鸡蛋清溶菌酶在淀粉样纤维化过程中的结构变化及其细胞毒性机理
|
摘要
目前有超过20种的人类疾病都与天然多肽转变为富含β折叠的淀粉样纤维有关,如老年痴呆症(Alzheimer's)、帕金森疾病以及二型糖尿病等等,我们把这些疾病定义为淀粉样变性(amyloidosis)疾病。这些疾病是由正常的可溶于血浆的蛋白转变为不溶的淀粉样纤维形态沉积于细胞外造成的,我们把这些纤维样聚集定义为淀粉样纤维(amyloid fibril)。它们几乎可以对所有的器官产生影响,因此具有一定的系统性,这也是临床诊断和治疗面临的一个主要挑战。迄今为止已发现至少有36种蛋白能发生淀粉样变性形成淀粉样沉积,造成心血管系统、神经系统以及泌尿系统损害,引发各种疾病。
从理论上来说,所有的蛋白质或多肽在一定的条件下,都可以自我组装形成淀粉样纤维。实验研究发现,许多蛋白包括一些非致病性的蛋白质和短肽都可以形成纤维。目前研究较多的有溶菌酶、Aβ肽、胰岛素、朊蛋白(prion)、核突触蛋白(synuclein)和微球蛋白等。淀粉样纤维的形成是一个复杂的过程,会产生一系列不同大小和形态的中间体,最后形成包括寡聚体(oligomers)、原纤维(protofilaments)等中间体和成熟纤维(mature fibrils)的淀粉样纤维混合物。它们通过聚集在器官或组织上形成淀粉样沉积,导致器官或组织的去功能化,从而导致相关病症的发生。目前对淀粉样蛋白的纤维化过程、淀粉样纤维的有毒成分及其毒性机理进行了很多研究,但是其确切机制仍然不清楚,仍然存在很多的问题和挑战,特别是在分子水平和蛋白结构方面,因此进一步进行研究是十分必要的。
本文中选用鸡蛋清溶菌酶作为淀粉样蛋白,以人类红细胞作为体外实验的细胞模型,研究了鸡蛋清溶菌酶淀粉样纤维化过程中的结构变化、细胞毒性的变化,淀粉样纤维混合物中各组分的细胞毒性的大小,并对溶菌酶淀粉样纤维造成红细胞损伤的毒性机理进行了研究,以寻找淀粉样纤维诱导细胞毒性的分子途径,为淀粉样疾病的治疗提供理论和实验依据。
主要方法与结论：
1.溶菌酶纤维生长的检测
采用了蛋白荧光标记技术(包括ThT荧光标记,ANS荧光标记)以及圆二色谱技术来检测溶菌酶纤维的生长动力学。结果表明,溶菌酶纤维在生长过程中,蛋白α螺旋减少,β折叠结构逐渐增多,内部的疏水区域也渐渐外露。
2.溶菌酶纤维形态的检测
通过刚果红染色后偏光显微镜观察和透射电子显微镜对淀粉样纤维进行观测。结果表明,孵育成熟的溶菌酶纤维在偏光显微镜下呈现黄绿色,这是淀粉样纤维的一个典型特征。在透射电子显微镜下,成熟的溶菌酶纤维呈纤维状,并相互聚集形成淀粉样沉积,淀粉样沉积是淀粉样疾病的一个主要表征。溶菌酶纤维的小分子SDS-PAGE说明了溶菌酶纤维的孵育制备过程中有少量淀粉样纤维聚集体的产生和部分酸水解产物的存在。
3.溶菌酶纤维的细胞毒性研究
溶菌酶纤维与红细胞共同孵育后,通过光吸收检测上清中血红蛋白的量。结果表明,随着纤维孵育时间的增加,其细胞毒性也增加,从第4天开始达到一个平台期。结合高速离心和超速离心,对成熟纤维的各组分进行了分离,并进行了蛋白含量测定和细胞毒性检测。结果表明,低聚物(寡聚体和原纤维)约占成熟淀粉样纤维混合物的一半,可溶性的低聚物在纤维组分中具有较高的细胞毒性。
4.溶菌酶纤维的细胞毒性机理研究
溶菌酶纤维与红细胞膜共同孵育后,用SDS-PAGE进行分离,结果显示在上样孔和高分子量区域出现了明显的大分子聚集,同时细胞膜蛋白的各个条带变浅,这些大分子是由溶菌酶纤维与膜蛋白共同形成的。接着对蛋白电泳的浓缩胶部分(含上样孔)和高分子量区域进行了Western Blotting免疫印迹检测。其结果清楚地显示了溶菌酶纤维参与了大分子聚集物的形成,这些大分子聚集物主要集中在上样孔和浓缩胶部分。合并质谱解析结果,我们得出结论,溶菌酶淀粉样纤维与膜蛋白通过二硫键交联形成了大分子的聚集物,这些大分子聚集物沉积于红细胞膜上,从而对红细胞造成损伤,引起红细胞的破裂。这表明了二硫键在溶菌酶纤维的细胞毒性中起着关键的作用。
5.溶菌酶纤维化过程中二硫键的外露情况
在本研究中,对天然溶菌酶分子及其淀粉样纤维的表面二硫键进行测定,(1)通过mBBr荧光标记技术进行荧光检测；(2)用DTT把外露的二硫键还原成自由巯基,然后用NEM对自由的巯基进行共价封闭,结合ESI-MS/MS质谱分析二硫键的外露情况。结果表明溶菌酶纤维在孵育过程中的确伴有分子内二硫键的外露,通过质谱分析得出了溶菌酶中8个半胱氨酸外露的难易程度。这些结果进一步证实了二硫键在溶菌酶纤维的细胞毒性中的主导作用。
More than 20 human diseases, such as Alzheimer's disease, Parkinson disease, TypeⅡdiabetes, are associated with the conversion of proteins/polypeptides from their native state into highly ordered and P-sheet-enriched aggregates known as amyloid fibrils. We defined these human diseases as amyloidosis, which result from the extracellular deposition of normal proteins which are dissoluble in plasma convert into insoluble amyloid fibril morphology. This deposition can cause damage to almost of all the organs and tissues, therefore, amyloidosis have systematic characters. Amyloidosis can actually occur anywhere of human body and cause dysfunctions of cardiovascular system, nervous system, and urinary system. Challenges are still remained for scientists to develop novel methods and medications for the diagnosis and treatment of amyloidosis.
Theoritically all of the proteins or peptides are potential to form amyloid fibrils through self-assembly under certain conditions. It has been found that many proteins, including some non-pathogenic proteins and short peptides can form amyloid fibrils. The list of amyloidogenic proteins and peptides includes lysozyme, Aβpeptide, insulin, prion protein, synuclein,β-micro-globulin etc. The formation of amyloid fibrils is a complex process, in which a series of intermediates with different size and morphology can be produced. The mature amyloid fibrils usually include oligomers, protofilaments, long fibrils and some amorphous aggragates. Although many attempts have been made to elucidate the molecular pathways of amyloidogenesis, the exact mechanism is still obscure and remains further explored.
In the present study, hen egg white lysozyme was used as a model protein to produce amyloid fibrils and human erythrocytes were used as an in vitro model to investigate the fibrillar cytotoxicity. Investigations were performed in terms of the changes of molecular conformation and the cytotoxicity of lysozyme during the course of amyloid fibrillation, and the molecular pathway of erythrocyte damage caused by the amyloid fibrils. The results provide a noval insight into the amyloidosis of proteins/peptides both on the theoretical and experimental basis.
Methods, results, and conclusions:
1. Kinetics of lysozyme amyloid fibrillation
The kinetics of lysozyme amyloid fibrillation was monitored by using the fluorescent probes ThT and ANS, and circular dichroism. The results showed that the conformation of lysozyme was changed along with the incubation of the protein, including increases of the content ofβ-sheet structures and the surfacial hydrophobicity, and a decrease of the content of a-helices.
2. The morphology of lysozyme fibrils
The morphologies lysozyme fibrils were observed under a transmission electron microscopy and a polarizing microscopy after the Congo-red stainning. The results showed that lysozyme mature fibril demonstrated yellow-green under polarizing microscopy, which is a typical feature of amyloid fibrils. The transmission electron microscope image showed that the mature lysozyme fibrils were long fibrous structures; together with bundles and aggregates of the fibrillar assemblies. SDS-PAGE in tricine buffer showed that there was a small amount of the protein hydrolyzed during the process of amyloid fibrillation.
3. Lysozyme fibril Cytotoxicity
The cytotoxicity of lysozyme fibrils was investigated in two aspects. In the first assay, human red blood cells were separated from blood and incubated with lysozyme fibrls. Spectrophotometric data showed that hemoglobin was released from the cells upon incubation with the fibrils, indicating that the fibrils induced cell damage. The hemolytical effect of the fibrils increased along with the process of fibrillar maturation. Both the fibril-growth and the fibrllar cytotoxicity reached a plateau on the fourth day of the incubation. The pool of mature fibrils contained long fibrils and their large aggregates, oligomers, protofilaments, and some acid-hydrolyzed products, as demonstrated by high-speed centrifugation and ultracentrifugation. In a comparison with the fibrils and large aggregates, the oligomers and protofilaments showed higher cytotoxicity.
4. Cellullar membrane damages by lysozyme fibrils
In the second assay of the fibrillar cytotoxicity, membranes were separated from the cells and were incubated with lysozyme fibrils prior to SDS-PAGE. The results showed that some macromolecular aggregates appeared on the top of the gel lanes and the sample wells, and the bands of membrane proteins became weaker than the control. Western Blotting and mass spectrometry analyses showed that lysozyme molecules and/or its molecular species were incorporated into the high-molecular aggregates through an intermolecular disulfide cross-linking. This result indicates that the exchange of thiol-disulfide between the fibrilar species and membrane proteins plays a key role in the amyloid-induced cell damages.
5. The exposure of disulfide bonds during amyloid fibrillation
The solvent-accessible disulfide bonds of lysozyme were determined by two methods. The first assay involved in the mBBr fluorescent labeling technology. In another method, the exposed disulfides of lysozyme were reduced into free thiol groups by DTT and then blocked by covalent binding to NEM. The resultant protein was digested and then analyzed by ES1-MS/MS mass spectrometry to identify the labeled thiol groups, by which the solvent-accessible disulfide bonds can be deduced. The data showed that the lysozyme fibrillation was associated with the exposure of internal disulfide bonds to the solvent; therefore, the cytotoxicity of the protein assemblies was increased. This study strongly supports the hypothesis that cellular membrane damage by amyloid fibrils involves intermolecular disulfide cross-linking between lysozyme and membrane proteins.
从理论上来说,所有的蛋白质或多肽在一定的条件下,都可以自我组装形成淀粉样纤维。实验研究发现,许多蛋白包括一些非致病性的蛋白质和短肽都可以形成纤维。目前研究较多的有溶菌酶、Aβ肽、胰岛素、朊蛋白(prion)、核突触蛋白(synuclein)和微球蛋白等。淀粉样纤维的形成是一个复杂的过程,会产生一系列不同大小和形态的中间体,最后形成包括寡聚体(oligomers)、原纤维(protofilaments)等中间体和成熟纤维(mature fibrils)的淀粉样纤维混合物。它们通过聚集在器官或组织上形成淀粉样沉积,导致器官或组织的去功能化,从而导致相关病症的发生。目前对淀粉样蛋白的纤维化过程、淀粉样纤维的有毒成分及其毒性机理进行了很多研究,但是其确切机制仍然不清楚,仍然存在很多的问题和挑战,特别是在分子水平和蛋白结构方面,因此进一步进行研究是十分必要的。
本文中选用鸡蛋清溶菌酶作为淀粉样蛋白,以人类红细胞作为体外实验的细胞模型,研究了鸡蛋清溶菌酶淀粉样纤维化过程中的结构变化、细胞毒性的变化,淀粉样纤维混合物中各组分的细胞毒性的大小,并对溶菌酶淀粉样纤维造成红细胞损伤的毒性机理进行了研究,以寻找淀粉样纤维诱导细胞毒性的分子途径,为淀粉样疾病的治疗提供理论和实验依据。
主要方法与结论：
1.溶菌酶纤维生长的检测
采用了蛋白荧光标记技术(包括ThT荧光标记,ANS荧光标记)以及圆二色谱技术来检测溶菌酶纤维的生长动力学。结果表明,溶菌酶纤维在生长过程中,蛋白α螺旋减少,β折叠结构逐渐增多,内部的疏水区域也渐渐外露。
2.溶菌酶纤维形态的检测
通过刚果红染色后偏光显微镜观察和透射电子显微镜对淀粉样纤维进行观测。结果表明,孵育成熟的溶菌酶纤维在偏光显微镜下呈现黄绿色,这是淀粉样纤维的一个典型特征。在透射电子显微镜下,成熟的溶菌酶纤维呈纤维状,并相互聚集形成淀粉样沉积,淀粉样沉积是淀粉样疾病的一个主要表征。溶菌酶纤维的小分子SDS-PAGE说明了溶菌酶纤维的孵育制备过程中有少量淀粉样纤维聚集体的产生和部分酸水解产物的存在。
3.溶菌酶纤维的细胞毒性研究
溶菌酶纤维与红细胞共同孵育后,通过光吸收检测上清中血红蛋白的量。结果表明,随着纤维孵育时间的增加,其细胞毒性也增加,从第4天开始达到一个平台期。结合高速离心和超速离心,对成熟纤维的各组分进行了分离,并进行了蛋白含量测定和细胞毒性检测。结果表明,低聚物(寡聚体和原纤维)约占成熟淀粉样纤维混合物的一半,可溶性的低聚物在纤维组分中具有较高的细胞毒性。
4.溶菌酶纤维的细胞毒性机理研究
溶菌酶纤维与红细胞膜共同孵育后,用SDS-PAGE进行分离,结果显示在上样孔和高分子量区域出现了明显的大分子聚集,同时细胞膜蛋白的各个条带变浅,这些大分子是由溶菌酶纤维与膜蛋白共同形成的。接着对蛋白电泳的浓缩胶部分(含上样孔)和高分子量区域进行了Western Blotting免疫印迹检测。其结果清楚地显示了溶菌酶纤维参与了大分子聚集物的形成,这些大分子聚集物主要集中在上样孔和浓缩胶部分。合并质谱解析结果,我们得出结论,溶菌酶淀粉样纤维与膜蛋白通过二硫键交联形成了大分子的聚集物,这些大分子聚集物沉积于红细胞膜上,从而对红细胞造成损伤,引起红细胞的破裂。这表明了二硫键在溶菌酶纤维的细胞毒性中起着关键的作用。
5.溶菌酶纤维化过程中二硫键的外露情况
在本研究中,对天然溶菌酶分子及其淀粉样纤维的表面二硫键进行测定,(1)通过mBBr荧光标记技术进行荧光检测；(2)用DTT把外露的二硫键还原成自由巯基,然后用NEM对自由的巯基进行共价封闭,结合ESI-MS/MS质谱分析二硫键的外露情况。结果表明溶菌酶纤维在孵育过程中的确伴有分子内二硫键的外露,通过质谱分析得出了溶菌酶中8个半胱氨酸外露的难易程度。这些结果进一步证实了二硫键在溶菌酶纤维的细胞毒性中的主导作用。
More than 20 human diseases, such as Alzheimer's disease, Parkinson disease, TypeⅡdiabetes, are associated with the conversion of proteins/polypeptides from their native state into highly ordered and P-sheet-enriched aggregates known as amyloid fibrils. We defined these human diseases as amyloidosis, which result from the extracellular deposition of normal proteins which are dissoluble in plasma convert into insoluble amyloid fibril morphology. This deposition can cause damage to almost of all the organs and tissues, therefore, amyloidosis have systematic characters. Amyloidosis can actually occur anywhere of human body and cause dysfunctions of cardiovascular system, nervous system, and urinary system. Challenges are still remained for scientists to develop novel methods and medications for the diagnosis and treatment of amyloidosis.
Theoritically all of the proteins or peptides are potential to form amyloid fibrils through self-assembly under certain conditions. It has been found that many proteins, including some non-pathogenic proteins and short peptides can form amyloid fibrils. The list of amyloidogenic proteins and peptides includes lysozyme, Aβpeptide, insulin, prion protein, synuclein,β-micro-globulin etc. The formation of amyloid fibrils is a complex process, in which a series of intermediates with different size and morphology can be produced. The mature amyloid fibrils usually include oligomers, protofilaments, long fibrils and some amorphous aggragates. Although many attempts have been made to elucidate the molecular pathways of amyloidogenesis, the exact mechanism is still obscure and remains further explored.
In the present study, hen egg white lysozyme was used as a model protein to produce amyloid fibrils and human erythrocytes were used as an in vitro model to investigate the fibrillar cytotoxicity. Investigations were performed in terms of the changes of molecular conformation and the cytotoxicity of lysozyme during the course of amyloid fibrillation, and the molecular pathway of erythrocyte damage caused by the amyloid fibrils. The results provide a noval insight into the amyloidosis of proteins/peptides both on the theoretical and experimental basis.
Methods, results, and conclusions:
1. Kinetics of lysozyme amyloid fibrillation
The kinetics of lysozyme amyloid fibrillation was monitored by using the fluorescent probes ThT and ANS, and circular dichroism. The results showed that the conformation of lysozyme was changed along with the incubation of the protein, including increases of the content ofβ-sheet structures and the surfacial hydrophobicity, and a decrease of the content of a-helices.
2. The morphology of lysozyme fibrils
The morphologies lysozyme fibrils were observed under a transmission electron microscopy and a polarizing microscopy after the Congo-red stainning. The results showed that lysozyme mature fibril demonstrated yellow-green under polarizing microscopy, which is a typical feature of amyloid fibrils. The transmission electron microscope image showed that the mature lysozyme fibrils were long fibrous structures; together with bundles and aggregates of the fibrillar assemblies. SDS-PAGE in tricine buffer showed that there was a small amount of the protein hydrolyzed during the process of amyloid fibrillation.
3. Lysozyme fibril Cytotoxicity
The cytotoxicity of lysozyme fibrils was investigated in two aspects. In the first assay, human red blood cells were separated from blood and incubated with lysozyme fibrls. Spectrophotometric data showed that hemoglobin was released from the cells upon incubation with the fibrils, indicating that the fibrils induced cell damage. The hemolytical effect of the fibrils increased along with the process of fibrillar maturation. Both the fibril-growth and the fibrllar cytotoxicity reached a plateau on the fourth day of the incubation. The pool of mature fibrils contained long fibrils and their large aggregates, oligomers, protofilaments, and some acid-hydrolyzed products, as demonstrated by high-speed centrifugation and ultracentrifugation. In a comparison with the fibrils and large aggregates, the oligomers and protofilaments showed higher cytotoxicity.
4. Cellullar membrane damages by lysozyme fibrils
In the second assay of the fibrillar cytotoxicity, membranes were separated from the cells and were incubated with lysozyme fibrils prior to SDS-PAGE. The results showed that some macromolecular aggregates appeared on the top of the gel lanes and the sample wells, and the bands of membrane proteins became weaker than the control. Western Blotting and mass spectrometry analyses showed that lysozyme molecules and/or its molecular species were incorporated into the high-molecular aggregates through an intermolecular disulfide cross-linking. This result indicates that the exchange of thiol-disulfide between the fibrilar species and membrane proteins plays a key role in the amyloid-induced cell damages.
5. The exposure of disulfide bonds during amyloid fibrillation
The solvent-accessible disulfide bonds of lysozyme were determined by two methods. The first assay involved in the mBBr fluorescent labeling technology. In another method, the exposed disulfides of lysozyme were reduced into free thiol groups by DTT and then blocked by covalent binding to NEM. The resultant protein was digested and then analyzed by ES1-MS/MS mass spectrometry to identify the labeled thiol groups, by which the solvent-accessible disulfide bonds can be deduced. The data showed that the lysozyme fibrillation was associated with the exposure of internal disulfide bonds to the solvent; therefore, the cytotoxicity of the protein assemblies was increased. This study strongly supports the hypothesis that cellular membrane damage by amyloid fibrils involves intermolecular disulfide cross-linking between lysozyme and membrane proteins.
引文
[1]J. D. Sipe, A. S. Cohen. Review:History of the amyloid fibril [J]. Journal of Structural Biology,2000,130:88～98.
[2]R. A. Kyle. Amyloidosis:a convoluted story [J]. British Journal of Haematology, 2001,114:529～538.
[3]J. C. Rochet, P. T. Jr. Lansbury. Amyloid fibrillogenesis:themes and variations [J]. Current Opinion in Structural Biology,2000,10:60～68.
[4]Y. Xing, K.Higuchi. Amyloid fibril proteins [J]. Mechanisms of Ageing and Development,2002,123:1625～1636.
[5]J. Guo, Y. Xu. Towards modeling of amyloid fibril structures [J]. Frontiers in Bioscience,2008,13:4039～4050.
[6]R. Tycko. Progress towards a molecular-level structural understanding of amyloid fibrils [J]. Current Opinion in Structural Biology,2004,14:96～103.
[7]Y. Goto, H. Yagi, K. Yamaguchi, E. Chatani, T. Ban. Structure,formation and propagation of amyloid fibrils [J]. Current Pharmaceutical Design,2008,14(30): 3205～3218.
[8]O. S. Makin, L. C. Serpell. Structures for amyloid fibrils [J]. FEBS Journal,2005, 272:5950～5961.
[9]S. Kasapis, I. T. Norton, J. B. Ubbink. Modern Biopolymer Science:bridging the divide between fundamental treatise and industrial application [M]. Academic Press,2009, CHAPTER 17:559～594.
[10]C. M. Dobson. Principles of protein folding, misfolding, and aggregation [J]. Seminars in Cell and Developmental Biology,2004,15:3～16.
[11]A. E. Langkilde, B. Vestergaard. Methods for structural characterization of prefibrillar intermediates and amyloid fibrils [J]. FEBS Letters,2009,583: 2600～2609.
[12]M. R. Nilsson. Techniques to study amyloid fibril formation in vitro [J]. Methods, 2004, 34:151～160.
[13]P. Hammarstrom, X. Jiang, A. R. Hurshman, E. T. Powers, J. W. Kelly. Sequence dependent denaturation energetics:A major determinant in amyloid disease diversity [J]. Proceedings of the National Academy of Sciences of the United States of America,2002,99(suppl.4):16427～16432.
[14]J. C. Rochet, P. T. Lansbury. Amyloid fibrillogenesis:themes and variations [J]. Current Opinion in Structural Biology,2000,10:60～68.
[15]C.M. Dobson. The structural basis of protein folding and its links with human disease [J]. Philosophical Transactions of the Royal Society B:Biological Sciences,2001,356:133～145.
[16]J. C. Rochet, P. T. Jr. Lansbury. Amyloid fibrillogenesis:themes and variations [J]. Current Opinion in Structural Biology,2000,10:60～68.
[17]R. Kayed, E. Head, J. L. Thompson, T. M. McIntire, S. C. Milton, C. W. Cotman, C. G. Glabe. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis [J]. Science,2003, Apr 18:486～489.
[18]E. T. Powers, D. L. Powers. The kinetics of nucleated polymerizations at high concentrations:amyloid fibril formation near and above the"supercritical concentration" [J]. Biophysical Journal,2006,91:122～132.
[19]S. Jain, J. B. Udgaonkar. Evidence for step wise formation of amyloid fibrils by the mouse prion protein [J]. Journal of Molecular Biology,2008,382:1228～1241.
[20]C. C. Lee, A. Nayak, A. Sethuraman, G. Belfort, G. J. McRae. A three-stage kinetic model of amyloid fibrillation [J]. Biophysical Journal,2007,92: 3448～3458.
[21]V. N. Uversky. Amyloidogenesis of natively unfolded proteins [J]. Current Alzheimer Research,2008, June 5(3):260～287.
[22]N. Phuong Humblet-Hua, L. M. Sagis, E. van der Linden. Effects of flow on Hen Egg White Lysozyme (HEWL) fibril formation:length distribution, flexibility, and kinetics [J]. Journal Agricultural and Food Chemistry,2008,56:11875～11882.
[23]P. Shahi, R. Sharma, S. Sanger, I. Kumar, R. S. Jolly. Formation of amyloid fibrils via longitudinal growth of oligomers [J]. Biochemistry,2007,46:7365～7373.
[24]H. J. Lachmann, P. N. Hawkins. Systemic amyloidosis [J]. Current Opinion in Pharmacology,2006,6:214～220.
[25]K. H. Mok, J. Pettersson, S. Orrenius, C. Svanborg. HAMLET, protein folding, and tumor cell death [J]. Biochemical and Biophysical Research Communications, 2007,354:1～7.
[26]J. P. Kastberg, S. Aits, L. Gustafsson, A. Mossberg, P. Storm, M. Trulsson, F. Persson, K. H. Mok, C. Svanborg. Can misfolded proteins be beneficial? The HAMLET case [J]. Annals of Medicine,2009,41:162～176.
[27]曾彩虹,刘志红.遗传性淀粉样变性[J].肾脏病与透析肾移植杂志,2007,6(1): 70～75.
[28]陈艳,江明锋,叶煜辉,刘勇涛,李生伟.溶菌酶的研究进展[J].生物学杂志,2009,26(2)：64～66.
[29]G. Merlini, V. Bellotti. Lysozyme:A paradigmatic molecule for the investigation of protein structure, function and misfolding [J]. Clinica Chimica Acta,2005, 357:168～172.
[30]E. Frare, P. Polverino de Laureto, J. Zurdo, C. M. Dobson, A. Fontana. A highly amyloidogenic region of hen Lysozyme [J]. Journal of Molecular Biology,2004, 340:1153～1165.
[31]S. S. Wang, S. W. Chou, K. N. Liu, C. H. Wu. Effects of glutathione on amyloid fibrillation of hen egg-white lysozyme [J]. International Journal of Biological Macromolecules,2009,45:321～329.
[32]A. Cao, D. Hu, L. Lai. Formation of amyloid fibrils from fully reduced hen egg white lysozyme [J]. Protein Science,2004,13 (2):319～324.
[33]R. Mishra, K. Sorgjerd, S. Nystrom, A. M. Nordigarden, Y. C. Yu, P. Hammarstrom. Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion [J]. Journal of Molecular Biology,2007,366(3):1029～1044.
[34]卢义钦,刘俊凡.红细胞膜蛋白与膜骨架[J].生命科学研究,2005,9(4)：283～291.
[35]M. E. Reid, N. Mohandas. Red blood cell blood group antigens:structure and function [J]. Seminars in Hematology,2004,4:93～117.
[36]D. J. Anstee. Blood group antigens defined by the amino acid sequences of red cell surface proteins [J]. Transfusion Medicine,1995,5:1～13.
[37]M. J. Telen.Erythrocyte blood group antigens:not so simple after all [J]. Blood. 1995,85:299～306.
[38]J. P. Cartron, Y. Colin. Structural and functional diversity of blood group antigens [J]. Transfusion Clinique et Biologique,2001,8:163～199.
[39]V. Nicolas, C. Le Van Kim, P. Gane, C. Birkenmeier, J. P. Cartron, Y. Colin, I. M. Chanteloup. Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation [J]. The Journal of Biological Chemistry,2003,278:25526～25533.
[40]M. Salomao, X. Zhang, Y. Yang, S. Lee, H. John. Hartwig, Joel Anne Chasis, Narla Mohandas, and Xiuli An.Protein 4.1R-dependent multiprotein complex: new insights into the structural organization of the red blood cell membrane [J]. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(23):8026～8031.
[41]N. Mohandas, P. G. Gallagher. Red cell membrane:past, present, and future [J]. Blood,2008,112(10):9329～3948.
[42]M. Stefani, C.M. Dobson. Protein aggregation and aggregate toxicity:new insight into protein folding, misfolding diseases and biological evolution [J]. Journal of Molecular Medicine.2003,81:678～699.
[43]M. Stefani. Protein misfolding and aggregation:new examples in medicine and biology of the dark side of the protein world [J]. Biochimica et Biophysica Acta, 2004,1739:5～25.
[44]J. P. Cleary, D. M. Walsh, J. J. Hofmeister, G. M. Shankar, M. A. Kuskowski, D. J. Selkoe, K. H. Ashe. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function [J]. Nature Neuroscience,2005,8:79～84.
[45]M. Hoshi, M. Sato, S. Matsumoto, A. Noguchi, K. Yasutake, N. Yoshida, K. Sato. Spherical aggregates of {beta}-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase～3{beta} [J]. Proceedings of the National Academy of Sciences of the United States of America, 2003,100:6370～6375.
[46]H. Mukai, T. Isagawa, E. Goyama, S. Tanaka, N. F. Bence, A. Tamura, Y. Ono, R. R. Kopito. Formation of morphologically similar globular aggregates from diverse aggregation prone proteins in mammalian cells [J]. Proceedings of the National Academy of Sciences of the United States of America,2005,102: 10887～10892.
[47]V. Novitskaya, O. V. Bocharova, I. Bronstein, I. V. Baskakov. Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons [J]. Journal of Biological Chemistry,2006,281:13828～13836.
[48]L. Pieri, M. Bucciantini, D. Nosi, L. Formigli, J. Savistchenko, R. Melki, M. Stefani. The yeast prion Ure2p native-like assemblies are toxic to mammalian cells regardless to their aggregation state [J]. Journal of Biological Chemistry, 2006,281:15337～15344.
[49]I. Sirangelo, C. Malmo, C. Iannuzzi, A. Mezzogiorno, M. R. Bianco, M. Papa, G. Irace. Fibrillogenesis and cytotoxic activity of the amyloidforming apomyoglobin mutant W7FW14F [J]. Journal of Biological Chemistry,2004,279:13183～13189.
[50]M. Bucciantini, S. Rigacci, A. Berti, L. Pieri, C. Cecchi, D. Nosi, L. Formigli, F Chiti, M. Stefani. Patterns of cell death triggered in two different cell lines by HypF-N prefibrillar aggregates [J]. FASEB Journal,2005,19:437～439.
[51]S. Grudzielanek, A. Velkova, A. Shukla, V. Smirnovas, M. TatarekNosso, H. Rehage, A. Kapurniotu, R. Winter. Cytotoxicity of insulin within its self-assembly and amyloidogenic pathways [J]. Journal of Molecular Biology,2007,370: 372～384.
[52]M. M. Sousa, I. Cardoso, R. Fernandes, A. Guimaraes, M. J. Saraiva. Deposition of transthyretin in early stages of familial amyloidotic polyneuropathy:Evidence for toxicity of non-fibrillar aggregates [J]. American Journal of Pathology,2001, 159:1993～2000.
[53]R. Bader, R. Bamford, J. Zurdo, B. F. Luisi, C. Dobson. Probing the Mechanism of amyloldogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation [J]. Journal of Molecular Biology,2006,356:189～208.
[54]M. Bucciantini, G. Calloni, F. Chiti, L. Formigli, D. Nosi, C. M. Dobson, M. Stefani. Prefibrillar amyloid protein aggregates share common features of cytotoxicity [J]. Journal of Biological Chemistry,2004,279:31374～31382.
[55]M. T. Pastor, N. Kummerer, V. Schubert, A. E. Chopo, C G. Dotti, M. Lopez de la Paz, L. Serrano. Amyloid toxicity is independent of polypeptide sequence, length and chirality [J]. Journal of Molecular Biology,2008,375:695～707.
[56]P. Cizas, R. Budvytyte, R. Morkuniene, R. Moldovan, M. Broccio, M. Losche, G. Niaura, G. Valinciu, V. Borutaite. Size-dependent neurotoxicity of β-amyloid oligomers [J]. Archives of Biochemistry and Biophysics, Available online 11 February 2010.
[57]N. Gregersen, L. Bolund, P. Bross. Protein misfolding, aggregation, and degradation in disease [J]. Methods in Molecular Biology,2003,232:3～16.
[58]M. Bucciantini, E. Giannoni, F. Chiti, F. Baroni, L. Formigli, J. S. Zurdo, N. Taddie, G. Ramponi, C. M. Dobson, M. Stefani. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases [J]. Nature,2002, 416:507～511.
[59]G. P. Gorbenko, P. K. Kinnunen. The role of lipid-protein interactions in amyloid-type protein fibril formation [J]. Chemistry and Physics of Lipids,2006, 141:72～82.
[60]D. Hall, H. Edskes. A model of amyloid's role in disease based on fibril fracture [J]. Biophysical Chemistry,2009,145:17～28.
[61]B. L. Kagan, Y. Hirakura, R. Azimov, R. Azimova, M. C. Lin. The channel hypothesis of Alzheimer's disease:Current status [J]. Peptides,2002,23: 1311～1315.
[62]R. Kayed, Y. Sokolov, B. Edmonds, T. M. Mclntire, S. C. Milton, J. E. Hall, C. G. Glabe. Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases [J]. Journal of Biological Chemistry,2004,279:6363～6366.
[63]M. E. Bamberger, M. E. Harris, D. R. McDonald, J. Husemann, G. E. Landreth. A cell surface receptor complex for fibrillar β-amyloid mediates microglial activation [J]. Journal of Neuroscience,2003,23:2665～2674.
[64]E. Sparr, M. F. Engel, D. V. Sakharov, M. Sprong, J.Jacobs, B. de Kruijff, J. W. Hoppener, J. A. Killian. Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers [J]. FEBS Letters,2004,577: 117～120.
[65]J. R. Brender, U. H. N. Durr, D. Heyl, M. B. Budarapu, A. Ramamoorthy. Membrane fragmentation by an amyloidogenic fragment of human Islet Amyloid Polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes [J]. Biochimica et Biophysica Acta,2007,1768:2026～2029.
[66]D. G. Smith, R. Cappai, K. J. Barnham. The redox chemistry of the Alzheimer's disease amyloid P peptide [J]. Biochimica et Biophysica Acta,2007,1768: 1976～1990.
[67]D. A. Butterfield, C. M. Lauderback. Lipid peroxidation and protein oxidation in Alzheimer's disease brain:Potential causes and consequences involving amyloid β-peptide-associated free radical oxidative stress [J]. Free Radical Biology & Medicine,2002,32:1050～1060.
[68]P. H. Reddy. Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease [J]. Experimental Neurology,2009,218:286～292.
[69]B. Su,X. Wang, A. Nunomura, P. I. Moreira, H. G. Lee, G. Perry, M. A. Smith, X. Zhu. Oxidative stress signaling in Alzheimer's disease [J]. Current Alzheimer Researsh,2008,5:525～532.
[70]D. Allsop, J. Mayes, S. Moore, A. Masad, B. J. Tabner. Metal-dependent generati-on of reactive oxygen species from amyloid proteins implicated in neurode-generative disease [J]. Biochemical Society Transaction,2008,36:1293～1298.
[71]D. A. Butterfield, R. Sultana. Redox proteomics identification of oxidative-lymodified brain proteins in Alzheimer's disease and mild cognitive impairment: Insights into the progression of this dementing disorder [J]. Journal of Alzheimer's Disease,2007,12:61～72.
[72]A. A. Koksoy. A unifying hypothesis for explaining the mechanism of amyloid formation under conditions of increased oxidative stress [J]. Bioscience Hypotheses,2008,1:209～212.
[73]I. V. Murray, M. E. Sindoni, P. H. Axelsen. Promotion of oxidative lipid membrane damage by amyloid β proteins [J]. Biochemistry,2005,44: 12606～12613.
[74]B. I. Giasson, H. Ischiropoulos, V. M. Lee, J. Q. Trojanowski. The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer's and Parkinson's diseases.Free Radical [J]. Free Radical Biology & Medicine,2002,32: 1264～1275.
[75]L. N. Arnaudov, R. De Vries… Thermally induced fibrillar aggregation of hen egg white lysozyme [J]. Biophysical Journal,2005,88:515～526.
[76]A.Harada, H. Azakami, A. Kato. Amyloid fibril formation of hen Lysozyme depends on the instability of the C-Helix (88-99) [J]. Bioscience Biotechnology & Biochemistry.,2008,72 (6):1523～1530.
[77]M. R. Nilsson. Techniques to study amyloid fibril formation in vitro [J]. Methods, 2004,34:151～160.
[78]M. Groenning, M. Norrman, J. M.Flink, M. van de Weert, J. T. Bukrinsky, G. Schluckebier, S. Frokjaer. Binding mode of Thioflavin T in insulin amyloid fibrils [J]. Journal of Structural Biology,2007,159:483～497.
[79]M. Biancalana, K. Makabe, A. Koide, S. Koide. Molecular mechanism of Thioflavin-T binding to the surface of β-rich peptide self-assemblies [J]. Journal of Molecular Biology,2009,385(4):1052～1063.
[80]D. H. A. Correal, C. H. I. Ramos. The use of circular dichroism spectroscopy to study protein folding, form and function[J]. African Journal of Biochemistry Research,2009,3 (5):164～173.
[81]J. Y. Chang, L. Li. The unfolding mechanism and the disulfide structures of denatured Lysozyme [J]. FEBS Letters,2002,511:73-78.
[82]T. Sivaraman, T. K. S. Kumar, G. Jayaraman, C. Yu. The mechanism of 2,2,2-trichloroacetic acid induced protein precipitation [J]. JournaL of Protein Chemistry,1997,16:291～297.
[83]L. Jiang, L. He, M. F. Lakis. Comparison of protein precipitation methods for sample preparation prior to proteomic anaLysis [J]. Journal of Chromatography A, 2004,1023:317～320.
[84]N. Patsoukis, C. D. Georgiou. Fluorometric determination of thiol redox state [J]. Analytical and Bioanalytical Chemistry,2005,383:923～929.
[85]J. T. Mason. Disulfide dithiol redox titrations of proteins [D]. USA:The Graduate Faculty of Texas Tech University,2005.
[86]R. Lafelice, S. Cristoni, D. Caccia, R. Russo, L. R. Bernardi, K. C. Lowe, M. Perrella. Identification of the sites of deoxyhaemoglobin PEGylation [J]. Biochemical Journal,2007,403:189-196.
[87]M. Z. Moghaddam, T. Kawamura, E. Yatagai, S. Niwayama Electrospray ionization mass spectroscopic analysis of peptides modified with N-ethylmaleimide or iodoacetanilide [J]. Bioorganic & Medicinal Chemistry Letters,2008,18:4891～4895.
[88]N. Berova, K. Nakanishi, R. W. Woody (Eds.). Circular Dichroism:Principles and Applications [M].2nd ed R.New York:Wiley～VCH,2000,877 pp.
[89]M. Holley, C. Eginton, D. Schaefer, L. R. Brown. Characterization of amyloidogenesis of hen egg lysozyme in concentrated ethanol solution [J]. Biochemical and Biophysical Research Communications,2008,373:164～168.
[90]D. H. A. Correal, C. H. I. Ramos. The use of circular dichroism spectroscopy to study protein folding, form and function [J]. African Journal of Biochemistry Research,2009,3 (5):164～173.
[91]S. Khrapunov. Circular dichroism spectroscopy has intrinsic limitations for protein secondary structure analysis [J]. Analytical Biochemistry,2009,389: 174～176.
[92]N. Chaudhary, R. Nagaraj. Hen lysozyme amyloid fibrils induce aggregation of erythrocytes and lipid vesicles [J]. Molecular and Cellular Biochemistry,2009,328: 209～215.
[93]B. Huang, J. He, J. Ren, X. Y. Yan, C. M. Zeng. Cellular membrane disruption by amyloid fibrils involved intermolecular disulfide cross-linking [J]. Biochemistry, 2009,48:5794～5800.
[94]R. Kayed, E. Head, J. L. Thompson, T. M. McIntire, S. C. Milton, C. W. Cotman, C. G. Glabe. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis [J]. Science,2003,300:486～489.
[95]B. Caughey, P. T. Lansbury. Protofibrils, pores, fibrils, and neurodegeneration: Separating the responsible protein aggregates from the innocent bystanders [J]. Annual Review Neuroscience,2003,26:267～298.
[96]E. V. Petrotchenko, D. Pasek, P. Elms, N. V. Dokholyan, G. Meissner, C. H. Borchers. Combining fluorescence detection and mass spectrometric analysis for comprehensive and quantitative analysis of redox-sensitive cysteines in native membrane proteins [J]. Analytical Chemistry,2006,78:7959～7966.
[97]A. L. Gharibyan, V. Zamotin, K. Yanamandra, O. S. Moskaleval, B. A. Margulis, I. A. Kostanyan, L. A. Morozova Roche. Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways [J]. Journal of Molecular Biology,2007,365:1337～1349.
[98]S. Kumar, V. K. Ravi, R. Swaminathan. How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates? [J]. Biochemical Journal.2008,415:275～288.
[99]K. Yamamoto, H. Yagi, D. Ozawa, K. Sasahara, H. Naiki, Y. Goto. Thiol compounds inhibit the formation of amyloid fibrils by beta 2-microglobulin at neutral ph [J]. Journal of Molecular Biology,2008,376:258～268.
[100]S. S. Wang, K. N. Liu, B. W. Wang. Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme [J]. European Biophysics Journal,2010, published online:February 07.
[101]S. S. Wang, S. W. Chou, K. N. Liu, C. H. Wu. Effects of glutathione on amyloid fibrillation of hen egg-white lysozyme [J]. International Journal of Biological Macromolecules,2009,45:321～329.
[102]E. Frare, M. F. Mossuto, P. Polverino de Laureto, M. Dumoulin, C. M. Dobson, A. Fontana. Identification of the core structure of Lysozyme amyloid fibrils by proteolysis [J]. Journal of Molecular Biology,2006,361:551～561.
[103]S. V. Rajkuma, M. A. Gertz. Advances in the treatment of amyloidosis [J]. The New England Journal of Medicine,2007,356:2413～2415.
[104]S. S. Wang, K. N. Liu, T. C. Han. Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants [J]. Biochimica et Biophysica Acta, 2010, Accepted 16 February 2010.
[2]R. A. Kyle. Amyloidosis:a convoluted story [J]. British Journal of Haematology, 2001,114:529～538.
[3]J. C. Rochet, P. T. Jr. Lansbury. Amyloid fibrillogenesis:themes and variations [J]. Current Opinion in Structural Biology,2000,10:60～68.
[4]Y. Xing, K.Higuchi. Amyloid fibril proteins [J]. Mechanisms of Ageing and Development,2002,123:1625～1636.
[5]J. Guo, Y. Xu. Towards modeling of amyloid fibril structures [J]. Frontiers in Bioscience,2008,13:4039～4050.
[6]R. Tycko. Progress towards a molecular-level structural understanding of amyloid fibrils [J]. Current Opinion in Structural Biology,2004,14:96～103.
[7]Y. Goto, H. Yagi, K. Yamaguchi, E. Chatani, T. Ban. Structure,formation and propagation of amyloid fibrils [J]. Current Pharmaceutical Design,2008,14(30): 3205～3218.
[8]O. S. Makin, L. C. Serpell. Structures for amyloid fibrils [J]. FEBS Journal,2005, 272:5950～5961.
[9]S. Kasapis, I. T. Norton, J. B. Ubbink. Modern Biopolymer Science:bridging the divide between fundamental treatise and industrial application [M]. Academic Press,2009, CHAPTER 17:559～594.
[10]C. M. Dobson. Principles of protein folding, misfolding, and aggregation [J]. Seminars in Cell and Developmental Biology,2004,15:3～16.
[11]A. E. Langkilde, B. Vestergaard. Methods for structural characterization of prefibrillar intermediates and amyloid fibrils [J]. FEBS Letters,2009,583: 2600～2609.
[12]M. R. Nilsson. Techniques to study amyloid fibril formation in vitro [J]. Methods, 2004, 34:151～160.
[13]P. Hammarstrom, X. Jiang, A. R. Hurshman, E. T. Powers, J. W. Kelly. Sequence dependent denaturation energetics:A major determinant in amyloid disease diversity [J]. Proceedings of the National Academy of Sciences of the United States of America,2002,99(suppl.4):16427～16432.
[14]J. C. Rochet, P. T. Lansbury. Amyloid fibrillogenesis:themes and variations [J]. Current Opinion in Structural Biology,2000,10:60～68.
[15]C.M. Dobson. The structural basis of protein folding and its links with human disease [J]. Philosophical Transactions of the Royal Society B:Biological Sciences,2001,356:133～145.
[16]J. C. Rochet, P. T. Jr. Lansbury. Amyloid fibrillogenesis:themes and variations [J]. Current Opinion in Structural Biology,2000,10:60～68.
[17]R. Kayed, E. Head, J. L. Thompson, T. M. McIntire, S. C. Milton, C. W. Cotman, C. G. Glabe. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis [J]. Science,2003, Apr 18:486～489.
[18]E. T. Powers, D. L. Powers. The kinetics of nucleated polymerizations at high concentrations:amyloid fibril formation near and above the"supercritical concentration" [J]. Biophysical Journal,2006,91:122～132.
[19]S. Jain, J. B. Udgaonkar. Evidence for step wise formation of amyloid fibrils by the mouse prion protein [J]. Journal of Molecular Biology,2008,382:1228～1241.
[20]C. C. Lee, A. Nayak, A. Sethuraman, G. Belfort, G. J. McRae. A three-stage kinetic model of amyloid fibrillation [J]. Biophysical Journal,2007,92: 3448～3458.
[21]V. N. Uversky. Amyloidogenesis of natively unfolded proteins [J]. Current Alzheimer Research,2008, June 5(3):260～287.
[22]N. Phuong Humblet-Hua, L. M. Sagis, E. van der Linden. Effects of flow on Hen Egg White Lysozyme (HEWL) fibril formation:length distribution, flexibility, and kinetics [J]. Journal Agricultural and Food Chemistry,2008,56:11875～11882.
[23]P. Shahi, R. Sharma, S. Sanger, I. Kumar, R. S. Jolly. Formation of amyloid fibrils via longitudinal growth of oligomers [J]. Biochemistry,2007,46:7365～7373.
[24]H. J. Lachmann, P. N. Hawkins. Systemic amyloidosis [J]. Current Opinion in Pharmacology,2006,6:214～220.
[25]K. H. Mok, J. Pettersson, S. Orrenius, C. Svanborg. HAMLET, protein folding, and tumor cell death [J]. Biochemical and Biophysical Research Communications, 2007,354:1～7.
[26]J. P. Kastberg, S. Aits, L. Gustafsson, A. Mossberg, P. Storm, M. Trulsson, F. Persson, K. H. Mok, C. Svanborg. Can misfolded proteins be beneficial? The HAMLET case [J]. Annals of Medicine,2009,41:162～176.
[27]曾彩虹,刘志红.遗传性淀粉样变性[J].肾脏病与透析肾移植杂志,2007,6(1): 70～75.
[28]陈艳,江明锋,叶煜辉,刘勇涛,李生伟.溶菌酶的研究进展[J].生物学杂志,2009,26(2)：64～66.
[29]G. Merlini, V. Bellotti. Lysozyme:A paradigmatic molecule for the investigation of protein structure, function and misfolding [J]. Clinica Chimica Acta,2005, 357:168～172.
[30]E. Frare, P. Polverino de Laureto, J. Zurdo, C. M. Dobson, A. Fontana. A highly amyloidogenic region of hen Lysozyme [J]. Journal of Molecular Biology,2004, 340:1153～1165.
[31]S. S. Wang, S. W. Chou, K. N. Liu, C. H. Wu. Effects of glutathione on amyloid fibrillation of hen egg-white lysozyme [J]. International Journal of Biological Macromolecules,2009,45:321～329.
[32]A. Cao, D. Hu, L. Lai. Formation of amyloid fibrils from fully reduced hen egg white lysozyme [J]. Protein Science,2004,13 (2):319～324.
[33]R. Mishra, K. Sorgjerd, S. Nystrom, A. M. Nordigarden, Y. C. Yu, P. Hammarstrom. Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion [J]. Journal of Molecular Biology,2007,366(3):1029～1044.
[34]卢义钦,刘俊凡.红细胞膜蛋白与膜骨架[J].生命科学研究,2005,9(4)：283～291.
[35]M. E. Reid, N. Mohandas. Red blood cell blood group antigens:structure and function [J]. Seminars in Hematology,2004,4:93～117.
[36]D. J. Anstee. Blood group antigens defined by the amino acid sequences of red cell surface proteins [J]. Transfusion Medicine,1995,5:1～13.
[37]M. J. Telen.Erythrocyte blood group antigens:not so simple after all [J]. Blood. 1995,85:299～306.
[38]J. P. Cartron, Y. Colin. Structural and functional diversity of blood group antigens [J]. Transfusion Clinique et Biologique,2001,8:163～199.
[39]V. Nicolas, C. Le Van Kim, P. Gane, C. Birkenmeier, J. P. Cartron, Y. Colin, I. M. Chanteloup. Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation [J]. The Journal of Biological Chemistry,2003,278:25526～25533.
[40]M. Salomao, X. Zhang, Y. Yang, S. Lee, H. John. Hartwig, Joel Anne Chasis, Narla Mohandas, and Xiuli An.Protein 4.1R-dependent multiprotein complex: new insights into the structural organization of the red blood cell membrane [J]. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(23):8026～8031.
[41]N. Mohandas, P. G. Gallagher. Red cell membrane:past, present, and future [J]. Blood,2008,112(10):9329～3948.
[42]M. Stefani, C.M. Dobson. Protein aggregation and aggregate toxicity:new insight into protein folding, misfolding diseases and biological evolution [J]. Journal of Molecular Medicine.2003,81:678～699.
[43]M. Stefani. Protein misfolding and aggregation:new examples in medicine and biology of the dark side of the protein world [J]. Biochimica et Biophysica Acta, 2004,1739:5～25.
[44]J. P. Cleary, D. M. Walsh, J. J. Hofmeister, G. M. Shankar, M. A. Kuskowski, D. J. Selkoe, K. H. Ashe. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function [J]. Nature Neuroscience,2005,8:79～84.
[45]M. Hoshi, M. Sato, S. Matsumoto, A. Noguchi, K. Yasutake, N. Yoshida, K. Sato. Spherical aggregates of {beta}-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase～3{beta} [J]. Proceedings of the National Academy of Sciences of the United States of America, 2003,100:6370～6375.
[46]H. Mukai, T. Isagawa, E. Goyama, S. Tanaka, N. F. Bence, A. Tamura, Y. Ono, R. R. Kopito. Formation of morphologically similar globular aggregates from diverse aggregation prone proteins in mammalian cells [J]. Proceedings of the National Academy of Sciences of the United States of America,2005,102: 10887～10892.
[47]V. Novitskaya, O. V. Bocharova, I. Bronstein, I. V. Baskakov. Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons [J]. Journal of Biological Chemistry,2006,281:13828～13836.
[48]L. Pieri, M. Bucciantini, D. Nosi, L. Formigli, J. Savistchenko, R. Melki, M. Stefani. The yeast prion Ure2p native-like assemblies are toxic to mammalian cells regardless to their aggregation state [J]. Journal of Biological Chemistry, 2006,281:15337～15344.
[49]I. Sirangelo, C. Malmo, C. Iannuzzi, A. Mezzogiorno, M. R. Bianco, M. Papa, G. Irace. Fibrillogenesis and cytotoxic activity of the amyloidforming apomyoglobin mutant W7FW14F [J]. Journal of Biological Chemistry,2004,279:13183～13189.
[50]M. Bucciantini, S. Rigacci, A. Berti, L. Pieri, C. Cecchi, D. Nosi, L. Formigli, F Chiti, M. Stefani. Patterns of cell death triggered in two different cell lines by HypF-N prefibrillar aggregates [J]. FASEB Journal,2005,19:437～439.
[51]S. Grudzielanek, A. Velkova, A. Shukla, V. Smirnovas, M. TatarekNosso, H. Rehage, A. Kapurniotu, R. Winter. Cytotoxicity of insulin within its self-assembly and amyloidogenic pathways [J]. Journal of Molecular Biology,2007,370: 372～384.
[52]M. M. Sousa, I. Cardoso, R. Fernandes, A. Guimaraes, M. J. Saraiva. Deposition of transthyretin in early stages of familial amyloidotic polyneuropathy:Evidence for toxicity of non-fibrillar aggregates [J]. American Journal of Pathology,2001, 159:1993～2000.
[53]R. Bader, R. Bamford, J. Zurdo, B. F. Luisi, C. Dobson. Probing the Mechanism of amyloldogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation [J]. Journal of Molecular Biology,2006,356:189～208.
[54]M. Bucciantini, G. Calloni, F. Chiti, L. Formigli, D. Nosi, C. M. Dobson, M. Stefani. Prefibrillar amyloid protein aggregates share common features of cytotoxicity [J]. Journal of Biological Chemistry,2004,279:31374～31382.
[55]M. T. Pastor, N. Kummerer, V. Schubert, A. E. Chopo, C G. Dotti, M. Lopez de la Paz, L. Serrano. Amyloid toxicity is independent of polypeptide sequence, length and chirality [J]. Journal of Molecular Biology,2008,375:695～707.
[56]P. Cizas, R. Budvytyte, R. Morkuniene, R. Moldovan, M. Broccio, M. Losche, G. Niaura, G. Valinciu, V. Borutaite. Size-dependent neurotoxicity of β-amyloid oligomers [J]. Archives of Biochemistry and Biophysics, Available online 11 February 2010.
[57]N. Gregersen, L. Bolund, P. Bross. Protein misfolding, aggregation, and degradation in disease [J]. Methods in Molecular Biology,2003,232:3～16.
[58]M. Bucciantini, E. Giannoni, F. Chiti, F. Baroni, L. Formigli, J. S. Zurdo, N. Taddie, G. Ramponi, C. M. Dobson, M. Stefani. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases [J]. Nature,2002, 416:507～511.
[59]G. P. Gorbenko, P. K. Kinnunen. The role of lipid-protein interactions in amyloid-type protein fibril formation [J]. Chemistry and Physics of Lipids,2006, 141:72～82.
[60]D. Hall, H. Edskes. A model of amyloid's role in disease based on fibril fracture [J]. Biophysical Chemistry,2009,145:17～28.
[61]B. L. Kagan, Y. Hirakura, R. Azimov, R. Azimova, M. C. Lin. The channel hypothesis of Alzheimer's disease:Current status [J]. Peptides,2002,23: 1311～1315.
[62]R. Kayed, Y. Sokolov, B. Edmonds, T. M. Mclntire, S. C. Milton, J. E. Hall, C. G. Glabe. Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases [J]. Journal of Biological Chemistry,2004,279:6363～6366.
[63]M. E. Bamberger, M. E. Harris, D. R. McDonald, J. Husemann, G. E. Landreth. A cell surface receptor complex for fibrillar β-amyloid mediates microglial activation [J]. Journal of Neuroscience,2003,23:2665～2674.
[64]E. Sparr, M. F. Engel, D. V. Sakharov, M. Sprong, J.Jacobs, B. de Kruijff, J. W. Hoppener, J. A. Killian. Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers [J]. FEBS Letters,2004,577: 117～120.
[65]J. R. Brender, U. H. N. Durr, D. Heyl, M. B. Budarapu, A. Ramamoorthy. Membrane fragmentation by an amyloidogenic fragment of human Islet Amyloid Polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes [J]. Biochimica et Biophysica Acta,2007,1768:2026～2029.
[66]D. G. Smith, R. Cappai, K. J. Barnham. The redox chemistry of the Alzheimer's disease amyloid P peptide [J]. Biochimica et Biophysica Acta,2007,1768: 1976～1990.
[67]D. A. Butterfield, C. M. Lauderback. Lipid peroxidation and protein oxidation in Alzheimer's disease brain:Potential causes and consequences involving amyloid β-peptide-associated free radical oxidative stress [J]. Free Radical Biology & Medicine,2002,32:1050～1060.
[68]P. H. Reddy. Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease [J]. Experimental Neurology,2009,218:286～292.
[69]B. Su,X. Wang, A. Nunomura, P. I. Moreira, H. G. Lee, G. Perry, M. A. Smith, X. Zhu. Oxidative stress signaling in Alzheimer's disease [J]. Current Alzheimer Researsh,2008,5:525～532.
[70]D. Allsop, J. Mayes, S. Moore, A. Masad, B. J. Tabner. Metal-dependent generati-on of reactive oxygen species from amyloid proteins implicated in neurode-generative disease [J]. Biochemical Society Transaction,2008,36:1293～1298.
[71]D. A. Butterfield, R. Sultana. Redox proteomics identification of oxidative-lymodified brain proteins in Alzheimer's disease and mild cognitive impairment: Insights into the progression of this dementing disorder [J]. Journal of Alzheimer's Disease,2007,12:61～72.
[72]A. A. Koksoy. A unifying hypothesis for explaining the mechanism of amyloid formation under conditions of increased oxidative stress [J]. Bioscience Hypotheses,2008,1:209～212.
[73]I. V. Murray, M. E. Sindoni, P. H. Axelsen. Promotion of oxidative lipid membrane damage by amyloid β proteins [J]. Biochemistry,2005,44: 12606～12613.
[74]B. I. Giasson, H. Ischiropoulos, V. M. Lee, J. Q. Trojanowski. The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer's and Parkinson's diseases.Free Radical [J]. Free Radical Biology & Medicine,2002,32: 1264～1275.
[75]L. N. Arnaudov, R. De Vries… Thermally induced fibrillar aggregation of hen egg white lysozyme [J]. Biophysical Journal,2005,88:515～526.
[76]A.Harada, H. Azakami, A. Kato. Amyloid fibril formation of hen Lysozyme depends on the instability of the C-Helix (88-99) [J]. Bioscience Biotechnology & Biochemistry.,2008,72 (6):1523～1530.
[77]M. R. Nilsson. Techniques to study amyloid fibril formation in vitro [J]. Methods, 2004,34:151～160.
[78]M. Groenning, M. Norrman, J. M.Flink, M. van de Weert, J. T. Bukrinsky, G. Schluckebier, S. Frokjaer. Binding mode of Thioflavin T in insulin amyloid fibrils [J]. Journal of Structural Biology,2007,159:483～497.
[79]M. Biancalana, K. Makabe, A. Koide, S. Koide. Molecular mechanism of Thioflavin-T binding to the surface of β-rich peptide self-assemblies [J]. Journal of Molecular Biology,2009,385(4):1052～1063.
[80]D. H. A. Correal, C. H. I. Ramos. The use of circular dichroism spectroscopy to study protein folding, form and function[J]. African Journal of Biochemistry Research,2009,3 (5):164～173.
[81]J. Y. Chang, L. Li. The unfolding mechanism and the disulfide structures of denatured Lysozyme [J]. FEBS Letters,2002,511:73-78.
[82]T. Sivaraman, T. K. S. Kumar, G. Jayaraman, C. Yu. The mechanism of 2,2,2-trichloroacetic acid induced protein precipitation [J]. JournaL of Protein Chemistry,1997,16:291～297.
[83]L. Jiang, L. He, M. F. Lakis. Comparison of protein precipitation methods for sample preparation prior to proteomic anaLysis [J]. Journal of Chromatography A, 2004,1023:317～320.
[84]N. Patsoukis, C. D. Georgiou. Fluorometric determination of thiol redox state [J]. Analytical and Bioanalytical Chemistry,2005,383:923～929.
[85]J. T. Mason. Disulfide dithiol redox titrations of proteins [D]. USA:The Graduate Faculty of Texas Tech University,2005.
[86]R. Lafelice, S. Cristoni, D. Caccia, R. Russo, L. R. Bernardi, K. C. Lowe, M. Perrella. Identification of the sites of deoxyhaemoglobin PEGylation [J]. Biochemical Journal,2007,403:189-196.
[87]M. Z. Moghaddam, T. Kawamura, E. Yatagai, S. Niwayama Electrospray ionization mass spectroscopic analysis of peptides modified with N-ethylmaleimide or iodoacetanilide [J]. Bioorganic & Medicinal Chemistry Letters,2008,18:4891～4895.
[88]N. Berova, K. Nakanishi, R. W. Woody (Eds.). Circular Dichroism:Principles and Applications [M].2nd ed R.New York:Wiley～VCH,2000,877 pp.
[89]M. Holley, C. Eginton, D. Schaefer, L. R. Brown. Characterization of amyloidogenesis of hen egg lysozyme in concentrated ethanol solution [J]. Biochemical and Biophysical Research Communications,2008,373:164～168.
[90]D. H. A. Correal, C. H. I. Ramos. The use of circular dichroism spectroscopy to study protein folding, form and function [J]. African Journal of Biochemistry Research,2009,3 (5):164～173.
[91]S. Khrapunov. Circular dichroism spectroscopy has intrinsic limitations for protein secondary structure analysis [J]. Analytical Biochemistry,2009,389: 174～176.
[92]N. Chaudhary, R. Nagaraj. Hen lysozyme amyloid fibrils induce aggregation of erythrocytes and lipid vesicles [J]. Molecular and Cellular Biochemistry,2009,328: 209～215.
[93]B. Huang, J. He, J. Ren, X. Y. Yan, C. M. Zeng. Cellular membrane disruption by amyloid fibrils involved intermolecular disulfide cross-linking [J]. Biochemistry, 2009,48:5794～5800.
[94]R. Kayed, E. Head, J. L. Thompson, T. M. McIntire, S. C. Milton, C. W. Cotman, C. G. Glabe. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis [J]. Science,2003,300:486～489.
[95]B. Caughey, P. T. Lansbury. Protofibrils, pores, fibrils, and neurodegeneration: Separating the responsible protein aggregates from the innocent bystanders [J]. Annual Review Neuroscience,2003,26:267～298.
[96]E. V. Petrotchenko, D. Pasek, P. Elms, N. V. Dokholyan, G. Meissner, C. H. Borchers. Combining fluorescence detection and mass spectrometric analysis for comprehensive and quantitative analysis of redox-sensitive cysteines in native membrane proteins [J]. Analytical Chemistry,2006,78:7959～7966.
[97]A. L. Gharibyan, V. Zamotin, K. Yanamandra, O. S. Moskaleval, B. A. Margulis, I. A. Kostanyan, L. A. Morozova Roche. Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways [J]. Journal of Molecular Biology,2007,365:1337～1349.
[98]S. Kumar, V. K. Ravi, R. Swaminathan. How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates? [J]. Biochemical Journal.2008,415:275～288.
[99]K. Yamamoto, H. Yagi, D. Ozawa, K. Sasahara, H. Naiki, Y. Goto. Thiol compounds inhibit the formation of amyloid fibrils by beta 2-microglobulin at neutral ph [J]. Journal of Molecular Biology,2008,376:258～268.
[100]S. S. Wang, K. N. Liu, B. W. Wang. Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme [J]. European Biophysics Journal,2010, published online:February 07.
[101]S. S. Wang, S. W. Chou, K. N. Liu, C. H. Wu. Effects of glutathione on amyloid fibrillation of hen egg-white lysozyme [J]. International Journal of Biological Macromolecules,2009,45:321～329.
[102]E. Frare, M. F. Mossuto, P. Polverino de Laureto, M. Dumoulin, C. M. Dobson, A. Fontana. Identification of the core structure of Lysozyme amyloid fibrils by proteolysis [J]. Journal of Molecular Biology,2006,361:551～561.
[103]S. V. Rajkuma, M. A. Gertz. Advances in the treatment of amyloidosis [J]. The New England Journal of Medicine,2007,356:2413～2415.
[104]S. S. Wang, K. N. Liu, T. C. Han. Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants [J]. Biochimica et Biophysica Acta, 2010, Accepted 16 February 2010.