含多麩胺酸的突變ataxin-3蛋白所導致的Bcl-2mRNA穩定度下降

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Title:	含多麩胺酸的突變ataxin-3蛋白所導致的Bcl-2mRNA穩定度下降
Other Titles:	The decrease of Bcl-2 mRNA stability affected by mutant ataxin-3, a polyglutamine protein
Authors:	田至良 Tien, Chih-Liang
Contributors:	謝明麗 Hsieh, Mingli 東海大學生命科學系
Keywords:	小腦脊髓萎縮運動失調症;CAG三核?酸 Spinocerebellar ataxias;Machado-Joseph disease (MJD) / SCA3;CAG trinucleotide;Bcl-2 mRNA
Date:	2006
Issue Date:	2011-04-15T01:33:38Z (UTC)
Abstract:	小腦脊髓萎縮運動失調症是一種神經性疾病，且有許多亞型已被找出。而其中第三型小腦脊髓萎縮運動失調症是最常見的一種。這個疾病是由於CAG三個核?酸重複片段擴張而造成，而這些CAG三個核?酸重複片段最後會被轉譯成多麩胺酸。而致病蛋白已被確認為突變的ataxin-3。之前的研究結果證實，不正常的ataxin-3蛋白會造成某些基因的表現改變，蛋白質間不正常的作用和蛋白質不正常摺疊，最後導致神經細胞的死亡。Bcl-2是控制細胞死亡的蛋白家族中典型的一種。已經知道過量表達Bcl-2可以保護神經細胞去對抗多種疾病的傷害。我們建立了有GFP標記且帶有全長MJD基因的細胞株COS7-MJD26-GFP和COS7-MJD78-GFP。西方墨點法的結果指出在與COS7-MJD26-GFP相較下，含突變ataxin-3細胞的Bcl-2蛋白表現量下降。同樣地，半定量RT-PCR的結果也顯示在COS7-MJD78-GFP細胞內bcl-2 mRNA 同樣呈現下降的現象。觀察到這樣的結果可以表示，Bcl-2蛋白的下降可能是因為在mRNA程度上的缺失。然而Bcl-2啟動子在COS7-MJD78-GFP細胞內的活性都遠高於COS7-MJD26- GFP細胞，這個結果顯示Bcl-2蛋白表達量的下降可能是因為mRNA穩定度上的缺失。因此，我們使用腺嘌呤類似物DRB去抑制mRNA的合成，並再利用半定量RT-PCR去估計bcl-2 mRNA的降解速率。我們的結果指出，在COS7-MJD78-GFP細胞內bcl-2 mRNA 的降解速度，約是在COS7-MJD26-GFP細胞中的五倍。在bcl-2 mRNA 3’端不轉錄區域內的AU-rich 序列對ARE結合蛋白是一段很重要的區域，ARE結合蛋白可以經由正向或是負向的方式去調節mRNA穩定度。首先我們研究nucleolin這個ARE結合蛋白在MJD細胞模型內的作用，然而從西方點墨法的結果顯示，nucleolin的表達在細胞表達MJD78-GFP或MJD26-GFP的質體，並沒有顯著差異。目前我們仍在進一步的分析，其他的ARE結合蛋白，是否會被突變的ataxin-3所影響。 Spinocerebellar ataxias are neurological disorders, and many subtypes were identified. Machado-Joseph disease (MJD) / SCA3 is the most common type. This disease is caused by CAG trinucleotide repeat expansion and the abnormal CAG repeats are transcribed and translated into polyglutamine. The pathogenic protein is defined as mutant ataxin-3. Previous studies demonstrated that mutant ataxin-3 causes altered gene expression, abnormal protein interaction and protein unfolding, eventually leading to neuronal cell death. Bcl-2 (B-cell lymphoma) is the prototype of a growing family of proteins which control cell death. It is known that overexpression of Bcl-2 protects neurons from a variety of pathological insults. COS7-MJD26-GFP and COS7-MJD78-GFP, which were stably transfected with full length MJD gene with GFP-tagged, were established in our laboratory. Western blot analysis revealed that Bcl-2 protein expression is decreased in cells containing mutant ataxin-3, when compared with that of COS7-MJD26- GFP cells. Consistently, results from semi-quantitative RT-PCR demonstrated that Bcl-2 mRNA expression is decreased in COS7- MJD78-GFP cells. This observation suggested that the down-regulation of Bcl-2 protein may be due to defects in mRNA level. However, the promoter activity in COS7-MJD78-GFP cells was much higher than that of COS7-MJD26-GFP, suggesting that the decrease of Bcl-2 expression may be due to defects in mRNA stability. Therefore, 5,6-Dichloro -benzimidazole 1-β-D-ribofuranoside (DRB), an adenosine analogue to inhibit mRNA synthesis, was used to estimate Bcl-2 mRNA degradation rate by semi-quantitative RT-PCR. Our results indicated that the bcl-2 mRNA decay in COS7-MJD78-GFP cells is about five-fold faster than that in COS7-MJD26-GFP. It is known that AU-rich elements (AREs) in 3’UTR of Bcl-2 mRNA are important regions for ARE binding proteins, which modulate the stability of ARE-containing mRNA in both positive and negative fashions. Effect of Nucleolin, an AU-rich element binding protein, was first investigated in our MJD cellular model. However, Western blot analysis revealed that nucleolin expression showed no difference between cells expressing with MJD78-GFP and MJD26-GFP. Further analysis to study which ARE binding protein affected by mutant ataxin-3 is under way.
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