銅離子在朊蛋白聚集過程及其致病機(jī)理中所發(fā)揮作用的研究
發(fā)布時(shí)間：2018-07-18 08:18
【摘要】： 朊蛋白是一類能侵染動物并在宿主細(xì)胞內(nèi)復(fù)制的無免疫性疏水蛋白質(zhì)。目前對于朊病毒疾病比較公認(rèn)的致病機(jī)制是正常的富含α螺旋細(xì)胞型朊蛋白(PrPc)構(gòu)象發(fā)生轉(zhuǎn)變,形成富含β折疊片層結(jié)構(gòu)并具有蛋白酶抗性的感染型朊蛋白(PrPSc)聚集。 許多證據(jù)證明大腦中淀粉樣蛋白沉積與朊病毒疾病有關(guān)。有研究已經(jīng)利用重組的PrP得到淀粉樣纖維。除了淀粉樣纖維聚集外,天然細(xì)胞型PrPc還能轉(zhuǎn)變成為一種穩(wěn)定的富含β折疊結(jié)構(gòu)的寡聚體形態(tài),并且這種來自于PrPSc的非纖維狀的寡聚體具有較強(qiáng)的毒性。近年的證據(jù)表明可溶性寡聚體在神經(jīng)退行性疾病中造成細(xì)胞功能的損傷。 已有的實(shí)驗(yàn)表明PrP能夠特異地和銅離子結(jié)合并在體內(nèi)可能作為一種銅離子結(jié)合蛋白存在。其中四個(gè)組氨酸殘基結(jié)合位點(diǎn)位于60到91位的氨基酸殘基區(qū)域,而最近的研究表明第96和111位的組氨酸殘基也在PrP與銅離子結(jié)合過程中起到重要作用。證據(jù)表明銅離子能夠調(diào)節(jié)朊病毒疾病的病理過程。 我們的研究結(jié)果發(fā)現(xiàn)銅離子是PrP寡聚過程中的關(guān)鍵因子。圓二色譜以及外源熒光實(shí)驗(yàn)結(jié)果揭示在弱酸性條件下銅離子促進(jìn)的了PrP向富含β折疊的結(jié)構(gòu)發(fā)生轉(zhuǎn)變,而在偏中性條件下銅離子促進(jìn)PrP形成無定型沉淀。通過分子篩色譜分離得到了PrP可溶性寡聚體并且利用原子力顯微鏡觀察了寡聚體的大小及形態(tài)。寡聚體的平均直徑為39±7nm。MTT實(shí)驗(yàn)和流式細(xì)胞術(shù)顯示PrP的可溶性寡聚體對神經(jīng)瘤母細(xì)胞SK-N-SH具有較強(qiáng)毒性并能誘導(dǎo)使其發(fā)生凋亡。熒光共聚焦顯微鏡顯示PrP的可溶性寡聚體能夠?qū)е录?xì)胞內(nèi)生的PrP發(fā)生聚集并轉(zhuǎn)運(yùn)到溶酶體中,并可能由此引發(fā)細(xì)胞凋亡信號。這些結(jié)果證明在生理酸性環(huán)境中,銅離子能夠促進(jìn)PrP形成具細(xì)胞毒性的可溶性寡聚體,并誘使神經(jīng)細(xì)胞發(fā)生凋亡。 此外,銅離子還對PrP淀粉樣纖維的形成起到調(diào)節(jié)作用。在接近生理的溫和實(shí)驗(yàn)條件下,銅離子抑制了PrP淀粉樣纖維的形成。光散射實(shí)驗(yàn)表明在銅離子參與情況下,PrP能夠更快的形成較大顆粒的聚集。而ThT實(shí)驗(yàn)顯示只有在pH7.0的中性環(huán)境中沒有銅離子參與的條件下,PrP能夠形成淀粉樣纖維。銅離子的存在抑制了該條件下PrP纖維的形成。而在弱酸性條件下ThT熒光沒有升高,表明沒有淀粉樣纖維形成。利用原子力顯微鏡觀察了PrP纖維樣聚集,其直徑大約15nm,長度為300nm左右。 通過以上的研究,我們證明銅離子在PrP的聚集過程中起到重要作用。一方面銅離子能夠促進(jìn)PrP形成神經(jīng)毒性的可溶性寡聚體,另一方面銅離子又能抑制體外典型PrP淀粉樣纖維的形成。我們的研究結(jié)果提示銅離子可能在朊病毒疾病不同的病理過程中發(fā)揮不同的作用,并且為研究朊病毒疾病以及其它蛋白質(zhì)構(gòu)象病的發(fā)病機(jī)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 偽 -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 鹵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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363
本文編號：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 偽 -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 鹵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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363
【共引文獻(xiàn)】
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