Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21583
標題: 人類脂蛋白朊E3及E4第158號精胺酸對蛋白質穩定性及功能之影響
Protein stability and functional variation in human apolipoprotein E3 and E4 Arginine 158
作者: 蘇國良
Su, Kuo-Liang
關鍵字: apolipoprotein E
脂蛋白朊
Alzheimer's disease
analytical ultracentrifugation
fluorescence
阿滋海默症
分析級超高速離心機
螢光
出版社: 生命科學系
摘要: 人類脂蛋白朊(ApoE)為分子量約34-kDa的蛋白質,由299個胺基酸組成,最主要功能是運送脂質與調節脂蛋白代謝,亦有維持及修補神經元作用。ApoE在一般人群中有三種常見的異構型ApoE2、ApoE3與ApoE4;這三種異構型的差異性,只有兩個胺基酸位置上不同,ApoE2的112號和158號胺基酸都是半胱胺酸,ApoE3的112號胺基酸是半胱胺酸而第158號胺基酸是精胺酸,ApoE4的112號和158號胺基酸都是精胺酸,其中ApoE4為阿滋海默症重要危險因子。本研究對ApoE3與ApoE4,以及ApoE3突變種ApoE3-R158A與ApoE4突變種ApoE4-R158A及ApoE4-R158E進行自由基耐受性試驗,結果發現ApoE4對自由基耐受性確實較ApoE3差,ApoE3突變種較ApoE3對自由基耐受性降低,而兩個ApoE4突變種均比ApoE4對自由基耐受性增加,證實158號精胺酸對ApoE3與ApoE4的穩定性扮演重要角色;接著進行ApoE蛋白質變性試驗,利用螢光分析三級結構變化,發現ApoE3在變性過程中具三種型態,ApoE4為兩種型態,顯示三級結構確實不同,ApoE3突變種仍維持三種型態,但穩定性略為降低,而兩個ApoE4突變種皆轉變成三種型態,顯示三級結構有很大改變;利用分析級超高速離心機的沉降速度實驗,以及連續性大小分布分析,發現ApoE3及ApoE3突變種與ApoE4分布情形相似,而ApoE4-R158A趨向大分子分布,ApoE4-R158E則是二元體的比例增加;接著觀察脂質結合能力的差異,進行ApoE蛋白質對二肉荳蔻磷脂醯膽濁度清除實驗,發現ApoE3清除能力較ApoE4略為好一些,ApoE3突變種與ApoE3清除能力差不多,而兩個ApoE4突變種起始的清除能力很強,但ApoE4-R158A在300分鐘以後,而ApoE4-R158E在660分鐘以後幾乎喪失清除能力,顯示ApoE4的158號精胺酸突變後對脂質結合能力確實有影響。目前ApoE4與形成阿滋海默症的病因一直未確定,希望藉由本實驗結果,將來也許可對ApoE蛋白質與阿滋海默症相關性的研究多一分瞭解。
Human apolipoprotein E(ApoE), a 34-kDa protein composed of 299 amino acids, plays an important role in lipid transport and plasma lipoprotein metabolism and is involved in the maintenance and repair of neurons. ApoE exists as one of three common isoforms, ApoE2, ApoE3 or ApoE4. These isoforms differ by cysteine and arginine at positions 112 and 158; ApoE3 contains cysteine and arginine at these positions, respectively. ApoE2 contains cysteine, and ApoE4 contains arginine at both positions. ApoE4 is a major risk factor for Alzheimer's disease (AD). In this thesis, we aimed to investigate the differential degradation and stability of ApoE3, ApoE4, ApoE3 mutant, ApoE3-R158A and two apoE4 mutants, ApoE4-R158A and ApoE4-R158E. Our data indicated that ApoE4 is more susceptible to free radical than ApoE3. ApoE3-R158A is also more sensitive to free radicals than ApoE3. Furthermore, apoE4 is more susceptible to free radicals than the two mutants, ApoE4-R158A and ApoE4-R158E, indicating that Arg158 plays an important rule in the stability of ApoE3 and ApoE4. The denaturation curves of showed ApoE3 an apparent three-state, and ApoE4 displayed a two-state in guanidine-HCl denaturation monitored by fluorescence. ApoE3 mutant was also remained three-state, but its stability was slightly lower than ApoE3. The denaturations of two ApoE4 mutants were from two-state to three-state, suggesting the very different tertiary structure after mutation of Arg158. Sedimentation velocity and continuous size distribution analysis using analytical ultracentrifugation exhibits that ApoE3, ApoE3 mutant and ApoE4 have very similar patterns. ApoE4-R158A had larger size distribution and ApoE4-R158E increases their trimers. In order to evaluate the effects of various ApoE proteins on the lipid-binding abilities, dimyristoylphosphatidycholine (DMPC) turbidity clearance assay is used. We found that ApoE3 has slightly better lipid-clearing abilities than ApoE4. ApoE3 mutant is the same as ApoE3. Two ApoE4 mutants have fine lipid-clearing abilities in the initial stage. After 300 minutes of ApoE4-R158A, and 660 minutes of ApoE4-R158E almost lost this abilities. These results indicated that Arg158 of ApoE4 was related with lipid-binding abilities. Although the rule of ApoE in the pathogenesis of AD is not entirely clear, we hope these results may contribute the understanding to how ApoE interacts with AD In the future.
URI: http://hdl.handle.net/11455/21583
Appears in Collections:生命科學系所

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