兰州大学机构库 >化学化工学院
α-突触核蛋白错误折叠、聚集以及与小分子抑制剂相互作用机制的分子模拟研究
Alternative TitleMolecular Dynamics Simulation of α-synuclein Misfolding and Aggregation Mechanisms and Interaction Mechanisms of Small Molecule Inhibitors
刘雪伟
Subtype博士
Thesis Advisor姚小军
2019-05-24
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Degree Discipline化学信息学
Keywordα-突触核蛋白 错误折叠与聚集 解聚 小分子抑制剂
Abstract帕金森病(Parkinson's disease,简称:PD)是仅次于阿尔茨海默病的神经退行性疾病。在年龄大于65岁的人群中,发病率高达2%。其主要的临床特征表现为:静止性震颤、姿势僵硬、运动迟缓等,晚期病例会出现痴呆和记忆力衰减的认知症状,严重影响着老年人的生活指数。多巴胺能神经元中以α-突触核蛋白为主要成分的路易小体以及路易神经炎的形成是其主要的病理标志。α-突触核蛋白的错误折叠与聚集形成淀粉样纤维是帕金森病的主要病理进程。由于其生化过程进行迅速且时间尺度小,实验研究很难揭示错误折叠与聚集这一病理学进程。而分子动力学模拟(Molecular Dynamics Simulation)则可以提供详细的结构动力学信息以研究蛋白质错误折叠与聚集及相关疾病的病理机制。在本论文中,我们应用分子动力学模拟研究α-突触核蛋白的错误折叠与聚集机制以及小分子抑制剂的抑制机理,为进一步阐明帕金森病的发病机制和新型合理药物设计提供理论支持。本论文第一章在帕金森病的发病机理、当前的诊断治疗策略,以及α-突触核蛋白结构和功能等方面进行了概述,同时介绍了α-突触核蛋白分子动力学模拟研究进展以及分子动力学模拟方法与原理。在其后的四个章节中,我们从不同结构层次探究了α-突触核蛋白的聚集机制以及相应小分子抑制剂的抑制机理。1)α-突触核蛋白的聚集延伸遵循模板诱导机制,即当聚集核seeds存在的条件下,α-突触核蛋白单体可以通过dock-lock的方式快速添加至seeds完成聚集延伸进一步形成纤维结构。肽段-syn47-56是-突触核蛋白的毒性核心,在α-突触核蛋白聚集折叠过程中起着关键作用,肽段的模板诱导研究可以揭示α-突触核蛋白错误折叠聚集分子机理。因此,我们进行了以α-突触核蛋白五聚体为模板,诱导肽段α-syn47-56错误折叠的常规分子动力学模拟研究。在第二章中,我们将肽段α-syn47-56分别置于α-突触核蛋白五聚体两侧,进行了400 ns的分子动力学模拟。研究发现,肽段α-syn47-56在五聚体两侧具有不同的延伸特性。在α-突触核蛋白五聚体左侧容易形成β-sheet结构完成模板诱导。而在右侧则不能形成β-sheet结构。此外,我们还发现α-突触核蛋白五聚体与单体间的氢键作用、静电相互作用以及范德华力在β-sheet延伸中起到重要作用。2)表达α-突触核蛋白的SNCA基因突变是引起家族性帕金森病的重要原因,实验研究表明E46K、H50Q突变可以促进α-突触核蛋白纤维化形成淀粉样斑块。然而其具体的分子机理仍未揭示。因此,我们针对α-突触核蛋白五聚体野生型及突变型E46K和H50Q的结构稳定性进行了500ns的加速分子动力学模拟研究。研究结果表明,突变型E46K显著改变了α-突触核蛋白五聚体的链间相互作用模式,溶剂化能在维持链间稳定性中起到重要作用。此外E46K突变可以很好的维持链间骨架氢键从而保持了边链的β-sheet二级结构,相对于野生型有序度增加。然而对于H50Q突变则表现为结合自由能混乱、有序度显著降低、链间骨架氢键近乎完全消失以及结构稳定性降低。因此,我们认为E46K突变与H50Q突变的致病机理不同。E46K突变可以改变链间相互作用及邻近残基的结构倾向性从而提高聚集体的稳定性。而H50Q突变的致病机理可能与α-突触核蛋白错误折叠聚集进程不相关。此外,肽段α-syn49-55的β-sheet二级结构稳定性在α-突触核蛋白聚集体整体结构稳定性中起到重要作用,这一关键折叠肽段的发现可以为针对α-突触核蛋白的新型治疗策略研发提供理论支持。3)α-突触核蛋白的聚集延伸形成淀粉样斑块是帕金森病的主要病理原因。α-突触核蛋白聚集分子机理的研究将为帕金森病的治疗提供理论依据。边界链解离是蛋白质错误折叠的逆过程,相对于α-突触核蛋白聚集进程,研究α-突触核蛋白边链解离过程所需的时间尺度小更容易研究。因此,第四章中,我们进行了α-突触核蛋白五聚体边链的解聚过程的拉伸分子动力学模拟研究。我们以α-突触核蛋白五聚体(残基44-97)为研究对象,研究其边链在一定拉力条件下的解离过程。研究发现,α-突触核蛋白解聚需要克服的主要相互作用是疏水相互作用。解离过程大概分为四步:1、肽段α-syn44-55的解离;2、肽段α-syn63-66的解离;3、肽段α-syn70-79的解离;4、肽段α-syn82-97的解离。其中,肽段α-syn63-66最难解离,因此该片段是解离的关键肽段,也是α-突触核蛋白聚集的关键肽段。4)通过小分子化合物抑制蛋白质低聚体形成和聚集是针对帕金森病的一种新型治疗策略。在这些抑制剂中,表没食子儿茶素没食子酸酯((-)-epigallocatechin-3-gallate,简称:EGCG)是绿茶中富于生物活性的化合物,可以抑制一系列淀粉样多肽的纤维化,如α-突触核蛋白、Aβ以及胰岛淀粉样多肽(IAPP)等。为了探究EGCG抑制α-突触核蛋白聚集的分子机理,在第五章中,我们进行了关于EGCG对α-突触核蛋白纤维影响的分子动力学模拟研究。我们应用两种不同大小的α-突触核蛋白纤维包括α-突触核蛋白五聚体和α-突触核蛋白十聚体作为研究模型。结果表明,EGCG可以降低α-突触核蛋白纤维的结构稳定性并且将其有序结构转换为无序结构。此外,研究发现EGCG在α-突触核蛋白纤维界面上的三个可能的结合位点。经过详细的分析结合模式后,确定S1和S3位点是最可能的结合位点并且分别拥有不同解构机制。即EGCG结合于α-突触核蛋白纤维通过破坏β-sheet二级结构降低有序度和破坏分子内氢键改变希腊花环式结构。此外,EGCG主要通过疏水作用和氢键相互作用结合于α-突触核蛋白纤维。在结合过程中,残基ASP 98,LYS 96,GLU 61,LYS 58 及 THR 64起到重要的作用。我们的工作所揭示的分子机制将有助于理解EGCG如何改变成熟纤维的结构并且为发现和设计α-突触核蛋白纤维的潜在抑制剂提供有价值的信息。
Other AbstractParkinson's disease is a second-ranked neurodegenerative disease, compared to Alzheimer's disease, which incidence is as high as 2 percent in people older than 65. Parkinsonian motor symptoms include bradykinesia, muscular rigidity and resting tremor, while non-motor ones include olfactory dysfunction, cognitive impairment, psychiatric symptoms and autonomic dysfunction. Microscopically, the specific degeneration of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies, which are brain deposits containing a substantial amount of α-synuclein, have been recognized.
It is the main pathological process of Parkinson's disease that misfolding and aggregation of α-synuclein to form oligomers and fibrils. Due to the rapid and small time scale of its biochemical processes, it is difficult to reveal the pathological process of misfolding and aggregation in experimental studies. Molecular dynamics (MD) simulation can provide detailed structural dynamics information to study protein misfolding and aggregation as well as the pathological mechanism of related diseases. In this paper, we applied molecular dynamics simulation to studying the misfolding and aggregation mechanism of α-synuclein and the inhibitory mechanism of small molecule inhibitors, providing theoretical support for further elucidation of the pathogenesis of Parkinson's disease and rational new drug design.
In the first chapter, we summarized the pathogenesis of Parkinson's disease, current diagnostic and therapeutic strategies, and the structure and function of α-synuclein and introduced the research progress of molecular dynamics simulation of α-synuclein, as well as the molecular dynamics simulation methods and principles methods. In the following four chapters, we explored the aggregation mechanism of α-synuclein and the inhibition mechanism of corresponding small molecule inhibitors from different structural levels.1)Peptide α-syn47-56 is the toxic core of α-synuclein and plays a key role in the process of aggregation and misfolding of α-synuclein. The template induction study of this peptide can reveal the molecular mechanism of α-synuclein misfolding and aggregation. Therefore, to reward the molecular mechanism of the peptide α-syn47-56 under the guidance of pentamer template of α-synuclein, we performed a molecular dynamics simulation of 400ns by placing the peptide α-syn47-56 on both sides of the α-synuclein pentamers in Chapter 2. It was found that there are different elongation characteristics on both sides of the pentamer in the peptide α-syn47-56. The β-sheet structure was easily formed on the left side of the α-synuclein pentamer to complete template induction while it is hard to get a β-sheet secondary structure on the right side of the α-synuclein pentamer. In addition, we also found that hydrogen bonding, electrostatic interaction and van der Waals force between α-synuclein pentamer and monomer play important roles in β-sheet extension.2) SNCA single gene mutation is an important cause of familial Parkinson's disease. Many experimental studies have shown that mutations E46K and H50Q can promote α-synuclein fibrosis to form amyloid plaques. However, its specific molecular mechanism has not yet been revealed. Therefore, Accelerated Molecular Dynamics (AMD) simulation  of 500ns for structural stability of wild-type and mutant E46K and H50Q of α-synuclein pentamer was conducted. The results showed that mutant E46K significantly changed the interchain interaction pattern of α-synuclein pentamer, and solvation could play an important role in maintaining interchain stability. In addition, E46K mutation can maintain the interchain skeleton hydrogen bond well and thus maintain the β-sheet secondary structure of the side chain, and the degree of order is increased compared with the wild type. However, for the mutation of H50Q, the binding free energy was confused, the degree of order was significantly reduced, the skeleton hydrogen bond between the chains almost completely disappeared, and the structural stability was reduced. Therefore, we believe that the pathogenesis of E46K mutation is different from that of H50Q mutation. E46K mutation can change the interaction between chains and the structural tendency of adjacent residues and improve the aggregate stability. However, the pathogenesis of H50Q mutation may not be related to the misfolding and aggregation process of α-synuclein. In addition, the secondary structural stability of β-sheet at α-syn49-55 plays an important role in the overall structural stability of α-synuclein aggregates, and the discovery of this key folded peptide can provide theoretical support for the research and development of new therapeutic strategies for α-synuclein.3)The α-synuclein side chain dissociation process is the reverse of this elongation process. Therefore, in chapter 4, we simulated the Steered Molecular Dynamics (SMD) of the depolymerization process of the side chain of the α-synuclein pentamer. We studied the dissociation of the side chain of the α-synuclein pentamer (residue from 44 to 97) and found that hydrophobic interaction plays an important role in the depolymerization of α-synuclein. Therefore, the corresponding van der Waals interaction is the main driver of α-synuclein aggregation. In addition, the depolymerization process of α-synuclein can be divided into four steps: 1. Depolymerization of peptide α-syn44-55; 2. Depolymerization of peptide α-syn63-66; 3. Depolymerization of peptide α-syn70-79; 4. Depolymerization of peptide α-syn82-97. The peptide α-syn63-66 is the most difficult one to depolymerize, so it is the key peptide of depolymerization and the corresponding key peptide of α-synuclein aggregation.4) Inhibition of protein oligomer formation and aggregation by small molecule compounds maybe an effective way to treat Parkinson's disease. Among these inhibitors, (-) -epigallocatechin-3-gallate (EGCG) is a bioactive compound in green tea that inhibits the fibrosis of a series of amyloid polypeptides, such as α-synuclein,  amyloid-β and international association for preventive pediatrics (IAPP). In order to explore the molecular mechanism of EGCG inhibiting a-synuclein aggregation, in chapter 5, we performed molecular dynamics simulation of the effect of EGCG on α-synuclein fibers. We took two different sizes of α-synuclein fibers including α-synuclein pentamer and α-synuclein decamerer as our aggregates models. The results showed that EGCG can reduce the structural stability of α-synuclein fibers and convert the ordered structure into disordered structure. In addition, three possible binding sites of EGCG at the interface of α-synuclein fibers were identified. After a detailed analysis of the binding mode, it is determined that S1 and S3 are the most likely binding sites with different remodeling mechanisms. In other words, EGCG binds to α-synuclein fibers and changes the structure by breaking the β-sheet secondary structure and intramolecular hydrogen bonds to destroy the Greek structure. In addition, EGCG is bound to α-synuclein fibers by hydrophobic interactions and hydrogen bond interactions. Finally, these residues: ASP 98, LYS 96, GLU 61, LYS 58 and THR 64 play important roles in EGCG binding. The molecular mechanisms revealed by our work will help to understand how EGCG remodels the structure of mature fibers and provide valuable information for the discovery and design of potential inhibitors of α-synuclein aggregates.
Pages112
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/340549
Collection化学化工学院
Affiliation
化学化工学院
First Author AffilicationCollege of Chemistry and Chemical Engineering
Recommended Citation
GB/T 7714
刘雪伟. α-突触核蛋白错误折叠、聚集以及与小分子抑制剂相互作用机制的分子模拟研究[D]. 兰州. 兰州大学,2019.
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