人体SOD1突变体在酵母细胞模型中的毒性功能的发现和研究

发布时间：2018-03-05 11:36

  本文选题：铜锌超氧化物歧化酶　切入点：家族性肌萎缩侧索硬化症　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：肌萎缩侧索硬化症（ALS）是一种致命的渐进型神经退行性疾病。它是由遗传缺陷或者非遗传性因素造成的。发病年龄通常在45岁至60岁，发病后的生存时间一般为1-5年，目前还没有可治愈的药物。已有多个基因缺陷被发现引起ALS，称作家族性肌萎缩侧索硬化症（fALS）。其中，最为著名的是由Cu,ZnSOD1的突变而造成的fALS。Cu,ZnSOD1因催化细胞中自由基的歧化反应而著称。它在细胞抵抗超氧化合物对细胞的损伤过程中起关键作用。目前已发现近150个Cu,ZnSOD1的突变能导致fALS。转基因小鼠研究结果表明fALS并不是因为突变使Cu,ZnSOD1失去活性，而是使Cu,ZnSOD1获得了一个新的有毒功能导致的。同时，也认为不同的突变是通过相同的机理引起fALS。但是，这一假说还未在实验上被完全证明。至于突变使Cu,ZnSOD1获得了什么性质的有毒新功能还有待被发现。 本论文选择在酵母细胞中研究在人体中引起fALS的Cu,ZnSOD1突变体A4V，G41D，G41S，G85R，G93C和I113T。我们实验室的研究结果表明酵母细胞也是研究fALS的理想模型。我们发现，当酵母细胞的H+-ATPase Pma1被过度表达时，酵母Cu,ZnSod1的G85R突变使其获得一个能使酵母细胞产生急剧形态变化和死亡的有毒新功能。酵母细胞和人体的Cu,ZnSod1相互间具有高度的同源性。因此，，本论文在酵母细胞中研究人体Cu,ZnSod1的这些突变体是否获得相同的有毒新功能。所得的实验结果表明人体的Cu,ZnSod1完全能够替代酵母细胞的Cu,ZnSod1抵抗H2O2的毒性，使酵母细胞能够在39℃温度下生长。人体Cu,ZnSod1也能够像酵母的Cu,ZnSod1那样使Pma1正确定位于细胞膜。突变体A4V和I113T也能够使sod1细胞抵抗H2O2的毒性和在39℃温度下生长。相比之下，其它G41D，G41S，G85R和G93C均在抗氧化和耐高温方面显示了不同程度的缺陷。尽管像A4V，G93C和I113T这样的突变体具有正常的催化歧化反应活性，然而在这些突变体细胞中Pma1-GFP仍然发生了定位错误。在G41D和G41S突变体细胞中，Pma1-GFP有正确显示细胞膜定位，也有发生了与A4V，G85R，G93C和I113T突体类似的定位错误。除了Pma1定位错误外，A4V和I113T突变体引起酵母细胞的体积发生了明显增大的变化。和酵母Cu,ZnSod1的G85R突变体细胞一样，人体的I113T突变体使酵母在YPD培养基上呈现棕黑色的皱纹形态，突变体细胞呈现细丝状形态。如此急剧的形态变化表明人体Cu,ZnSod1的I113T突变和酵母Cu,ZnSod1的G85R突变一样严重扰乱了细胞周期这一关键的生物过程。本论文所得实验结果首次利用酵母细胞模型证明在人体中能引起fALS疾病的I113T突变确实使Cu,ZnSod1获得了一个新的有毒功能，使酵母细胞产生了急剧的形态变化和死亡。这结果从而在酵母模型中证明了有关fALS的假设，也就是a toxic gain-of-function;进一步表明酵母细胞是研究人体fALS疾病的一个重要模型。
[Abstract]:Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease. It is caused by genetic defects or non-genetic factors. There is no cure yet. Several genetic defects have been found to cause ALS, known as familial amyotrophic lateral sclerosis. The most famous one is fALS.CuOZnSOD1, which is caused by the mutation of CuOZnSOD1. It plays a key role in cell resistance to the damage caused by superoxide compounds. Nearly 150 CuOZnSOD1 compounds have been found to catalyze the disproportionation of free radicals in cells. The results showed that fALS was not due to the inactivation of CuOZnSOD1, However, it is believed that different mutations cause fALSs by the same mechanism. This hypothesis has not yet been fully proved experimentally. The nature of the toxic new function obtained by the mutation of CuPZnSOD1 has yet to be discovered. In this paper, we chose to study in yeast cells the mutant A4VG41DG41SG85RG93C and I113T. our laboratory results showed that yeast cells were also ideal models for studying fALS. We found that when the H -ATPase Pma1 of yeast cells was overexpressed, The G85R mutation of yeast CuPZnSod1 enables it to obtain a new toxic function which can cause sharp morphological changes and death of yeast cells. There is a high homology between yeast cells and human Cu-ZnSod1. In this paper, we studied whether these mutants of human CuPZnSod1 have the same toxic new function in yeast cells. The experimental results show that the human CuPZnSod1 can completely replace the CuOZnSod1 of yeast cells to resist the toxicity of H _ 2O _ 2. The yeast cells were able to grow at 39 鈩

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