桑色素对鸡溶菌酶淀粉样纤维形成的影响

发布时间：2018-01-17 19:37

本文关键词：桑色素对鸡溶菌酶淀粉样纤维形成的影响　出处：《辽宁大学》2016年硕士论文　论文类型：学位论文

【摘要】：淀粉样纤维由淀粉样蛋白经错误折叠聚集形成,并沉积于体内不同器官或组织中,所以称之为“蛋白质错误折叠疾病”。到目前为止,已经证明有30多种蛋白所形成的淀粉样纤维涉及到人类淀粉样疾病。这类慢性退行性疾病包括阿尔兹海默症、帕金森疾病、Ⅱ型糖尿病以及一系列的系统淀粉样疾病。虽然不同蛋白质的氨基酸序列及三级结构会有差异,但在这些不同淀粉样纤维形成过程却发现了通有性质。因此,基于不同种淀粉样蛋白纤维具有相似结构,细胞毒性以及着色特性的特点使得研究发展针对淀粉样疾病的通用治疗方法成为可能。目前还没有能够完全治愈人类淀粉样疾病的方法,因此开发研究有关淀粉样疾病的防治方法和新型药物迫在眉睫。近年来已经有关于抑制淀粉样纤维形成甚至于解聚淀粉样前体的小分子的研究,大量研究显示天然多酚类小分子具有针对几种淀粉样蛋白形成淀粉样纤维的抑制作用。天然多酚类化合物是一种大量存在于食物和中草药的植物化学物质,人们通过每日三餐的饮食即可获得这些化合物。鸡溶菌酶在序列和结构上与导致系统性淀粉样疾病的人溶菌酶具有高度的相似性,并且由于鸡溶菌酶的易获得和在体外高温强酸条件下较易形成纤维的特性,从而使鸡溶菌酶成为理解此类疾病致病机制的经典研究模型。本研究首次发现桑色素对鸡溶菌酶这一经典淀粉样蛋白模型的纤维化具有显著性抑制作用。荧光光谱检测和计算机模拟结果显示桑色素通过与鸡溶菌酶p结构域的聚集倾向区域相结合来稳定蛋白分子的天然结构从而抑制形成鸡溶菌酶淀粉样纤维。透射电子显微镜观察证实了桑色素对鸡溶菌酶纤维的形成具有浓度依懒性的抑制作用,其中还观察到桑色素与鸡溶菌酶共同孵育所形成的无定形聚集体随着桑色素浓度的增加而变大,并通过MTT细胞毒性检测发现桑色素可以有效减少鸡溶菌酶纤维对细胞形成的毒性。我们提出了一个可能的抑制机制,即一定浓度的桑色素可以通过与鸡溶菌酶的凹槽相结合来稳定鸡溶菌酶的天然构象,而当桑色素超过这一浓度时,未与鸡溶菌酶结合的小分子会与蛋白表面相互作用,这样一来桑色素逐渐替代了原本结合在鸡溶菌酶表面的水分子,从而削弱了鸡溶菌酶与水分子之间的相互作用。这个机制不仅使我们在分子水平上更好地理解桑色素如何抑制鸡溶菌酶淀粉样纤维的形成,也对未来设计开发针对淀粉样纤维形成的新型抑制剂具有一定的借鉴和指导意义。
[Abstract]:Amyloid fibers are formed by misfolding and aggregation of amyloid proteins and deposited in different organs or tissues of the body, so they are called "protein misfolding diseases." so far. More than 30 proteins have been shown to form amyloid fibers involved in human amyloid diseases. These chronic degenerative diseases include Alzheimer's disease and Parkinson's disease. Type 2 diabetes mellitus and a series of systemic amyloid diseases. Although the amino acid sequences and tertiary structures of different proteins may differ, they are found to be common in the formation of these different amyloid fibers. Different amyloid fibers have similar structures. The characteristics of cytotoxicity and chromaticity make it possible to develop a universal treatment for amyloid disease. There is currently no cure for human amyloid disease. Therefore, it is urgent to develop methods and new drugs for the prevention and treatment of amyloid diseases. In recent years, there have been studies on the inhibition of the formation of amyloid fibers and even the depolymerization of amyloid precursors. A large number of studies have shown that natural polyphenols have inhibitory effects on the formation of amyloid fibers against several kinds of amyloid proteins. Natural polyphenols are a large number of phytochemicals found in food and Chinese herbal medicine. These compounds can be obtained by eating three meals a day. Chicken lysozyme is highly similar in sequence and structure to the human lysozyme that causes systemic amyloid disease. And because of the easy to obtain chicken lysozyme and in vitro high temperature strong acid conditions easier to form fiber characteristics. So that chicken lysozyme became a classic model to understand the pathogenesis of this disease. This study first found that Morin has a significant inhibitory effect on the fibrosis of chicken lysozyme, a classical amyloid model. The results of spectrum detection and computer simulation show that mulberry pigment can stabilize the natural structure of protein molecule by combining with the aggregation tendency region of chicken lysozyme p domain and thus inhibit the formation of chicken lysozyme amyloid fiber. Microscopical observation confirmed that mulberry pigment had a concentration-dependent inhibitory effect on the formation of chicken lysozyme fiber. It was also observed that the amorphous aggregates formed by the co-incubation of mulberry pigments with chicken lysozyme increased with the increase of mulberry pigment concentration. MTT cytotoxicity test showed that Morin could effectively reduce the cytotoxicity of chicken lysozyme fibers. We proposed a possible inhibition mechanism. That is to say, mulberry pigment of a certain concentration can stabilize the natural conformation of chicken lysozyme by combining with the groove of chicken lysozyme, and when mulberry pigment exceeds this concentration, it can stabilize the natural conformation of chicken lysozyme. Small molecules that do not bind to chicken lysozyme interact with the surface of the protein, so mulberry gradually replaces the water molecules that were bound to the surface of the chicken lysozyme. Therefore, the interaction between chicken lysozyme and water molecules is weakened. This mechanism not only enables us to better understand how Morin inhibits the formation of chicken lysozyme amyloid fibers at the molecular level. It also has some reference and guidance significance for the future design and development of new inhibitors for amyloid fiber formation.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R597.2

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