自吞噬途径在polyQ扩展突变型ataxin-3降解及SCA3/MJD发病机制中的作用Ataxin-3多克隆抗体的制备
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摘要
背景：
遗传性脊髓小脑型共济失调(spinocerebellar ataxia,SCAs)是一组以神经细胞变性为病理特征的神经系统遗传疾病,目前认为其发病机制与致病基因外显子中CAG拷贝数异常扩增产生带有多聚谷氨酰胺(polyQ)链的蛋白有关。脊髓小脑型共济失调三型/马查多-约瑟夫病(spinocerebellar ataxia 3/Machado-Joseph disease, SCA3/MJD)是SCAs中发病率较高的亚型,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,致病蛋白的降解在此类疾病的发病机制中具有重要的意义。
细胞内蛋白主要通过泛素-蛋白酶体途径(ubiquitin-proteasome pathway, UPP)和自吞噬途径(Autophagy)进行降解。其中自吞噬途径在降解清除细胞自身大分子蛋白复合物和衰老细胞器,维持细胞内环境稳定的过程中起着非常重要的作用,同时也是细胞适应外界环境变化的一种重要机制。目前研究提示,自吞噬途径参与降解阿尔茨海默病、多聚谷氨酸疾病(SCA1、Huntingtin病)、帕金森病等多种神经系统变性疾病相关的致病蛋白并参与其生理及发病过程。
参与调节自吞噬途径的信号分子种类繁多,其具体调节机制仍不清楚。现在已经证实的是mTOR (mammalian target of rapamycin)对于自吞噬信号转导途径的负性调节作用。近年来在对mTOR信号通路众多上游调节蛋白的研究中,LKB1-AMPK途径受到了广泛关注。目前认为LKB1是AMPK (AMP-activated protein kinase)重要的蛋白激酶,它能通过磷酸化AMPK第172位苏氨酸(Thr172)使之活化进而参与调节mTOR。亦有报道显示,LKB1-AMPK途径能通过介导细胞周期依赖性蛋白激酶抑制物p27kip1的磷酸化而直接参与调节自吞噬途径。
在前期研究中,我们构建了野生型ataxin-3蛋白和polyQ异常扩展突变型ataxin-3蛋白的真核表达载体：pCDNA3.1-Myc-His(-)B-ataxin-3-20Q和pCDNA3.1-Myc-His(-)B-ataxin-3-68Q。
目的：
拟开展自吞噬途径对于polyQ扩展突变型ataxin-3蛋白的降解、胞内聚合物形成、细胞活性影响的研究,以期较全面的探讨其在SCA3/MJD发病机制中的作用；进一步研究自吞噬途径可能的上游激酶LKB1能否通过自吞噬途径参与调节polyQ扩展突变型ataxin-3蛋白的降解。
方法：
1、应用免疫荧光-激光共聚焦技术、Western印迹技术明确自吞噬途径是否参与polyQ扩展突变型ataxin-3蛋白的降解；
2、应用Western印迹技术研究LKB1是否参与polyQ扩展突变型ataxin-3蛋白的降解；
3、应用免疫荧光-激光共聚焦技术、MTT法探讨自吞噬途径对polyQ扩展突变型ataxin-3蛋白细胞毒性的调节作用；
4、应用重组基因技术、Western-blot技术构建并检测野生型ataxin-3的pGEX-4T-2原核表达载体并诱导其表达；
5、应用亲和层析技术纯化融合蛋白,免疫新西兰兔制备了高效价的ataxin-3多克隆抗体；
结果：
1、免疫荧光-激光共聚焦结果显示：共转pCDNA3.1-Myc-His (B)-ataxin-3-68Q和pEGFP-C1-LC3细胞组中可见过表达的LC3与突变ataxin-3形成的胞内蛋白聚合体明显重叠。Western-blot实验结果显示：分别经3-MA、NH4CL处理的细胞,ataxin-3-68Q条带明显增粗加深,而转染后9小时加入rapamycin处理细胞后则ataxin-3-68Q条带变浅,这说明polyQ扩展突变型ataxin-3能够被自吞噬小体识别并包被,自吞噬途径参与ataxin-3-68Q的降解,抑制或早期促进自吞噬途径能有效减少或增加突变蛋白的降解。
2、Western-blot结果发现LKB1的表达能抑制转染细胞内polyQ异常扩展突变型ataxin-3的降解。
3、免疫荧光结果显示：3-MA处理组表达ataxin-3-68Q的细胞内可见大量的胞内蛋白聚合物的形成,从形态学上分析,聚合物不仅数量增多而且形态更大,大部分聚集在胞核周围；转染后9小时加入rapamycin处理组细胞中,ataxin-3-68Q形成的胞内聚合物则明显减少。进一步说明抑制自吞噬途径能增加细胞内ataxin-3-68Q蛋白聚合体的形成,反之亦然。经rapamycin处理的两组细胞中,转染后早期(9小时)处理组较对照组中ataxin-3-68Q表达减少,但转染后晚期(16小时)处理组目标蛋白表达变化不大。这说明在突变蛋白表达早期激活胞内自噬水平能加快聚合物的降解清除。
4、MTT分析结果显示,与ataxin-3-68Q对照组比较：3-MA组组聚合体阳性细胞数明显增加,细胞活性降低(P<0.05)；转染后9小时加入rapamycin组聚合体阳性细胞数明显减少,细胞活性有所增加(P<0.005)；转染后16小时加入rapamycin组,聚合体阳性细胞数差异无统计学意义(P>0.05),细胞活性反而降低(P＜0.01)。
5、DNA测序证实所构建的野生型ataxin-3氨基端的原核表达载体的目的基因位点均与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,说明原核表达载体构建成功。
6、Western-blot、免疫荧光结果均证实制备的多克隆抗体能够识别ataxin-3蛋白,具有较高特异性。
结论：
1证实自吞噬途径参与细胞内polyQ异常扩展突变型ataxin-3蛋白的降解；
2发现抑制或早期刺激自吞噬途径能分别抑制或促进胞内polyQ扩展突变型ataxin-3蛋白的降解；
3首次发现LKB1的表达抑制polyQ扩展突变型ataxin-3蛋白的降解；
4证实自吞噬途径能促进polyQ扩展突变型ataxin-3蛋白的降解,减少胞内聚合体的形成,减轻细胞毒性；
5制备了ataxin-3-N蛋白特异性抗体,可用于进一步研究其相关功能。
Background:
The hereditary spinocerebellar ataxias (SCAs) are a group of inherited neurodegenerative disorders, which is characterized by accumulation of aberrant protein aggregates in affected neurons. Genetic and transgenic datas suggest that these diseases are caused by codon reiteration mutations, where protein misfolding is mediated by the abnormal expansion of a tract of repeated amino acids. Among them, spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is the most common subtype. The pathogenic 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 MJD1 gene product, ataxin-3. However, 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 degeneration of the disease proteins plays a critical role in their physiological function and pathogenesis.
Eukaryotic cells have two major protein degradation pathways, the ubiquitin-proteasome pathway and autophagy, which are responsible for cellular homeostasis. Autophagy is a cellular catabolic mechanism mediating the turnover of intracellular long-lived proteins even entire organelles. It is a highly regulated process that plays a role in cellular maintenance and development, and has been implicated in a number of genetic diseases. Several studies have observed that autophagy is a major route for the degradation of the disease proteins in Alzheimer's disease, Huntington's disease (HD), spinocerebellar ataxia type 1 and Parkinson's disease.
Several signalling complexes and pathways are involved in the autophagic route in mammalian cells. And many of these genes remain to be poorly understood. It is widely agreed that the mTOR (mammalian target of rapamycin) signalling pathway negatively regulates autophagy, and LKB1-AMPK pathway plays a role in regulating mTOR. It has found that LKB1, which is the upstream kinase of autophagy, can efficiently phosphorylated AMPK (AMP-activated protein kinase) in vitro specifically at Thr 172 functioning as a upstream regulator of autophagy. The cyclin-dependent kinase inhibitor p27Kipl, is phosphorylated at Thr 198 downstream of the LKB1-AMPK pathway, directly linking sensing of LKB1 and autophagy.
In previous work, we have constructed the eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3:pCDNA3.1-Myc-His(-)B-ataxin-3-20Q and pCDNA3.1-Myc-His(-)B-ataxin-3-68Q by using recombinant DNA technology.
Objective:
To research the influence of autophagy on the protein degradation, formation of intracellular protein aggregates, and cell viability of polyQ-expanded ataxin-3; And discuss the role of autophagy on pathogenesis of spinocerebellar ataxia 3/Machado-Josephdisease (SCA3/MJD). Further more, to address the role of LKB1 in degradation of polyQ-expanded ataxin-3, and discuss the mechanism of it.
Methods:
1. Immunofluorescence-laser cofocalization and western-blot were undertaken to observe the degradation of ataxin-3 influenced by autophagy.
2. By Western-blot, we investigated the effect of LKB1 on the protein degradation of polyQ-expanded ataxin-3.
3. Immunofluorescence and MTT were used to detect the formation of ataxin-3 aggregates and cell viability modified by autophagy.
4. Recombinant DNA technology and Western-blot were undertaken to construct and detect the expression of pGEX-4T-2 prokaryotic expression plasmids of wild-type ataxin-3.
5. The expressed proteins were purified from total proteins with Glutathione sepharose 4B agarose column. The New Zealand rabbits were immunized with the purified fusion proteins to prepare polyclonal antiserum.
Results:
1. We found that ataxin-3-68Q in HEK293T cells expressed in cytoplasm and nucleus, and aggregated in cells which colocalized with GFP-LC3 by Immunofluorescence-laser cofocalization. By western-blot,3-MA or NH4CL treatment led to a significant increase in the levels of ataxin-3-68Q, whereas rapamycin had the opposite effect. These results suggested that the aggregates can be sequestered into autophagic vacuoles. The reduction of autophagy led to the increase of ataxin-3 with expanded polyQ.
2. The result from western blot showed that expressing of LKB1 led to a significant increase in the levels of ataxin-3-68Q.
3. We discovered by Immunofluorescence-laser that 3-MA treatment resulted in an obvious change in the appearance of the aggregates formed by ataxin-3-68Q constructs transiently transfected into HEK293T cells. In cells with aggregates,3-MA increased their apparent size and number. Treatment with rapamycin after 9 hours of transfection, resulted in a decrease in the proportions of aggregate-containing cells in HEK293T cells expressing ataxin-3-68Q. It indicated that inhibition of autophagy increased aggregate formation in HEK293T cells expressing ataxin-3-68Q, and vice versa. Treatment with rapamycin after 9 hours of transfection, resulted in a decrease in expressing of mutant ataxin-3 in HEK293T cells than that of control cells. But no significant change was observed in cells expressing ataxin-3-68Q treated with rapamycin after 16 hours of transfection. This observation counters the possibility that autophagy can degrade monomeric and oligomeric precursors of aggregates, rather than the large inclusions.
4. The MTT assay showed that the cell viability of group of 3-MA obviously decreased in contrast to control group (P<0.05). The cell viability of group of early treatment with rapamycin was higher than that of control group (P<0.05). But the aggregate-containing cells (%) of late treatment with rapamycin group and control group had no significantly different (P>0.05) and the cell viability of which was lower (P<0.01).
5. DNA sequencing confirmed that prokaryotic expression plasmids of wild-type ataxin-3-N matched with GenBank standard sequence (S75313). It suggested that prokaryotic expression plasmids constructed successfully when we checked the reading frame of the plasmids without any frameshift after purpose gene order insertion.
6. Western-blot and immunoflurescence analysis suggest that the polyclonal antibody raised in the rabbit could recognized ataxin-3.
Conclusion:
1. We confirm that autophagy is involved in the degradation of mutant ataxin-3.
2. We discover that inhibiting or inducing autophagy in early stage after transfection, results in a increase or a decrease in the level of polyglutamine-expanded ataxin-3, respectively.
3. For the first time, we find that expressing of LKB1 inhibits the degradation of polyglutamine-expanded ataxin-3.
4. We confirm that autophagy induces degradation of mutant ataxin-3, decreases the formation of aggregation and alleviates the protein toxity.
5. We prepare anti-ataxin-3 polyclonal antibody which could widely useful in researches of ataxin-3.
遗传性脊髓小脑型共济失调(spinocerebellar ataxia,SCAs)是一组以神经细胞变性为病理特征的神经系统遗传疾病,目前认为其发病机制与致病基因外显子中CAG拷贝数异常扩增产生带有多聚谷氨酰胺(polyQ)链的蛋白有关。脊髓小脑型共济失调三型/马查多-约瑟夫病(spinocerebellar ataxia 3/Machado-Joseph disease, SCA3/MJD)是SCAs中发病率较高的亚型,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,致病蛋白的降解在此类疾病的发病机制中具有重要的意义。
细胞内蛋白主要通过泛素-蛋白酶体途径(ubiquitin-proteasome pathway, UPP)和自吞噬途径(Autophagy)进行降解。其中自吞噬途径在降解清除细胞自身大分子蛋白复合物和衰老细胞器,维持细胞内环境稳定的过程中起着非常重要的作用,同时也是细胞适应外界环境变化的一种重要机制。目前研究提示,自吞噬途径参与降解阿尔茨海默病、多聚谷氨酸疾病(SCA1、Huntingtin病)、帕金森病等多种神经系统变性疾病相关的致病蛋白并参与其生理及发病过程。
参与调节自吞噬途径的信号分子种类繁多,其具体调节机制仍不清楚。现在已经证实的是mTOR (mammalian target of rapamycin)对于自吞噬信号转导途径的负性调节作用。近年来在对mTOR信号通路众多上游调节蛋白的研究中,LKB1-AMPK途径受到了广泛关注。目前认为LKB1是AMPK (AMP-activated protein kinase)重要的蛋白激酶,它能通过磷酸化AMPK第172位苏氨酸(Thr172)使之活化进而参与调节mTOR。亦有报道显示,LKB1-AMPK途径能通过介导细胞周期依赖性蛋白激酶抑制物p27kip1的磷酸化而直接参与调节自吞噬途径。
在前期研究中,我们构建了野生型ataxin-3蛋白和polyQ异常扩展突变型ataxin-3蛋白的真核表达载体：pCDNA3.1-Myc-His(-)B-ataxin-3-20Q和pCDNA3.1-Myc-His(-)B-ataxin-3-68Q。
目的：
拟开展自吞噬途径对于polyQ扩展突变型ataxin-3蛋白的降解、胞内聚合物形成、细胞活性影响的研究,以期较全面的探讨其在SCA3/MJD发病机制中的作用；进一步研究自吞噬途径可能的上游激酶LKB1能否通过自吞噬途径参与调节polyQ扩展突变型ataxin-3蛋白的降解。
方法：
1、应用免疫荧光-激光共聚焦技术、Western印迹技术明确自吞噬途径是否参与polyQ扩展突变型ataxin-3蛋白的降解；
2、应用Western印迹技术研究LKB1是否参与polyQ扩展突变型ataxin-3蛋白的降解；
3、应用免疫荧光-激光共聚焦技术、MTT法探讨自吞噬途径对polyQ扩展突变型ataxin-3蛋白细胞毒性的调节作用；
4、应用重组基因技术、Western-blot技术构建并检测野生型ataxin-3的pGEX-4T-2原核表达载体并诱导其表达；
5、应用亲和层析技术纯化融合蛋白,免疫新西兰兔制备了高效价的ataxin-3多克隆抗体；
结果：
1、免疫荧光-激光共聚焦结果显示：共转pCDNA3.1-Myc-His (B)-ataxin-3-68Q和pEGFP-C1-LC3细胞组中可见过表达的LC3与突变ataxin-3形成的胞内蛋白聚合体明显重叠。Western-blot实验结果显示：分别经3-MA、NH4CL处理的细胞,ataxin-3-68Q条带明显增粗加深,而转染后9小时加入rapamycin处理细胞后则ataxin-3-68Q条带变浅,这说明polyQ扩展突变型ataxin-3能够被自吞噬小体识别并包被,自吞噬途径参与ataxin-3-68Q的降解,抑制或早期促进自吞噬途径能有效减少或增加突变蛋白的降解。
2、Western-blot结果发现LKB1的表达能抑制转染细胞内polyQ异常扩展突变型ataxin-3的降解。
3、免疫荧光结果显示：3-MA处理组表达ataxin-3-68Q的细胞内可见大量的胞内蛋白聚合物的形成,从形态学上分析,聚合物不仅数量增多而且形态更大,大部分聚集在胞核周围；转染后9小时加入rapamycin处理组细胞中,ataxin-3-68Q形成的胞内聚合物则明显减少。进一步说明抑制自吞噬途径能增加细胞内ataxin-3-68Q蛋白聚合体的形成,反之亦然。经rapamycin处理的两组细胞中,转染后早期(9小时)处理组较对照组中ataxin-3-68Q表达减少,但转染后晚期(16小时)处理组目标蛋白表达变化不大。这说明在突变蛋白表达早期激活胞内自噬水平能加快聚合物的降解清除。
4、MTT分析结果显示,与ataxin-3-68Q对照组比较：3-MA组组聚合体阳性细胞数明显增加,细胞活性降低(P<0.05)；转染后9小时加入rapamycin组聚合体阳性细胞数明显减少,细胞活性有所增加(P<0.005)；转染后16小时加入rapamycin组,聚合体阳性细胞数差异无统计学意义(P>0.05),细胞活性反而降低(P＜0.01)。
5、DNA测序证实所构建的野生型ataxin-3氨基端的原核表达载体的目的基因位点均与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,说明原核表达载体构建成功。
6、Western-blot、免疫荧光结果均证实制备的多克隆抗体能够识别ataxin-3蛋白,具有较高特异性。
结论：
1证实自吞噬途径参与细胞内polyQ异常扩展突变型ataxin-3蛋白的降解；
2发现抑制或早期刺激自吞噬途径能分别抑制或促进胞内polyQ扩展突变型ataxin-3蛋白的降解；
3首次发现LKB1的表达抑制polyQ扩展突变型ataxin-3蛋白的降解；
4证实自吞噬途径能促进polyQ扩展突变型ataxin-3蛋白的降解,减少胞内聚合体的形成,减轻细胞毒性；
5制备了ataxin-3-N蛋白特异性抗体,可用于进一步研究其相关功能。
Background:
The hereditary spinocerebellar ataxias (SCAs) are a group of inherited neurodegenerative disorders, which is characterized by accumulation of aberrant protein aggregates in affected neurons. Genetic and transgenic datas suggest that these diseases are caused by codon reiteration mutations, where protein misfolding is mediated by the abnormal expansion of a tract of repeated amino acids. Among them, spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is the most common subtype. The pathogenic 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 MJD1 gene product, ataxin-3. However, 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 degeneration of the disease proteins plays a critical role in their physiological function and pathogenesis.
Eukaryotic cells have two major protein degradation pathways, the ubiquitin-proteasome pathway and autophagy, which are responsible for cellular homeostasis. Autophagy is a cellular catabolic mechanism mediating the turnover of intracellular long-lived proteins even entire organelles. It is a highly regulated process that plays a role in cellular maintenance and development, and has been implicated in a number of genetic diseases. Several studies have observed that autophagy is a major route for the degradation of the disease proteins in Alzheimer's disease, Huntington's disease (HD), spinocerebellar ataxia type 1 and Parkinson's disease.
Several signalling complexes and pathways are involved in the autophagic route in mammalian cells. And many of these genes remain to be poorly understood. It is widely agreed that the mTOR (mammalian target of rapamycin) signalling pathway negatively regulates autophagy, and LKB1-AMPK pathway plays a role in regulating mTOR. It has found that LKB1, which is the upstream kinase of autophagy, can efficiently phosphorylated AMPK (AMP-activated protein kinase) in vitro specifically at Thr 172 functioning as a upstream regulator of autophagy. The cyclin-dependent kinase inhibitor p27Kipl, is phosphorylated at Thr 198 downstream of the LKB1-AMPK pathway, directly linking sensing of LKB1 and autophagy.
In previous work, we have constructed the eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3:pCDNA3.1-Myc-His(-)B-ataxin-3-20Q and pCDNA3.1-Myc-His(-)B-ataxin-3-68Q by using recombinant DNA technology.
Objective:
To research the influence of autophagy on the protein degradation, formation of intracellular protein aggregates, and cell viability of polyQ-expanded ataxin-3; And discuss the role of autophagy on pathogenesis of spinocerebellar ataxia 3/Machado-Josephdisease (SCA3/MJD). Further more, to address the role of LKB1 in degradation of polyQ-expanded ataxin-3, and discuss the mechanism of it.
Methods:
1. Immunofluorescence-laser cofocalization and western-blot were undertaken to observe the degradation of ataxin-3 influenced by autophagy.
2. By Western-blot, we investigated the effect of LKB1 on the protein degradation of polyQ-expanded ataxin-3.
3. Immunofluorescence and MTT were used to detect the formation of ataxin-3 aggregates and cell viability modified by autophagy.
4. Recombinant DNA technology and Western-blot were undertaken to construct and detect the expression of pGEX-4T-2 prokaryotic expression plasmids of wild-type ataxin-3.
5. The expressed proteins were purified from total proteins with Glutathione sepharose 4B agarose column. The New Zealand rabbits were immunized with the purified fusion proteins to prepare polyclonal antiserum.
Results:
1. We found that ataxin-3-68Q in HEK293T cells expressed in cytoplasm and nucleus, and aggregated in cells which colocalized with GFP-LC3 by Immunofluorescence-laser cofocalization. By western-blot,3-MA or NH4CL treatment led to a significant increase in the levels of ataxin-3-68Q, whereas rapamycin had the opposite effect. These results suggested that the aggregates can be sequestered into autophagic vacuoles. The reduction of autophagy led to the increase of ataxin-3 with expanded polyQ.
2. The result from western blot showed that expressing of LKB1 led to a significant increase in the levels of ataxin-3-68Q.
3. We discovered by Immunofluorescence-laser that 3-MA treatment resulted in an obvious change in the appearance of the aggregates formed by ataxin-3-68Q constructs transiently transfected into HEK293T cells. In cells with aggregates,3-MA increased their apparent size and number. Treatment with rapamycin after 9 hours of transfection, resulted in a decrease in the proportions of aggregate-containing cells in HEK293T cells expressing ataxin-3-68Q. It indicated that inhibition of autophagy increased aggregate formation in HEK293T cells expressing ataxin-3-68Q, and vice versa. Treatment with rapamycin after 9 hours of transfection, resulted in a decrease in expressing of mutant ataxin-3 in HEK293T cells than that of control cells. But no significant change was observed in cells expressing ataxin-3-68Q treated with rapamycin after 16 hours of transfection. This observation counters the possibility that autophagy can degrade monomeric and oligomeric precursors of aggregates, rather than the large inclusions.
4. The MTT assay showed that the cell viability of group of 3-MA obviously decreased in contrast to control group (P<0.05). The cell viability of group of early treatment with rapamycin was higher than that of control group (P<0.05). But the aggregate-containing cells (%) of late treatment with rapamycin group and control group had no significantly different (P>0.05) and the cell viability of which was lower (P<0.01).
5. DNA sequencing confirmed that prokaryotic expression plasmids of wild-type ataxin-3-N matched with GenBank standard sequence (S75313). It suggested that prokaryotic expression plasmids constructed successfully when we checked the reading frame of the plasmids without any frameshift after purpose gene order insertion.
6. Western-blot and immunoflurescence analysis suggest that the polyclonal antibody raised in the rabbit could recognized ataxin-3.
Conclusion:
1. We confirm that autophagy is involved in the degradation of mutant ataxin-3.
2. We discover that inhibiting or inducing autophagy in early stage after transfection, results in a increase or a decrease in the level of polyglutamine-expanded ataxin-3, respectively.
3. For the first time, we find that expressing of LKB1 inhibits the degradation of polyglutamine-expanded ataxin-3.
4. We confirm that autophagy induces degradation of mutant ataxin-3, decreases the formation of aggregation and alleviates the protein toxity.
5. We prepare anti-ataxin-3 polyclonal antibody which could widely useful in researches of ataxin-3.
引文
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