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题名: 纳米富勒烯和取代多溴二苯醚神经毒性分子机制及构效关系
作者: 刘燕燕1
学位类别: 博士
答辩日期: 2017-05
授予单位: 中国科学院大学
授予地点: 北京
导师: 张爱茜
关键词: 神经毒性,神经保护作用,乙酰胆碱酯酶,凝血酶,取代多溴二苯醚,纳米富勒烯 ; neurotoxicity, neuroprotective effect, AChE, thrombin, substituted PBDEs,nano-C60
其他题名: Molecular Mechanism and Quantitative Structure-Activity Relationship for Neurotoxicity of Fullerene Nanoparticles and Substituted Polybrominated Diphenyl Ethers
学位专业: 环境科学
中文摘要: 神经系统发生紊乱将对人类健康造成巨大危害。近年来,越来越多的研究报道 很多持久性有机污染物和人工纳米材料等新型环境污染物可引发各种神经系统相 关的生物学效应和毒性。多溴二苯醚(polybrominated diphenyl ethers ,简称 PBDEs) 和富勒烯分别作为持久性有机污染物和人工纳米材料的典型代表,被多次报道在动 物实验中表现出对中枢神经系统的影响。例如,多溴二苯醚具有脑损伤等毒性,富 勒烯具有神经保护作用和抗脑梗塞等功能。但是它们神经毒性和神经保护作用的分 子机制尚未清晰解析。作为神经毒性的生物标志物分子之一,乙酰胆碱酯酶 (acetylcholinesterase,简称 AChE)很可能是环境污染物神经毒性机制所涉及的靶 点之一。而凝血酶在脑组织中的少量分布与脑梗塞病理的发生密不可分。所以,本 研究着眼于乙酰胆碱酯酶和凝血酶两个作用靶点,从分子水平考察取代多溴二苯醚 和纳米富勒烯对中枢神经系统影响的分子机制。本研究主要包括以下三部分内容: 1. 5’-OH-BDE-99经后门机制抑制 AChE活性 PBDEs的神经毒性报道很多,但是作用机制一直尚不明确。本论文选取神经毒 性标志物分子 AChE作为潜在靶点,研究取代 PBDEs与 AChE分子间的相互作用机 制。研究发现取代 PBDEs是可以通过与 AChE直接结合而发生酶活抑制的,说明 AChE很可能是其神经毒性效应作用的靶点之一。有趣的是,不同取代数目、不同 取代位置的取代 PBDEs与 AChE分子间的作用机制不尽相同。多数取代 PBDEs是 通过与 AChE催化活性中心进行结合而引发酶活抑制,而 5’-OH-BDE-99则是通过 与参与后门机制的 Trp86残基建立分子间氢键而引发酶活抑制,这一发现为将来设 计 AChE后门机制抑制剂分子提供了结构指引。 2.提出并证实非催化位点结合的新型 AChE竞争性抑制机制 通过实验分析和理论预测的方法考察了表面修饰富勒烯的 AChE活性抑制和 分子作用机制。由于特殊的尺寸,纳米富勒烯不能进入 AChE的活性口袋。酶促反 应动力学实验暗示富勒烯很可能结合在 AChE的活性口袋入口阴离子位点 PAS处, 限定了产物进入口袋,与此同时阻碍了底物离开口袋,最终降低了酶的活性。到目 前为止,很少有研究报道纳米材料与蛋白在分子水平上的作用机制。在本研究中, 分子模拟结合实验定点突变技术为我们提供了纳米富勒烯在 AChE的 PAS上特异 性的作用位点的直接证据。分子对接结果证明纳米富勒烯是通过与 Tyr72和 Trp286 形成 π-π堆积作用而与 AChE结合,并且定点突变也证实了两个残基的重要性。本 研究提出并证实了从未被报道的非催化位点结合的 AChE竞争性抑制机制,首次在 分子水平上阐明了富勒烯可通过非催化位点依赖的外周阴离子位点结合而竞争性 地抑制 AChE从而起到神经保护作用。 3.亲水修饰的富勒烯可通过结合在凝血酶催化位点而竞争抑制其酶活 不同于其它的碳基纳米材料,纳米富勒烯被报道可以引发抗凝效应,但是具体 的分子机理尚不明确。本研究通过考察表面改性的纳米富勒烯与凝血酶分子间的相 互作用,发现具有氢键供受体基团修饰的富勒烯是可以通过与底物和凝血酶的已知 抑制剂竞争,从而结合在凝血酶的催化活性位点上。通过分子对接手段发现,表面 修饰的富勒烯是通过表面分子与凝血酶催化三联体形成分子间氢键而抑制其酶活。 说明纳米富勒烯通过改性可以与凝血酶发生特异性地结合,为揭示富勒烯纳米材料 的神经保护机制提供了潜在的靶点和作用途径。 综上所述,论文的研究证实乙酰胆碱酯酶和凝血酶很可能是富勒烯纳米材料和 取代多溴二苯醚神经保护作用和神经毒性效应的靶点之一。然而,富勒烯纳米材料 选择性识别这两个靶标所需的结构特征迥异,疏水性富勒烯纳米材料可竞争性抑制 乙酰胆碱酯酶,但凝血酶需要水溶性相对较高的富勒烯纳米材料与其催化位点作用。 这说明两个靶标在富勒烯纳米材料神经保护作用中的作用是相互关联的并在一定 程度上由纳米富勒烯的表面性质决定。
英文摘要: Many human diseases are related to neurological disorders. Recently, an increasing number of studies reported that environmental pollutants, including persistent organic pollutants(POPs) and engineering nanomarterials(ENPs), could cause various effect on neural systems. Both polybrominated diphenyl ethers (PBDEs), as typical POPs,and fullerene,as a representative of ENPs, have been reported to show neurotoxicity or neuroprotective effect in animals. For example, PBDEs have induce brain damage in murine. Moreover, fullerene has been proved to play a role in neuroprotection and cerebral infarction resistence. However, the mechanisms of their effect on central neural system is still unknown. As a typical biomarker in neurotoxicity,acetylcholinesterase(AChE) is a potential target protein for environmental pollutants.Besides, thrombin has a great effect in cerebral infarction development since there is low expression of thrombin in brain tissue. Herein, the work focused on two target proteins (AChE and thrombin), respectively, to investigates the mechanisms of neuroprotection or neurotoxicity for fullerene and substituted PBDEs at molecular level. This dissertation mainly contains the following three parts: 1. 5’-OH-BDE-99 may inhibit AChE activity via back door mechanism We selected AChE as a target protein and investigated the interaction menchanisms between AChE and substituted PBDEs. The tested substituted PBDEs were found to induce enzyme activity inhibition through directly binding with AChE, indicating that AChE was likely a potential target protein of neurotoxicity of PBDEs. There are differentiated mechanisms for the substituted PBDEs with different substitution mode. Different from other substituted PBDEs directly bound to the catalysis active site (CAS)of AChE, 5’-OH-BDE-99 could bind with Trp86 which involved in back door mechanism of AChE, thus providing a guide for the design of tnovel AChE inhibitors. 2. A novel AChE competitive inhibition mechanism without CAS binding AChE inhibition of surface-modified C60 and its molecular mechanisms have been investigated by in vitro assays and in silico prediction method. Due to the distinct size, nano-C60 cannot enter into the AChE active site gorge. The competitive inhibition analysis provided information that nano-C60 might bind to the peripheral anionic sites (PAS) of AChE and subsequently prevented the substrate entering into the gorge and meanwhile restrained the product exiting, resulting in enzyme inhibition. A quantitative nanostructure-activity relationship (QNAR) model with statistical significance was established to reveal the determinant structure factor for AChE inhibition difference, which provided critical support for the design of safe nano-C60 in future. To date, little has been reported about the interaction mechanism between nanoparticles and protein at molecular level. In this work, however, molecular simulation combined with site-directed mutagenesis provided the direct evidence of specific binding of nano-C60 onto PAS of AChE. The surflex-dock results identified that the driving force for the interaction between nano-C60 and AChE was π-π stacking interaction, and the identified key residues Tyr 72 and Trp 286 were confirmed by mutagenesis. Briefly speaking, a novel AChE competitive inhibition mechanism without CAS binding was proposed and verified. Moreover, AChE inhibition via PAS binding is assumed to be one possible mechanism for the neuroprotective effect of nano-C60, which has not been reported before. 3. Competitive inhibition of thrombin by selected hydrophilic surface-modified nano-C60 We investigated a series of interactions between surface-modified nano-C60 and thrombin using in vitro assays combined with in silico analysis. Different from other carbon-based nanoparticles, nano-C60 has been reported to have anticoagulant effect. However, the related molecular mechanism is still unclear. In the study, we found selected nano-C60 modified with H-bond donor or acceptor groups could specifically bind into the active site of the enzyme by competing with the substrate and a known active site inhibitor. Nano-C60 can directly bind with active sites of thrombin via H-bond between surface group of nano-C60 and the catalytic triad. The result indicates that certain kinds of surface modification makes nano-C60 specifically bind with thrombin and also provides a potential target for revealing its neuroprotection mechanisms. In conclusion, the work proposed that AChE and thrombin were likely to be target proteins for neuroprotection and neurotoxicity of nano-C60 and PBDEs, respectively. However, the structural basis for molecular recognition between nano-C60 and two targets are totally different. Hydrophobic nano-C60 may competitively inhibit the AChE activity, while that of thrombin needs hydrophilic one to bind with. The roles of the two proteins in neuroprotection of nano-C60 largely depends on the surface feature of nano-C60.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38671
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
刘燕燕. 纳米富勒烯和取代多溴二苯醚神经毒性分子机制及构效关系[D]. 北京. 中国科学院大学. 2017.
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