ALS相关SOD1突变体的自噬性降解及Gemin3作为ALS相关FUS蛋白的结合蛋白的鉴定

发布时间：2018-04-06 22:40

本文选题：肌萎缩侧索硬化症(ALS)　切入点：铜锌超氧化物歧化酶(SOD1)　出处：《西北农林科技大学》2014年博士论文

【摘要】：肌萎缩侧索硬化症(ALS)是一种由运动神经元死亡引起的,成年发作的,致命的神经退行性疾病。绝大多数ALS病例为发病原因不明的散发性ALS (sALS),其余约10%具有家族史,称为家族性ALS(fALS)。目前已经发现了多种家族性ALS致病基因,SOD1(铜锌超氧化物歧化酶)和FUS (fused in sarcoma)是其中的两种。为了了解ALS的发生机制,并最终为ALS的治疗提供新途径,本实验研究了SOD1的自噬降解途径；鉴定了一种新型的FUS结合蛋白。 (1)ALS相关的SOD1突变体的自噬性降解 SOD1是最早发现的ALS致病基因,约占家族性ALS病例的20%。SOD1突变体对运动神经元的损伤不是功能缺失而是获得性毒性引起的。因此,了解SOD1突变体的降解清除途径可以为ALS的发生及治疗提供新的线索。通过比较野生型SOD1和ALS相关SOD1突变体(A4V, G93A)在细胞内的表达,发现SOD1突变体的表达水平较野生型明显下降,并且形成大的聚集体,表现出明显的去垢剂不溶性。研究证明多种神经退行性疾病致病蛋白通过自噬途径降解。为了验证ALS相关的SOD1突变体是否通过该途径降解,利用多种自噬调节剂,如rapamycin抑制mTOR诱导自噬发生,Beclinl通过与PI3KⅢ (也称为hVps34)结合增强其激酶活性诱导自噬起始,3-MA抑制PI3KⅢ活性从而抑制自噬小体的形成,Bafilomycin Al和NH4C1抑制自噬小体和溶酶体的融合,E64d和pepstatin A抑制溶酶体中组织蛋白酶活性等调控自噬过程,结果显示诱导自噬可以促进SOD1的降解,而抑制自噬则导致SOD1发生堆积。此外,利用一种pH敏感的双荧光标签发现SOD1突变体可以被包裹进自噬溶酶体中。因此,ALS相关SOD1突变体经自噬途径降解。 (2) SOD1G93A突变体对自噬活性的影响作为机体在病理条件下清除异常蛋白的一种潜在机制,自噬可以被多种聚集体蛋白活化。通过检测不同发病阶段的ALS小鼠体内的自噬小体标记物LC3Ⅱ的表达水平,发现其在发病末期(125d)较对照组显著升高。细胞模型中,利用Bafilomycin A1抑制自噬小体-溶酶体融合,发现SOD1G93A突变体可以提高内源性LC3Ⅱ的水平,促进共转染的EGFP-LC3Ⅰ向EGFP-LC3Ⅱ转化,提示在细胞模型中SOD1突变体可以诱导自噬小体的形成。为了探讨其可能的机制,检测了SOD1G93A对自噬起始关键因子Beclinl与其结合蛋白(ATG14L、Rubicon及UVRAG)结合能力的影响。结果显示,与野生型对照组相比,SOD1G93A对ATG14L, Rubicon和UVRAG与Beclin1的结合能力无明显影响,说明SOD1G93A可能通过其它途径影响了自噬的发生。 (3) Gemin3作为ALS相关FUS蛋白的结合蛋白的鉴定 FUS是最近发现的一种新型ALS致病蛋白。作为一种RNA/DNA结合蛋白,FUS参与mRNA转录,剪接,运输及成熟等过程。因此,FUS突变引起的RNA代谢异常可能在ALS的发生过程中起关键作用,但目前对其致病机制的了解还很有限。为了揭示FUS突变体可能的致病机制,利用质谱蛋白组学的方法,发现了Gemin3(也称为DP103或DDX20),一种SMN复合体组分,是FUS的结合蛋白。GST-pulldown和Co-IP实验证实了野生型FUS和ALS相关的FUS突变体均可与Gemin3相互作用,且野生型与突变体之间没有明显差异。利用FUS和Gemin3的截断突变体发现FUS结合于Gemin3的C末端(438-824氨基酸),Gemin3结合于FUS的QSYG区(1-165氨基酸)。细胞中过表达FUS突变体使Gemin3形成与FUS聚集体共定位的大的细胞质斑点,抑制细胞核内Gem小体的形成,并且引起Gemin3在去垢剂不溶部分发生堆积。此外,FUS突变体可以下调细胞内U snRNP的水平,引起E1A报告基因的剪接异常。因此,结果表明FUS突变体可能通过将Gemin3包裹进聚集体中引起U snRNP功能缺陷,使随后的rnRNA前体剪接发生改变。总之,实验结果表明ALS相关的SOD1通过自噬途径降解,为开发自噬调控因子以清除体内的SOD1突变体作为ALS治疗的手段提供了依据；鉴定了FUS与Gemin3之间的相互作用,为FUS相关的ALS的发生的研究提供了线索。
[Abstract]:Amyotrophic lateral sclerosis (ALS) is caused by a motor neuron death, adult onset, fatal neurodegenerative diseases. Most of the ALS cases for the unknown etiology of sporadic ALS (sALS), the remaining 10% with a family history, called familial ALS (fALS) has. Found a variety of familial ALS disease gene, SOD1 (superoxide dismutase) and FUS (fused in sarcoma) is one of the two. In order to understand the mechanism of ALS, and provides a new way for the treatment of ALS, the experimental study on the autophagy of SOD1; identification of a novel the FUS binding protein.
(1) autophagic degradation of ALS associated SOD1 mutants
SOD1 is the earliest discovered ALS gene, about 20%.SOD1 mutant familial ALS cases of motor neuron injury but not loss of function caused by acquired toxicity. Therefore, understanding the degradation of mutant SOD1 pathways may provide new clues for the pathogenesis and therapy of ALS. Through the comparison of wild type SOD1 and ALS SOD1 mutants (A4V, G93A) expression in cells and found that the expression level of SOD1 mutants compared to the wild type was significantly decreased, and the formation of large aggregates, showed obvious detergent insoluble. Studies show that a variety of neurodegenerative disease proteins through the autophagy pathway degradation. In order to verify whether the SOD1 mutant ALS by the degradation, using a variety of autophagy regulators, such as rapamycin inhibited mTOR induced autophagy by Beclinl and PI3K III (also known as hVps34) with the kinase activity induced by self enhancement The initial formation of 3-MA macrophages, inhibiting the activity of PI3K III inhibits autophagosome fusion, Bafilomycin Al and NH4C1 inhibited the autophagosome and lysosome, E64d and pepstatin A in inhibition of cathepsin activity of lysosomal regulation of the autophagic process, the result indicated that the degradation induced autophagy can promote SOD1, and inhibition of autophagy leads to the occurrence of SOD1 accumulation. In addition, the use of double a pH sensitive fluorescence label SOD1 mutant can be wrapped into autophagolysosomes. Therefore, ALS related SOD1 mutant by autophagy pathway degradation.
(2) effect of SOD1G93A mutant on autophagy activity
As a potential mechanism for the body to eliminate abnormal protein in pathological conditions, autophagy can be activated by protein aggregates. The expression level of autophagosome marker LC3 in ALS mice at different stages of the detection of the disease, the end (125d) was significantly higher than the control group. The cell model, using Bafilomycin A1 inhibition of autophagy lysosomefusion, SOD1G93A mutant can increase endogenous LC3 II level, promote the transformation of EGFP-LC3 co transfected to EGFP-LC3 I II, suggesting that the formation of the cell model in SOD1 mutants can induce autophagic bodies. In order to explore the possible mechanism of SOD1G93A detection of autophagy initiation key transcription factor Beclinl binding protein (ATG14L Rubicon, and UVRAG) affect the binding capacity. The results showed that, compared with the wild type control group, SOD1G93A of ATG14L, Rubicon and UVRAG and Beclin1 Binding ability had no significant effect, indicating that SOD1G93A may influence through other ways of autophagy.
(3) identified as a ALS binding protein related protein FUS Gemin3
FUS is a new type of ALS pathogenic protein discovered recently. As a RNA/DNA binding protein, FUS participates in mRNA transcription, splicing, transport and maturation process. Therefore, the mutation of FUS RNA caused by abnormal metabolism may play a key role in the process of ALS, but its pathogenesis remains unclear in order to reveal the pathogenic mechanism of FUS mutants may, by using the method of mass spectrometry proteomics, found Gemin3 (also known as DP103 or DDX20), a component of SMN complex, FUS binding protein.GST-pulldown and Co-IP experiments showed that the wild type FUS and ALS related FUS mutants can interact with Gemin3, no obvious the difference between wild type and mutant. And the use of truncated mutants FUS and Gemin3 found C terminal FUS binding to Gemin3 (438-824 amino acids), Gemin3 binding to FUS QSYG (1-165 amino acids) in cells overexpressing FUS mutation. To the formation of Gemin3 co localized with FUS aggregates of cytoplasmic spots, inhibit the formation of nuclei of Gem bodies, and caused Gemin3 insoluble part accumulation in detergent. In addition, the FUS mutant can lower intracellular U levels of snRNP, E1A by reporter gene splicing. Therefore, the results show that the FUS mutant may pass the Gemin3 packages in U snRNP aggregates caused by defects, the subsequent pre rnRNA splicing change.
In conclusion, the experimental results show that ALS related SOD1 degradation by autophagy pathway, and provide a basis for the development of autophagy regulation factor to clear the SOD1 mutant in vivo as ALS treatment; the interaction between FUS and Gemin3 were identified, study the occurrence of FUS related ALS provided clues.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R744.8

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