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研究生: 許鈺暄
Hsu, Yu-Hsuan
論文名稱: 以果蠅模式研究皰疹病毒出核蛋白BFRF1減緩蛋白質包涵體毒性之機制
Herpesvirus nucleoprotein BFRF1 in Drosophila melanogaster models to elucidate mechanisms mitigating protein aggregation toxicity
指導教授: 蘇銘燦
Su, Ming-Tsan
口試委員: 蘇銘燦
Su, Ming-Tsan
林炎壽
Lin, Yen-Shou
李重霈
Lee, Chung-Pei
口試日期: 2024/07/30
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2024
畢業學年度: 112
語文別: 英文
論文頁數: 51
中文關鍵詞: 第三型脊髓小腦共濟失調多麩醯胺酸Atxn3自噬作用BFRF1
英文關鍵詞: Spinocerebellar Ataxia Type 3 (SCA3), Polyglutamine, Atxn3, Autophagy, BFRF1
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202401410
論文種類: 學術論文
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  • 常見多麩醯胺酸異常擴增蛋白質引起的神經退化性疾病,有脊髓小腦共濟失調症、亨丁頓舞蹈症、齒狀紅核蒼白球萎縮症和脊髓延髓性肌肉萎縮症,這些神經退化性疾病的共同病理特徵,為神經細胞中多麩醯胺酸擴增蛋白所形成包涵體的累積,不正常蛋白折疊所形成包涵體的角色,是為疾病的成因或結果尚有爭議,但許多研究發現減少錯誤折疊蛋白質的合成,促進清除蛋白質聚集體,或降低蛋白質聚集體所產生的毒性,皆可有效減緩這類神經退化性疾病。先前研究發現,人類疱疹病毒第四型的 BFRF1 蛋白可誘導的囊泡形成,選擇性地吞噬細胞核內蛋白包涵體,促進神經母細胞瘤細胞核中突變 Huntingtin (Htt)蛋白包涵體的運送和清除。為了進一步了解 BFRF1 蛋白質能否在動物體中有類似效果,本研究利用第三型脊髓小腦共濟失調果蠅模式,測試BFRF1蛋白是否可減緩多麩醯胺酸擴增包涵體的蛋白毒性。我們發現過量表現BFRF1蛋白質,本身並不影響果蠅複眼發育,而 Atxn3 蛋白質所引起的果蠅視網膜神徑細胞退化,可因BFRF1蛋白質表現而緩解,BFRF1 蛋白質也可減少果蠅中可溶性和不溶性的 Atxn3 蛋白質。儘管壽命沒改善,但 BFRF1 蛋白質可增加第三型脊髓小腦共濟失調果蠅模式的運動功能。這些結果說明 BFRF1 蛋白質能在果蠅中抑制突變 Atxn3 蛋白的毒性,其作用可能藉誘導囊泡形成,透過細胞自噬途徑,清除選吞噬蛋白質包涵體。

    Spinocerebellar Ataxias (SCAs), Huntington's Disease (HD), Spinal and Bulbar Muscular Atrophy (SBMA), and Dentatorubral Pallidoluysian atrophy (DRPLA) are a group of neurodegenerative diseases caused by the aberrant formation and accumulation of the misfolded polyglutamine (polyQ) containing proteins in the neurons. The common pathological hallmark of these neurodegenerations is characterized by the presence of the polyQ-expanded inclusion body. The role of the inclusion body in the disease is inconclusive, many studies have shown that intervening biosynthesis, resolution, and clearance of the misfolded proteins can alleviate the progression of the neuronal disorders. Previous studies have found that the BFRF1 protein of the Epstein-Barr virus (EBV) promotes the translocation and clearance of nuclear mutant Huntingtin (Htt) inclusions in neuroblastoma cells. To investigate whether BFRF1 exhibits a similar effect in animals, BFRF1 was overexpressed in a Drosophila model of SCA3. We found that expression of BFRF1 did not cause damage to the development of Drosophila compound eyes. The retinal degeneration induced by polyQ-expanded Ataxin 3 (Atxn3) proteins was alleviated in flies overexpressing BFRF1. Overexpression of BFRF1 also reduced both soluble and insoluble Atxn3 proteins in flies. Although lifespan extension in SCA3 flies was not observed, overexpression of BFRF1 enhanced motor function in flies. These results suggest that BFRF1 could suppress the toxicity mutant Atxn3 in flies. It is plausible that BFRF1-containing vesicles engulf the protein aggregates and selectively fuse with autophagosomes as seen in neuroblasma cells. Our study indicates that the beneficial effects of BFRF1 in SCA3 flies could be mediated through autophagy.

    Acknowledgement i 中文摘要 ii Abstract iii Introduction 1 Materials and methods 9 Fly Stocks 9 Morphology 10 Fluorescence quantification 11 Lifespan analysis 13 Locomotor activity 14 Protein extraction 14 Immunoblotting 15 Results 17 Overexpression of BFRF1 in the Drosophila model of SCA3 17 RNA interference of bchs 21 Effects of BFRF1 in Drosophila models of AD 23 Discussion 26 References 29 Appendix 36 PSEBV BFRF1 (AJ507799) DNA sequence (1008 bp) 36 Protein sequence (M. W. 37.6 kd) 37 Expression of BFRF1 in Drosophila; Cloning in pBID-UASC vector for transgenesis 37 Primers for cloning: order from mdbio 38 Figures 39

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