铜离子在朊蛋白聚集过程及其致病机理中所发挥作用的研究
发布时间：2018-07-18 08:18
【摘要】： 朊蛋白是一类能侵染动物并在宿主细胞内复制的无免疫性疏水蛋白质。目前对于朊病毒疾病比较公认的致病机制是正常的富含α螺旋细胞型朊蛋白(PrPc)构象发生转变,形成富含β折叠片层结构并具有蛋白酶抗性的感染型朊蛋白(PrPSc)聚集。 许多证据证明大脑中淀粉样蛋白沉积与朊病毒疾病有关。有研究已经利用重组的PrP得到淀粉样纤维。除了淀粉样纤维聚集外,天然细胞型PrPc还能转变成为一种稳定的富含β折叠结构的寡聚体形态,并且这种来自于PrPSc的非纤维状的寡聚体具有较强的毒性。近年的证据表明可溶性寡聚体在神经退行性疾病中造成细胞功能的损伤。 已有的实验表明PrP能够特异地和铜离子结合并在体内可能作为一种铜离子结合蛋白存在。其中四个组氨酸残基结合位点位于60到91位的氨基酸残基区域,而最近的研究表明第96和111位的组氨酸残基也在PrP与铜离子结合过程中起到重要作用。证据表明铜离子能够调节朊病毒疾病的病理过程。 我们的研究结果发现铜离子是PrP寡聚过程中的关键因子。圆二色谱以及外源荧光实验结果揭示在弱酸性条件下铜离子促进的了PrP向富含β折叠的结构发生转变,而在偏中性条件下铜离子促进PrP形成无定型沉淀。通过分子筛色谱分离得到了PrP可溶性寡聚体并且利用原子力显微镜观察了寡聚体的大小及形态。寡聚体的平均直径为39±7nm。MTT实验和流式细胞术显示PrP的可溶性寡聚体对神经瘤母细胞SK-N-SH具有较强毒性并能诱导使其发生凋亡。荧光共聚焦显微镜显示PrP的可溶性寡聚体能够导致细胞内生的PrP发生聚集并转运到溶酶体中,并可能由此引发细胞凋亡信号。这些结果证明在生理酸性环境中,铜离子能够促进PrP形成具细胞毒性的可溶性寡聚体,并诱使神经细胞发生凋亡。 此外,铜离子还对PrP淀粉样纤维的形成起到调节作用。在接近生理的温和实验条件下,铜离子抑制了PrP淀粉样纤维的形成。光散射实验表明在铜离子参与情况下,PrP能够更快的形成较大颗粒的聚集。而ThT实验显示只有在pH7.0的中性环境中没有铜离子参与的条件下,PrP能够形成淀粉样纤维。铜离子的存在抑制了该条件下PrP纤维的形成。而在弱酸性条件下ThT荧光没有升高,表明没有淀粉样纤维形成。利用原子力显微镜观察了PrP纤维样聚集,其直径大约15nm,长度为300nm左右。 通过以上的研究,我们证明铜离子在PrP的聚集过程中起到重要作用。一方面铜离子能够促进PrP形成神经毒性的可溶性寡聚体,另一方面铜离子又能抑制体外典型PrP淀粉样纤维的形成。我们的研究结果提示铜离子可能在朊病毒疾病不同的病理过程中发挥不同的作用,并且为研究朊病毒疾病以及其它蛋白质构象病的发病机理提供有益的思路。
[Abstract]:Prion proteins are non-immune hydrophobic proteins that infect animals and replicate in host cells. At present, the commonly accepted pathogenetic mechanism for prion diseases is the conformation transformation of 伪 -rich helical cell-type prion protein (PrPc), which forms an infective prion protein (PrPSc) aggregation with 尾 -fold lamellar structure and protease resistance. There is much evidence that amyloid deposition in the brain is associated with prion disease. Studies have been conducted to obtain amyloid fibers using recombinant PrP. In addition to the aggregation of amyloid fibers, the natural cell-type PrPc can be transformed into a stable 尾 -folded structure rich in oligomer morphology, and this non-fibrous oligomer from PrPSc is highly toxic. Recent evidence suggests that soluble oligomers can damage cell function in neurodegenerative diseases. It has been shown that PrP can specifically bind to copper ion and may exist as a copper ion binding protein in vivo. Four of the histidine residues are located at amino acid residues from 60 to 91, and recent studies have shown that the 96 and 111 histidine residues also play an important role in the binding of PrP to copper ions. Evidence suggests that copper ions regulate the pathological process of prion disease. Our results show that copper ion is a key factor in the process of PrP oligomerization. The results of circular dichroism and fluorescence experiments showed that copper ions promoted the transition of PrP to 尾 -rich folding structures under weak acid conditions, while copper ions promoted the formation of amorphous precipitates under neutral conditions. PrP soluble oligomers were separated by molecular sieve chromatography and the size and morphology of the oligomers were observed by atomic force microscope. The average diameter of oligodeoxynucleotides was 39 卤7nm.MTT and flow cytometry showed that PrP soluble oligomer was highly toxic to SK-N-SH and could induce apoptosis of neuroblastoma cells. Fluorescence confocal microscopy showed that the soluble oligomer of PrP could cause intracellular PrP aggregation and transport into lysosome, which might induce apoptosis signal. These results suggest that copper ions can promote PrP to form cytotoxic soluble oligomers and induce neuronal apoptosis in physiological acidic environment. In addition, copper ions also play a regulatory role in the formation of PrP amyloid fibers. Copper ions inhibited the formation of PrP amyloid fibers under mild experimental conditions. Light scattering experiments show that PrP can form larger particles faster in the presence of copper ions. ThT experiments showed that PrP could form amyloid fibers only when copper ions were not involved in pH 7.0 neutral environment. The presence of copper ions inhibited the formation of PrP fibers under these conditions. However, ThT fluorescence did not increase under weak acid condition, indicating that no amyloid fibers were formed. Atomic force microscopy (AFM) was used to observe the aggregation of PrP fibers with a diameter of about 15 nm and a length of about 300nm. Through the above studies, we prove that copper ions play an important role in the aggregation of PrP. On the one hand, copper ions can promote the formation of neurotoxic soluble oligomers of PrP, on the other hand, copper ions can inhibit the formation of typical PrP amyloid fibers in vitro. Our results suggest that copper ions may play different roles in different pathological processes of prion diseases and provide useful ideas for studying the pathogenesis of prion diseases and other protein conformation diseases.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363
本文编号：2131304
[Abstract]:Prion proteins are non-immune hydrophobic proteins that infect animals and replicate in host cells. At present, the commonly accepted pathogenetic mechanism for prion diseases is the conformation transformation of 伪 -rich helical cell-type prion protein (PrPc), which forms an infective prion protein (PrPSc) aggregation with 尾 -fold lamellar structure and protease resistance. There is much evidence that amyloid deposition in the brain is associated with prion disease. Studies have been conducted to obtain amyloid fibers using recombinant PrP. In addition to the aggregation of amyloid fibers, the natural cell-type PrPc can be transformed into a stable 尾 -folded structure rich in oligomer morphology, and this non-fibrous oligomer from PrPSc is highly toxic. Recent evidence suggests that soluble oligomers can damage cell function in neurodegenerative diseases. It has been shown that PrP can specifically bind to copper ion and may exist as a copper ion binding protein in vivo. Four of the histidine residues are located at amino acid residues from 60 to 91, and recent studies have shown that the 96 and 111 histidine residues also play an important role in the binding of PrP to copper ions. Evidence suggests that copper ions regulate the pathological process of prion disease. Our results show that copper ion is a key factor in the process of PrP oligomerization. The results of circular dichroism and fluorescence experiments showed that copper ions promoted the transition of PrP to 尾 -rich folding structures under weak acid conditions, while copper ions promoted the formation of amorphous precipitates under neutral conditions. PrP soluble oligomers were separated by molecular sieve chromatography and the size and morphology of the oligomers were observed by atomic force microscope. The average diameter of oligodeoxynucleotides was 39 卤7nm.MTT and flow cytometry showed that PrP soluble oligomer was highly toxic to SK-N-SH and could induce apoptosis of neuroblastoma cells. Fluorescence confocal microscopy showed that the soluble oligomer of PrP could cause intracellular PrP aggregation and transport into lysosome, which might induce apoptosis signal. These results suggest that copper ions can promote PrP to form cytotoxic soluble oligomers and induce neuronal apoptosis in physiological acidic environment. In addition, copper ions also play a regulatory role in the formation of PrP amyloid fibers. Copper ions inhibited the formation of PrP amyloid fibers under mild experimental conditions. Light scattering experiments show that PrP can form larger particles faster in the presence of copper ions. ThT experiments showed that PrP could form amyloid fibers only when copper ions were not involved in pH 7.0 neutral environment. The presence of copper ions inhibited the formation of PrP fibers under these conditions. However, ThT fluorescence did not increase under weak acid condition, indicating that no amyloid fibers were formed. Atomic force microscopy (AFM) was used to observe the aggregation of PrP fibers with a diameter of about 15 nm and a length of about 300nm. Through the above studies, we prove that copper ions play an important role in the aggregation of PrP. On the one hand, copper ions can promote the formation of neurotoxic soluble oligomers of PrP, on the other hand, copper ions can inhibit the formation of typical PrP amyloid fibers in vitro. Our results suggest that copper ions may play different roles in different pathological processes of prion diseases and provide useful ideas for studying the pathogenesis of prion diseases and other protein conformation diseases.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363
【共引文献】
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