SUMO-1修饰ataxin-3蛋白对其泛素化、降解、包涵体形成、细胞凋亡等的影响
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摘要
背景:
遗传性脊髓小脑型共济失调(hereditary spinocerebellar ataxia,SCAs)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28种基因型,其中18个疾病基因已被克隆,脊髓小脑型共济失调3型/马查多-约瑟夫病(Spinocerebellar ataxia type 3/Machado-JoesphDisease,SCA3/MJD)为最常见亚型。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蛋白的生理功能尚不明确,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)是新近发现的一类重要的蛋白质翻译后修饰因子。SUMO家族成员包括SUMO-1,SUMO-2,SUMO-3,SUMO-4等至少四个家族成员,它们主要功能是对底物蛋白进行苏素化(SUMOylation)修饰。与泛素化通路相似,SUMO-1修饰其底物包含一系列酶的级联反应。底物蛋白被SUMO-1修饰主要与底物蛋白的亚细胞定位、转录调控、蛋白质稳定性的调节等方面密切相关。泛素修饰底物蛋白主要导致底物蛋白被蛋白酶体降解,由于SUMO-1与泛素存在相同的靶蛋白结合位点—赖氨酸残基,苏素化与泛素化还具有拮抗作用,从而可能影响底物蛋白通过泛素蛋白酶体途径的降解。目前研究发现SUMO-1存在于阿尔茨海默病、多系统萎缩、多聚谷氨酰胺疾病(SCA1、SBMA、DRPLA、Huntingtin病)、帕金森病等多种神经退行性疾病的包涵体中。同时,很多与神经退行性疾病相关的蛋白被发现是SUMO-1的底物蛋白,苏素化可能参与了其生理及病理过程。
在前期工作中,沈璐等应用酵母双杂交技术,以ataxin-3蛋白为Bait筛选成人脑cDNA文库,发现并证实ataxin-3蛋白以其氨基端与SUMO-1相互作用。汤建光等应用免疫荧光-激光共聚焦技术、免疫共沉淀技术,在真核细胞水平上进一步证实ataxin-3蛋白与SUMO-1存在相互作用,说明ataxin-3蛋白是SUMO-1的底物蛋白;并发现含polyQ异常扩展突变型ataxin-3蛋白经SUMO-1修饰后,可能使其细胞毒性加重,而RNAi下调SUMO-1的表达能缓解含polyQ异常扩展突变型ataxin-3蛋白的细胞毒性。廖书胜等应用在线SUMOplot分析程序(http://www.abgent.com.cn/doc/sumoplot/login.asp)分析,发现ataxin-3蛋白在第166位赖氨酸(K166)位点具有苏素化修饰模块位点(165VKD168),能被SUMO-1修饰;并应用Ni-NTA沉淀富集蛋白结合Western-blot技术证实SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白的关键氨基酸位点为K166;免疫荧光实验初步证实SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白并未改变其亚细胞定位。这些前期的研究工作结果:证实了ataxin-3蛋白是SUMO-1的底物蛋白,SUMO-1/苏素化参与了SCA3/MJD的发病过程。
目的:
从真核细胞水平探讨SUMO-1修饰对野生型、含polyQ异常扩展突变型ataxin-3蛋白的泛素化水平、降解情况、核内包涵体形成、细胞凋亡率的影响;进一步探讨SUMO-1修饰对野生型、含polyQ异常扩展突变型ataxin-3蛋白的胞浆/胞核分布的影响。
方法:
1.应用重组基因技术、Western-blot技术、GFP荧光技术构建并检测存在或缺失SUMO-1修饰位点的野生型、含polyQ异常扩展突变型ataxin-3的pEGFP-N1真核表达载体及其表达。
2.应用亚细胞组分分离技术研究存在或缺失苏素化修饰的野生型、含polyQ异常扩展突变型ataxin-3蛋白胞浆/胞核分布的差异。
3.应用免疫共沉淀技术分析过苏素化或缺失苏素化修饰位点的野生型、含polyQ异常扩展突变型ataxin-3蛋白泛素化水平的差异。
4.应用追踪实验技术研究存在或缺失苏素化修饰位点的野生型、含polyQ异常扩展突变型ataxin-3蛋白降解水平的差异。
5.应用荧光技术、PI/Annexin-V-FITC双染流式细胞仪技术分析存在或缺失苏素化修饰的野生型、含polyQ异常扩展突变型ataxin-3蛋白的核内包涵体形成及细胞凋亡率的差异。
结果:
1.DNA测序证实所构建的野生型、含polyQ异常扩展突变型ataxin-3的pEGFP-N1真核表达载体除了K166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异常扩展突变型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、含polyQ异常扩展突变型ataxin-3的pEGFP-N1真核表达载体均能在HEK 293T细胞中正常表达;荧光结果显示野生型ataxin-3及其K166R突变体在细胞中呈弥散分布,含polyQ异常扩展突变型ataxin-3及其K166R突变体能形成蛋白聚集物。
3.亚细胞组分分离发现存在或缺失SUMO-1修饰位点对野生型、含polyQ异常扩展突变型ataxin-3蛋白的胞浆/胞核分布无明显影响。
4.免疫共沉淀结果显示在外源性过表达SUMO-1或缺失SUMO-1修饰情况下,野生型与含polyQ异常扩展突变型ataxin-3蛋白的多聚泛素化水平可见明显差别,含polyQ异常扩展突变型ataxin-3蛋白的多聚泛素化水平更高;在外源性过表达SUMO-1或缺失SUMO-1修饰情况下,野生型ataxin-3蛋白的多聚泛素化水平无明显改变;同样,含polyQ异常扩展突变型ataxin-3蛋白的多聚泛素化水平也无明显改变。
5.追踪实验发现野生型ataxin-3及其K166R突变体随时间延长蛋白逐渐降解,条带变化基本一致,未发现明显的差别;而含polyQ异常扩展突变型ataxin-3蛋白在第7小时、第15小时的条带均明显比含polyQ异常扩展突变型ataxin-3-68Q-K166R突变体同时间点的条带浓,说明含polyQ异常扩展突变型ataxin-3蛋白在存在SUMO-1修饰时变得更为稳定,降解明显减慢。
6.随机双盲试验分析野生型、含polyQ异常扩展突变型ataxin-3蛋白核内包涵体有明显差异(P＜0.005),说明polyQ的次数增加可导致核内包涵体的形成;在野生型组、含polyQ异常扩展突变型ataxin-3组内比较时,有或无外源性过SUMO-1修饰、存在或缺失SUMO-1修饰位点时,核内包涵体的数目无明显差异。
7.PI/Annexin V-FITC双染流式细胞仪结果显示:含polyQ异常扩展突变型ataxin-3组与其K166R突变体组比较,含polyQ异常扩展突变型ataxin-3组的早期凋亡率要高于含polyQ异常扩展突变型ataxin-3-K166R突变体组,存在显著性差别(P＜0.005),说明SUMO-1修饰含polyQ异常扩展突变型ataxin-3蛋白对细胞有毒性作用,增加了细胞的凋亡率;野生型ataxin-3及其K166R突变体组间早期凋亡率未见明显差别(P＞0.005),说明SUMO-1修饰野生型ataxin-3蛋白无明显细胞毒性作用。
结论:
1.构建成功野生型、含polyQ异常扩展突变型ataxin-3及其K166R突变体的pEGFP-N1真核表达载体。
2.进一步证实SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白不能改变其胞浆/胞核分布。
3.证明SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白的K166位点不是同时被泛素修饰。
4.发现SUMO-1修饰含polyQ异常扩展突变型ataxin-3蛋白能延缓其降解。
5.发现SUMO-1修饰含polyQ异常扩展突变型ataxin-3蛋白后增加了细胞毒性,但对核内包涵体形成无明显影响。
Background:
The hereditary spinocerebellar ataxias(SCAs) are a heterogeneous group of neurodegenerative disorders.To date,at least 28 gene loci responsible for SCAs have been mapped,in which 18 pathogenic genes have been cloned.Among them,spinocerebellar ataxia type 3/Machado -Joseph disease(SCA3/MJD) is the most common subtype.The gene for SCA3/MJD has been cloned and designated as MJD1.SCA3/MJD is one of the polyglutamine(polyQ) diseases caused by an expansion of a polyQ stretch near the C-terminus of the MJD-1 gene product,ataxin-3.Up to now,the physiological function of ataxin-3 is unknown,and the pathogenesis of the expansion of a polyQ stretch near the C-terminus is still not well illuminated.Studies have found that post-translational modification of the disease proteins plays a critical role in their physiological function and pathogenesis.
Ten years after its discovery,the small ubiquitin-like protein modifier(SUMO) has emerged as an important post-translational modification factor.SUMO family contains SUMO-1,SUMO-2, SUMO-3,SUMO-4 isoforms.Similar to ubiquitin,SUMO attachment to proteins which referred to as "SUMOylation",enters a multi-step enzymatic pathway.This reversible pathway provides a rapid and efficient way to modulate much prominent and basic function,such as subcellular localization,nuclear transport,transcriptional regulation,and protein stability.Similar to ubiquitin,SUMO are linked directly to the amino sidechains of lysine residues and,in some instances,both modifiers target the same substrate.This suggests a dynamic interplay between the related ubiquitination and SUMOylation pathways.SUMO immunoreactivity has been observed within inclusions in numerous neurodegenerative diseases including Alzheimer disease,multiple system atrophy,polyQ diseases(SCA1、SBMA、DRPLA、Huntingtin disease) and Parkinson's disease.The identification of huntingtin,ataxin-1,tau andα-synuclein as SUMO substrates further supports the involvement of SUMOylation in the pathogenesis of neurodegenerative diseases.
We have found that the N-terminus of ataxin-3 interacted with SUMO-1 by yeast two-hybrid techniques screening human brain cDNA library,then confirmed the interaction in eukaryocyte by immunofluorescence-laser cofocalization and co-immunoprecipitation, and SUMO-1 modification might have toxic effect on polyQ-expanded ataxin-3.By online SUMOylation analysis protocol,we found that ataxin-3 contains SUMOylation motif(165VKGD168) at K166.Then by Ni-NTA precipitation and western-blot,we detected that K166 was the key amino acid of wild-type and polyQ-expanded ataxin-3 for SUMO-1 modification.We found that SUMO-1 modification didn't change the subcellular localization of wild-type and polyQ-expanded ataxin-3 using immunofluorescence initially.Our previous data indentified that ataxin-3 was the substrate of SUMO-1,and SUMOylation participated the pathogenesis of SCA3/MJD.
Objective:
To research the influence of SUMO-1 modification on the ubiquitination,protein degradation,formation of intranuclear inclusions, and apoptosis of wild-type and polyQ-expanded ataxin-3;And explore the effect on cytoplasmic/nuclear distribution further in eukaryocyte level.
Methods:
1.Recombinant DNA technology,Western-blot,GFP fluorescence technique were undertaken to construct and detect the expression of pEGFP-N1 eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3.
2.Subcellular fractionation was used to observe the effect of SUMO-1 modification on the cytoplasmic/nuclear distribution of wild-type and polyQ-expanded ataxin-3.
3.Co-immunoprecipitation was utilized to analyze the influence of SUMO-1 modifcation on the ubiquitination of wild-type and polyQ-expanded ataxin-3.
4.Chase experiment was undertaken to investigate the effect of SUMO-1 modification on the protein degradation of wild-type and polyQ-expanded ataxin-3.
5.Fluorescence technique,flow cytometry of PI/Annexin-V-FITC were used to study the effect of SUMO-1 modification on the formation of intranuclear inclusions,and apoptosis of wild-type and polyQ- expanded ataxin-3.
Results:
1.DNA sequencing confirmed that eukaryotic expression plasmids of the wild-type and polyQ-expanded ataxin-3 were altered on the designed mutation loci,and the repeat number of CAG trinucleotide were 20 and 68 on wild-type and polyQ-expanded respectively,which the other loci matched with GenBank standard sequence(S75313).It suggested that eukaryotic expression plasmids constructed successfully when we checked the reading frame of the plasmids without any frame shift after purpose gene order insertion.
2.We identified eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3 was expressed in HEK 293T cell by Western-blot.Using GFP fluorescence technique,we found that ataxin-3-20Q and ataxin-3-20Q-K166R distributed scattered;on the contrast,ataxin-3-68Q and ataxin-3-68Q-K166R aggregated.
3.We found SUMO-1 modification had no influence on the cytoplasmic/nuclear distribution of wild-type and polyQ- expanded ataxin-3 by subcellular fractionation.
4.By co-immunoprecipitation,our study suggested that over-expression of SUMO-1 or SUMO-1 modification of missing,the polyubiquitination level between the wild-type and polyQ-expanded ataxin-3 protein was significantly different, including polyQ-expanded ataxin-3 protein polyubiquitination higher level.Over-expression of SUMO-1 or SUMO-1 modification of missing,the wild-type ataxin-3 protein polyubiquitination level had no difference,and the result was similar for polyQ-expanded ataxin-3 protein.
5.We discovered by chase experiment that the bands weren't significantly different between ataxin3-20Q and ataxin3-20Q-K166R in 0h,1h,3h,7h,and 15h;but the bands of ataxin3-68Q were predominantly thicker than ataxin-3-68Q-K166R in 7h and 15h,which suggested that SUMO-1 modification resulted in stabilization of ataxin3-68Q.
6.We analyzed the quantitation of intranuclear inclusion of the wild-type and polyQ-expanded ataxin-3 had significant different (P＜0.005),indicated that polyQ expansion could induce formation of the intranuclear inclusion;the means of wild-type ataxin-3 group or polyQ-expanded ataxin-3 group weren't significantly different between with/without SUMO-1 or excessive/native SUMO-1 modification.
7.The result from flow cytometry of PI/Annexin-V/FITC showed the early apoptosis rate of ataxin-3-68Q group was higher than ataxin-3-68Q-K166R group(P＜0.005),which suggested that SUMO-1 modification had toxic effect;but the early apoptosis rate of ataxin-3-20Q group and ataxin-3-20Q-K166R group wasn't significantly different(P＞0.005).
Conclusion:
1.We construct pEGFP-N1 eukaryotic expression plasmids of the wild-type and polyQ-expanded ataxin-3 firstly,and obtain 14 HEK 293T cell strains.
2.We confirm that SUMO-1 modification has no influence on the cytoplasmic/nuclear distribution of the wild-type and polyQ-expanded ataxin-3.
3.We identify that K166 is not the ubiquitination locus of wild-type and polyQ-expanded ataxin-3.
4.We discover that SUMO-1 modification resulted in stabilization of polyQ-expanded ataxin-3.
5.We disclose that SUMO-1 modification of polyQ-expanded ataxin-3 had toxic effect,but didn't have influence on the nuclear inclusion formation.
遗传性脊髓小脑型共济失调(hereditary spinocerebellar ataxia,SCAs)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28种基因型,其中18个疾病基因已被克隆,脊髓小脑型共济失调3型/马查多-约瑟夫病(Spinocerebellar ataxia type 3/Machado-JoesphDisease,SCA3/MJD)为最常见亚型。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蛋白的生理功能尚不明确,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)是新近发现的一类重要的蛋白质翻译后修饰因子。SUMO家族成员包括SUMO-1,SUMO-2,SUMO-3,SUMO-4等至少四个家族成员,它们主要功能是对底物蛋白进行苏素化(SUMOylation)修饰。与泛素化通路相似,SUMO-1修饰其底物包含一系列酶的级联反应。底物蛋白被SUMO-1修饰主要与底物蛋白的亚细胞定位、转录调控、蛋白质稳定性的调节等方面密切相关。泛素修饰底物蛋白主要导致底物蛋白被蛋白酶体降解,由于SUMO-1与泛素存在相同的靶蛋白结合位点—赖氨酸残基,苏素化与泛素化还具有拮抗作用,从而可能影响底物蛋白通过泛素蛋白酶体途径的降解。目前研究发现SUMO-1存在于阿尔茨海默病、多系统萎缩、多聚谷氨酰胺疾病(SCA1、SBMA、DRPLA、Huntingtin病)、帕金森病等多种神经退行性疾病的包涵体中。同时,很多与神经退行性疾病相关的蛋白被发现是SUMO-1的底物蛋白,苏素化可能参与了其生理及病理过程。
在前期工作中,沈璐等应用酵母双杂交技术,以ataxin-3蛋白为Bait筛选成人脑cDNA文库,发现并证实ataxin-3蛋白以其氨基端与SUMO-1相互作用。汤建光等应用免疫荧光-激光共聚焦技术、免疫共沉淀技术,在真核细胞水平上进一步证实ataxin-3蛋白与SUMO-1存在相互作用,说明ataxin-3蛋白是SUMO-1的底物蛋白;并发现含polyQ异常扩展突变型ataxin-3蛋白经SUMO-1修饰后,可能使其细胞毒性加重,而RNAi下调SUMO-1的表达能缓解含polyQ异常扩展突变型ataxin-3蛋白的细胞毒性。廖书胜等应用在线SUMOplot分析程序(http://www.abgent.com.cn/doc/sumoplot/login.asp)分析,发现ataxin-3蛋白在第166位赖氨酸(K166)位点具有苏素化修饰模块位点(165VKD168),能被SUMO-1修饰;并应用Ni-NTA沉淀富集蛋白结合Western-blot技术证实SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白的关键氨基酸位点为K166;免疫荧光实验初步证实SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白并未改变其亚细胞定位。这些前期的研究工作结果:证实了ataxin-3蛋白是SUMO-1的底物蛋白,SUMO-1/苏素化参与了SCA3/MJD的发病过程。
目的:
从真核细胞水平探讨SUMO-1修饰对野生型、含polyQ异常扩展突变型ataxin-3蛋白的泛素化水平、降解情况、核内包涵体形成、细胞凋亡率的影响;进一步探讨SUMO-1修饰对野生型、含polyQ异常扩展突变型ataxin-3蛋白的胞浆/胞核分布的影响。
方法:
1.应用重组基因技术、Western-blot技术、GFP荧光技术构建并检测存在或缺失SUMO-1修饰位点的野生型、含polyQ异常扩展突变型ataxin-3的pEGFP-N1真核表达载体及其表达。
2.应用亚细胞组分分离技术研究存在或缺失苏素化修饰的野生型、含polyQ异常扩展突变型ataxin-3蛋白胞浆/胞核分布的差异。
3.应用免疫共沉淀技术分析过苏素化或缺失苏素化修饰位点的野生型、含polyQ异常扩展突变型ataxin-3蛋白泛素化水平的差异。
4.应用追踪实验技术研究存在或缺失苏素化修饰位点的野生型、含polyQ异常扩展突变型ataxin-3蛋白降解水平的差异。
5.应用荧光技术、PI/Annexin-V-FITC双染流式细胞仪技术分析存在或缺失苏素化修饰的野生型、含polyQ异常扩展突变型ataxin-3蛋白的核内包涵体形成及细胞凋亡率的差异。
结果:
1.DNA测序证实所构建的野生型、含polyQ异常扩展突变型ataxin-3的pEGFP-N1真核表达载体除了K166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异常扩展突变型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、含polyQ异常扩展突变型ataxin-3的pEGFP-N1真核表达载体均能在HEK 293T细胞中正常表达;荧光结果显示野生型ataxin-3及其K166R突变体在细胞中呈弥散分布,含polyQ异常扩展突变型ataxin-3及其K166R突变体能形成蛋白聚集物。
3.亚细胞组分分离发现存在或缺失SUMO-1修饰位点对野生型、含polyQ异常扩展突变型ataxin-3蛋白的胞浆/胞核分布无明显影响。
4.免疫共沉淀结果显示在外源性过表达SUMO-1或缺失SUMO-1修饰情况下,野生型与含polyQ异常扩展突变型ataxin-3蛋白的多聚泛素化水平可见明显差别,含polyQ异常扩展突变型ataxin-3蛋白的多聚泛素化水平更高;在外源性过表达SUMO-1或缺失SUMO-1修饰情况下,野生型ataxin-3蛋白的多聚泛素化水平无明显改变;同样,含polyQ异常扩展突变型ataxin-3蛋白的多聚泛素化水平也无明显改变。
5.追踪实验发现野生型ataxin-3及其K166R突变体随时间延长蛋白逐渐降解,条带变化基本一致,未发现明显的差别;而含polyQ异常扩展突变型ataxin-3蛋白在第7小时、第15小时的条带均明显比含polyQ异常扩展突变型ataxin-3-68Q-K166R突变体同时间点的条带浓,说明含polyQ异常扩展突变型ataxin-3蛋白在存在SUMO-1修饰时变得更为稳定,降解明显减慢。
6.随机双盲试验分析野生型、含polyQ异常扩展突变型ataxin-3蛋白核内包涵体有明显差异(P＜0.005),说明polyQ的次数增加可导致核内包涵体的形成;在野生型组、含polyQ异常扩展突变型ataxin-3组内比较时,有或无外源性过SUMO-1修饰、存在或缺失SUMO-1修饰位点时,核内包涵体的数目无明显差异。
7.PI/Annexin V-FITC双染流式细胞仪结果显示:含polyQ异常扩展突变型ataxin-3组与其K166R突变体组比较,含polyQ异常扩展突变型ataxin-3组的早期凋亡率要高于含polyQ异常扩展突变型ataxin-3-K166R突变体组,存在显著性差别(P＜0.005),说明SUMO-1修饰含polyQ异常扩展突变型ataxin-3蛋白对细胞有毒性作用,增加了细胞的凋亡率;野生型ataxin-3及其K166R突变体组间早期凋亡率未见明显差别(P＞0.005),说明SUMO-1修饰野生型ataxin-3蛋白无明显细胞毒性作用。
结论:
1.构建成功野生型、含polyQ异常扩展突变型ataxin-3及其K166R突变体的pEGFP-N1真核表达载体。
2.进一步证实SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白不能改变其胞浆/胞核分布。
3.证明SUMO-1修饰野生型、含polyQ异常扩展突变型ataxin-3蛋白的K166位点不是同时被泛素修饰。
4.发现SUMO-1修饰含polyQ异常扩展突变型ataxin-3蛋白能延缓其降解。
5.发现SUMO-1修饰含polyQ异常扩展突变型ataxin-3蛋白后增加了细胞毒性,但对核内包涵体形成无明显影响。
Background:
The hereditary spinocerebellar ataxias(SCAs) are a heterogeneous group of neurodegenerative disorders.To date,at least 28 gene loci responsible for SCAs have been mapped,in which 18 pathogenic genes have been cloned.Among them,spinocerebellar ataxia type 3/Machado -Joseph disease(SCA3/MJD) is the most common subtype.The gene for SCA3/MJD has been cloned and designated as MJD1.SCA3/MJD is one of the polyglutamine(polyQ) diseases caused by an expansion of a polyQ stretch near the C-terminus of the MJD-1 gene product,ataxin-3.Up to now,the physiological function of ataxin-3 is unknown,and the pathogenesis of the expansion of a polyQ stretch near the C-terminus is still not well illuminated.Studies have found that post-translational modification of the disease proteins plays a critical role in their physiological function and pathogenesis.
Ten years after its discovery,the small ubiquitin-like protein modifier(SUMO) has emerged as an important post-translational modification factor.SUMO family contains SUMO-1,SUMO-2, SUMO-3,SUMO-4 isoforms.Similar to ubiquitin,SUMO attachment to proteins which referred to as "SUMOylation",enters a multi-step enzymatic pathway.This reversible pathway provides a rapid and efficient way to modulate much prominent and basic function,such as subcellular localization,nuclear transport,transcriptional regulation,and protein stability.Similar to ubiquitin,SUMO are linked directly to the amino sidechains of lysine residues and,in some instances,both modifiers target the same substrate.This suggests a dynamic interplay between the related ubiquitination and SUMOylation pathways.SUMO immunoreactivity has been observed within inclusions in numerous neurodegenerative diseases including Alzheimer disease,multiple system atrophy,polyQ diseases(SCA1、SBMA、DRPLA、Huntingtin disease) and Parkinson's disease.The identification of huntingtin,ataxin-1,tau andα-synuclein as SUMO substrates further supports the involvement of SUMOylation in the pathogenesis of neurodegenerative diseases.
We have found that the N-terminus of ataxin-3 interacted with SUMO-1 by yeast two-hybrid techniques screening human brain cDNA library,then confirmed the interaction in eukaryocyte by immunofluorescence-laser cofocalization and co-immunoprecipitation, and SUMO-1 modification might have toxic effect on polyQ-expanded ataxin-3.By online SUMOylation analysis protocol,we found that ataxin-3 contains SUMOylation motif(165VKGD168) at K166.Then by Ni-NTA precipitation and western-blot,we detected that K166 was the key amino acid of wild-type and polyQ-expanded ataxin-3 for SUMO-1 modification.We found that SUMO-1 modification didn't change the subcellular localization of wild-type and polyQ-expanded ataxin-3 using immunofluorescence initially.Our previous data indentified that ataxin-3 was the substrate of SUMO-1,and SUMOylation participated the pathogenesis of SCA3/MJD.
Objective:
To research the influence of SUMO-1 modification on the ubiquitination,protein degradation,formation of intranuclear inclusions, and apoptosis of wild-type and polyQ-expanded ataxin-3;And explore the effect on cytoplasmic/nuclear distribution further in eukaryocyte level.
Methods:
1.Recombinant DNA technology,Western-blot,GFP fluorescence technique were undertaken to construct and detect the expression of pEGFP-N1 eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3.
2.Subcellular fractionation was used to observe the effect of SUMO-1 modification on the cytoplasmic/nuclear distribution of wild-type and polyQ-expanded ataxin-3.
3.Co-immunoprecipitation was utilized to analyze the influence of SUMO-1 modifcation on the ubiquitination of wild-type and polyQ-expanded ataxin-3.
4.Chase experiment was undertaken to investigate the effect of SUMO-1 modification on the protein degradation of wild-type and polyQ-expanded ataxin-3.
5.Fluorescence technique,flow cytometry of PI/Annexin-V-FITC were used to study the effect of SUMO-1 modification on the formation of intranuclear inclusions,and apoptosis of wild-type and polyQ- expanded ataxin-3.
Results:
1.DNA sequencing confirmed that eukaryotic expression plasmids of the wild-type and polyQ-expanded ataxin-3 were altered on the designed mutation loci,and the repeat number of CAG trinucleotide were 20 and 68 on wild-type and polyQ-expanded respectively,which the other loci matched with GenBank standard sequence(S75313).It suggested that eukaryotic expression plasmids constructed successfully when we checked the reading frame of the plasmids without any frame shift after purpose gene order insertion.
2.We identified eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3 was expressed in HEK 293T cell by Western-blot.Using GFP fluorescence technique,we found that ataxin-3-20Q and ataxin-3-20Q-K166R distributed scattered;on the contrast,ataxin-3-68Q and ataxin-3-68Q-K166R aggregated.
3.We found SUMO-1 modification had no influence on the cytoplasmic/nuclear distribution of wild-type and polyQ- expanded ataxin-3 by subcellular fractionation.
4.By co-immunoprecipitation,our study suggested that over-expression of SUMO-1 or SUMO-1 modification of missing,the polyubiquitination level between the wild-type and polyQ-expanded ataxin-3 protein was significantly different, including polyQ-expanded ataxin-3 protein polyubiquitination higher level.Over-expression of SUMO-1 or SUMO-1 modification of missing,the wild-type ataxin-3 protein polyubiquitination level had no difference,and the result was similar for polyQ-expanded ataxin-3 protein.
5.We discovered by chase experiment that the bands weren't significantly different between ataxin3-20Q and ataxin3-20Q-K166R in 0h,1h,3h,7h,and 15h;but the bands of ataxin3-68Q were predominantly thicker than ataxin-3-68Q-K166R in 7h and 15h,which suggested that SUMO-1 modification resulted in stabilization of ataxin3-68Q.
6.We analyzed the quantitation of intranuclear inclusion of the wild-type and polyQ-expanded ataxin-3 had significant different (P＜0.005),indicated that polyQ expansion could induce formation of the intranuclear inclusion;the means of wild-type ataxin-3 group or polyQ-expanded ataxin-3 group weren't significantly different between with/without SUMO-1 or excessive/native SUMO-1 modification.
7.The result from flow cytometry of PI/Annexin-V/FITC showed the early apoptosis rate of ataxin-3-68Q group was higher than ataxin-3-68Q-K166R group(P＜0.005),which suggested that SUMO-1 modification had toxic effect;but the early apoptosis rate of ataxin-3-20Q group and ataxin-3-20Q-K166R group wasn't significantly different(P＞0.005).
Conclusion:
1.We construct pEGFP-N1 eukaryotic expression plasmids of the wild-type and polyQ-expanded ataxin-3 firstly,and obtain 14 HEK 293T cell strains.
2.We confirm that SUMO-1 modification has no influence on the cytoplasmic/nuclear distribution of the wild-type and polyQ-expanded ataxin-3.
3.We identify that K166 is not the ubiquitination locus of wild-type and polyQ-expanded ataxin-3.
4.We discover that SUMO-1 modification resulted in stabilization of polyQ-expanded ataxin-3.
5.We disclose that SUMO-1 modification of polyQ-expanded ataxin-3 had toxic effect,but didn't have influence on the nuclear inclusion formation.
引文
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