异源蛋白对胰岛素淀粉样纤维化及其细胞毒性的抑制作用

【作者】 张磊；

【导师】 曾成鸣；

【作者基本信息】 陕西师范大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 蛋白质淀粉样变性以及由此引发的多种人类疾病日益受到人们的关注,已知20多种人类疾病如阿尔兹海默症,亨廷顿症都与蛋白质错误折叠有关。一些与疾病不相关的蛋白质、肽类甚至多聚氨基酸也可以形成淀粉样沉积。淀粉样变性是蛋白质的一种共性,理论上所有蛋白质在特定的条件下都能够发生错误折叠并聚集形成淀粉样纤维。胰岛素是一种富含α螺旋的短肽,由两条链组成,分为A链(21AA)和B链(30AA),A链与B链之间由二硫键连接。胰岛素作为研究淀粉样变性的模型分子具有一级结构简单、分子量小等优势:其作为临床用药,在合成和储存的过程中产生的淀粉样沉积可能产生免疫原性,使其作用功效减弱,因此对于胰岛素淀粉样变性的分子机制的研究和抑制剂的筛选具有重要的理论意义和实际应用价值。胰岛素在高温,低pH条件下容易发生淀粉样变性,一些小分子化合物和氨基酸能够影响它的成纤维过程。有研究发现,胰岛素的纤维化过程可以被自身形成的同源纤维极大的加速,这可能是由于加入的胰岛素纤维扮演了成核寡聚体的角色,使胰岛素成纤维过程的延滞期缩短甚至无延滞期。那么,是否异源蛋白也具有这种作用?已经有报道一些不相关蛋白可以影响另一种蛋白的淀粉样纤维的形成,如蚕丝纤维和Sup35可以导致血清淀粉样蛋白(SAA)的纤维形成,富含谷氨酸和天冬氨酸的肽段可以促进Sup35朊蛋白淀粉样纤维的形成。异源蛋白与胰岛素分子之间存在一级结构和分子构象上的差异,这种差异是否会对胰岛素淀粉样变性过程产生不同的影响?本文基于以上问题,筛选出4种蛋白,鸡卵清溶菌酶(Lys)、人血红蛋白(Hem)、辣根过氧化物酶(HRP)和牛血清白蛋白(BSA),对异源蛋白存在条件下胰岛素淀粉样纤维化进行了深入研究。采用的主要实验方法包括:ThT和ANS荧光监测胰岛素纤维化及分子表面疏水性变化,异源蛋白的分子伴侣活性检测,原子力显微镜和透射电镜对纤维进行形态分析,以及检测淀粉样纤维对细胞通透性的影响等。结果表明,鸡卵清溶菌酶、人血红蛋白Hem、辣根过氧化物酶HRP和牛血清白蛋白BSA这四种异源蛋白能够在较小的浓度范围(质量比5%-20%)抑制胰岛素淀粉样纤维形成,并使纤维的细胞毒性减弱。溶菌酶在一定条件(pH2.0,65℃)下经过孵育形成淀粉样纤维,孵育5天的成熟溶菌酶纤维对胰岛素淀粉样纤维的形成和细胞毒性同样具有抑制作用,不成熟溶菌酶纤维则没有明显抑制作用。溶菌酶纤维可使胰岛素的疏水性发生变化。分子伴侣活性分析发现,辣根过氧化物酶,牛血清白蛋白具有分子伴侣活性,能够抑制胰岛素B链的聚集。但溶菌酶和血红蛋白无类似的分子伴侣作用,这表明了异源蛋白对胰岛素纤维化的抑制作用可能与分子伴侣活性无关。四种异源蛋白对胰岛素淀粉样纤维的形成无一例外都具有抑制作用,这种作用与其降低胰岛素分子在溶液中的“有效浓度”有关。蛋白质的淀粉样纤维具有一种相对有序的三维结构,异源蛋白分子由于组成和结构不一样,难以整合入胰岛素的纤维结构。胰岛素在纤维化过程中,需要经历一个时间较长的成核过程,即延迟时间。在这个时段,胰岛素经分子间的碰撞形成多聚体,进而形成纤维核心并组装成纤维。异源蛋白的加入使胰岛素蛋白处于一种含有其他大分子的“拥挤环境”中,降低了胰岛素的分子扩散速率,减少了胰岛素分子之间发生碰撞的机会,导致胰岛素的成核时间被延长。结论:本文研究了四种异源蛋白对胰岛素淀粉样变性的影响,发现四种异源蛋白对胰岛素纤维化和细胞毒性具有抑制作用,这种作用与异源蛋白的分子伴侣作用无关,而与降低胰岛素的分子扩散速率进而抑制蛋白质分子的自组装有关。本文的结果对于探索异源蛋白在淀粉样变性过程中所扮演的角色,阐明淀粉样变性的机制以及筛选蛋白淀粉样纤维化抑制剂提供了新的实验和理论依据。更多还原

【Abstract】 Protein amyloidogenesis, which is associated with more than 20 human diseases such as Alzheimer’s and Huntington’s disease, hemodialysis-related amyloid deposition and a number of systemic amyloidoses, has become a critical subject in scientific research and health concerns. In addition to the proteins related to diseases, it has been demonstrated that some proteins, peptides and poly-amino acids that are not related to disease can also form amyloid fibrils. Theoretically all proteins are able to rearrange their backbones to assemble into amyloid fibrils upon unfolding under certain circumstances. Therefore amyloid formation is recognized as a common property of proteins and peptides.Insulin is a predominantlyα-helical structured protein that can undergo amyloid fibril formation in destabilizing conditions. It is consisting of a 21-residue A chain and a 30-residue B chain which are linked together by two interchain disulfide bonds. The pharmaceutical preparation of insulin can form amyloid aggregates in the processes of production, delivery, storage and clinical application. These aggregates may contain immune epitopes and side-effects can be triggered. Therefore it is of great significance to elucidate the molecular mechanism and to screen inhibitors for insulin fibrillation. In this study, insulin has been used as a model molecule in order to explore the kinetics of fibrillation and the fibrillar cytotoxicity of a protein in the presence of heterogeneous proteins.Insulin tends to form amyloid fibrils under high temperature and low pH. It has been reported that some low molecular compounds such as amino acids are able to affect the fibrillation of insulin. It is well-established that the kinetics of fibrillation can be significantly accelerated by the addition of seed fibrils, which, at a sufficient concentration, will abolish the lag time completely. The seed fibrils serve as nucleation templates for insulin to assemble into amyloid fibrils. Whether heterogeneous protein or its amyloid fibrils affect the kinetics of insulin fibrillation? To address this question four proteins, hen egg white lysozyme (Lys), hemoglobin (Hem), horseradish peroxidase (HRP) and bovine serum albumin (BSA) have been selected to investigate the fibrillation kinetics and cytotoxicity of insulin in the presence of heterogeneous proteins.Experimental methods included ThT and ANS fluorescence to monitor the fibril growth and hydrophobic domain exposure, chaperone activity assay, characterization of the fibrillar structure by atomic force microscope and transmission microscope and evaluation of the fibrillar cytotoxicity by hemolytic assay. The results indicated that all the heterogeneous proteins inhibited insulin fibrillation in a dose range of 5% - 20%. The resultant insulin fibrils showed lower cytotoxicity than that of the control insulin fibrils. The inhibitory effect of lysozyme on insulin fibrillation was increased while lysozyme was applied in fibrillar forms. ANS fluorescence indicated that lysozyme fibrils promoted the exposure of hydrophobic domains of insulin. Chaperones activity assay revealed that HRP and BSA possessed chaperone activity to inhibit the B-chain aggregation of insulin. The fact that other two proteins Lys and Hem failed to inhibit insulin B-chain aggregation suggested that chaperone activity was not involved in the inhibitory effect of heterogeneous protein on insulin fibrillation. An alternative mechanism is suggested that the heterogeneous proteins provided a crowding environment that reduced the probability of insulin molecules assembled into aggregates.In conclusion, amyloid fibrillation and the fibrillar cytotoxicity of insulin were inhibited when insulin fibrils were prepared in the presence of heterogeneous proteins. Chaperone activity was not involved in this process. A crowding environment originated from the heterogeneous protein has been suggested to be the driving force in the inhibition of insulin fibrillation. The results of this study are of significance in elucidating amyloidogenesis of a protein and screening inhibitors for protein fibrillation.更多还原