68Q-K166R與SUMO-1共轉(zhuǎn)組中,均缺少與SUMO-1相結(jié)合的條帶;而野生型及polyQ擴展突變型ataxin-3-K8R、ataxin-3-K206R組則存在與SUMO-1相結(jié)合的條帶。說明ataxin-3的第166位賴氨酸是SUMO-1修飾的關(guān)鍵氨基酸位點,第8位賴氨酸以及第206位賴氨酸不與SUMO-1結(jié)合。 4、免疫熒光-激光共聚焦結(jié)果顯示ataxin-3-20Q蛋白、ataxin-3-20Q-K166R蛋白在HEK293T細胞及PC12細胞中彌散表達,兩者胞漿胞核分布無明顯差異,PC12細胞在NGF誘導下可見軸突生長;ataxin-3-68Q蛋白、ataxin-3-68Q-K166R蛋白在HEK293T細胞及PC12細胞的胞漿胞核均有表達,均能在核內(nèi)形成蛋白聚集物;說明ataxin-3-20Q及ataxin-3-68Q在有/無SUMO-1修飾時亞細胞定位無明顯變化。 5、螢光素酶活性分析結(jié)果顯示:與pCMV-BD組基本酶活性比較,pCMV-BD-ataxin-3-20Q組及pCMV-BD-ataxin-3-68Q組酶活性均明顯降低(P＜0.05),以后者尤為明顯;與pCMV-BD-ataxin-3-20Q組比較,pCMV-BD-ataxin-3-20Q-K166組酶活性明顯降低(P＜0.05);與pCMV-BD-ataxin-3-68Q組比較,pCMV-BD-ataxin-3-68Q-K166組酶活性降低,但差異無明顯統(tǒng)計學意義(P＞0.05)。 結(jié)論: 1、首次發(fā)現(xiàn)SUMO-1修飾野生型、polyQ擴展突變型ataxin-3的關(guān)鍵氨基酸位點為第166位賴氨酸,第8位賴氨酸以及第206位賴氨酸不是SUMO-1修飾ataxin-3的氨基酸位點; 2、首次發(fā)現(xiàn)SUMO-1修飾野生型、polyQ擴展突變型ataxin-3不能改變其亞細胞定位; 3、證實野生型、polyQ擴展突變型ataxin-3具有轉(zhuǎn)錄抑制活性,尤以polyQ異常擴展型ataxin-3為甚; 4、首次發(fā)現(xiàn)SUMO-1修飾能抑制野生型、polyQ擴展突變型ataxin-3的轉(zhuǎn)錄抑制活性。
[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 .

【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前1條

1 沈璐;唐北沙;湯建光;江泓;王成;房海燕;;脊髓小腦型共濟失調(diào)Ⅲ型ataxin-3相互作用蛋白的篩選[J];中南大學學報(醫(yī)學版);2006年01期

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本文編號：1670391