全氟化合物与脂肪酸靶蛋白的相互作用及干扰效应研究
文献类型：学位论文
| 作者 | 张连营 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郭良宏 |
| 关键词 | 全氟化合物 肝脏型脂肪酸结合蛋白 过氧化物酶体增殖剂激活受体 G-蛋白偶联受体40 信号通路 perfluorinated compounds liver fatty acid binding protein peroxisome proliferator-activated receptor γ G protein coupled receptor 40 signaling pathway. |
| 其他题名 | Studies on the Interactions of Perfluorinated Compounds with Fatty Acid Target Proteins and Subsequent Disruption Effects |
| 学位专业 | 环境科学 |
| 中文摘要 | 全氟类化合物 (PFCs) 是一类新型有机污染物，这类化合物应用广泛、在环境中难降解、能够远距离迁移，且随着食物链的传递在生物体内富集放大。在野生动物及人体中都检出了PFCs 的存在。PFCs 已造成明显的环境问题，其对生态环境和人类健康的影响也受到广泛关注。 PFCs 进入体内后可分布于全身多个脏器，其中肝脏、血液等组织中的含量最高。毒理学研究发现，PFCs 主要引起肝脏毒性和脂代谢毒性，同时PFCs 也具有内分泌毒性、生殖毒性、神经毒性、发育毒性以及免疫毒性等，被认为是一类具有全身多脏器毒性的环境污染物。流行病学调查发现PFCs 含量与脂肪酸代谢紊乱、糖尿病等疾病的发生率都有明显的相关性。PFCs 的毒理学数据多是根据实验动物暴露PFCs 后的生理指标及组织病理检测而获得的，对于PFCs产生这些毒性的机制尚不是很明确。 游离脂肪酸 (FFA) 不仅是人和动物体内的能量来源，也是一种重要的信号分子，在脂代谢、葡萄糖摄取、免疫等重要生理活动的调控中发挥重要作用。由于PFCs 的结构与脂肪酸相似，我们推测PFCs 可能会通过与脂肪酸竞争结合一些重要的结合蛋白或受体蛋白，从而产生毒性作用。基于此假设，本论文研究了PFCs 与脂肪酸结合蛋白及靶受体的相互作用，从肝脏型脂肪酸结合蛋白、PPAR核受体、GPR40 膜受体三个层面研究了PFCs 与相关蛋白质的直接结合作用，以及对其后续生物效应的影响作用，以期探讨PFCs 对这些功能蛋白生理作用的干扰机制。本论文研究内容主要包含以下三部分： 1. PFCs 与人类肝脏型脂肪酸结合蛋白 (hL-FABP) 的相互作用及正常功能的干扰效应。建立了研究配体与hL-FABP 相互作用的荧光竞争方法，并研究了PFCs 与hL-FABP 的结合特性。竞争结果显示PFCs 能够与hL-FABP 直接结合。对于≤11C 的全氟羧酸 (PFCAs) 来说，PFCs 与hL-FABP 结合能力随C 原子数的增加而增强；当＞11C 后，PFCAs 与hL-FABP 结合能力略微下降。末端基团也影响PFCs 与hL-FABP 的结合能力，表现为磺酸＞羧酸＞醇类。结构分析发现，PFCs 的结合能力是由hL-FABP 蛋白的空腔长度、PFCs 与hL-FABP 形成氢键的数目，这两个因素所决定的。基于本研究所测得的结合常数，我们评估了PFCs在人体内对hL-FABP 蛋白的干扰效应。发现对于普通人群来说，体内的PFCs 含量对hL-FABP/FFA 的结合几乎没有影响，但是对于职业暴露人群来说，PFCs对于体内hL-FABP/FFA 结合的竞争作用不容忽视。同时， PFCs 将脂肪酸竞争出hL-FABP，可能是PFCs 暴露导致肝脏内脂滴积累的原因之一。 2. PFCs 与过氧化物酶体增殖剂激活受体-γ (PPARγ) 的结合作用及信号通路激活效应。基于荧光偏振的荧光竞争方法研究了PFCs 与hPPARγ 受体的直接相互作用，分析了结合能力与PFCs 结构之间的构-效关系，发现二者的结合与PFCs的结构具有明显的相关性。中等链长PFCs 结合能力最强。PFCs 的结合能够改变hPPARγ 受体的构象，且能够在细胞内激活hPPARγ 信号通路，激活效应与其结合能力基本一致。计算模拟发现，不同链长的PFCs 在受体空腔中的位置不同，短链PFCs 匹配在受体的arm I 空腔，而长链PFCs 的非极性尾巴则伸向外侧的arm II 和arm III 区域。 PFCs 与Tyr-473、His-323 和His-449 三个重要残基形成氢键的数目也是影响其能否与PPARγ 受体结合及激活的重要因素。本部分研究明确了PFCs 可以激活PPARγ 信号通路。 3. PFCs 与G 蛋白偶联受体40 (GPR40) 的结合作用及激活效应。建立了检测配体与GPR40 直接结合作用的流式细胞分析方法 (FCM)，并研究了PFCs 与GPR40 的结合作用。结果显示中长链的PFOA、PFDA 和PFDoA 能够与GPR40直接结合，其中PFDA 的结合能力最强，约为油酸结合能力的1/14，PFDoA 和PFOA 与GPR40 的结合能力很弱；碳链太短或太长的PFCs 都未发现与GPR40结合。PFDA 能够通过GPR40 的结合而诱导细胞内的[Ca2+]i 升高，进一步激活ERK1/2 信号通路。本研究证明PFCs 类污染物不仅可以通过进入细胞后产生毒性作用，还可以在细胞外与膜受体结合，通过第二信使对细胞的正常生理功能产生干扰效应。PFCs 对GPR40 的干扰作用，可能是PFCs 诱发人类糖尿病的一个可能机制。 |
| 英文摘要 | Perfluorinated compounds (PFCs) have been widely used in industrial application and consumer products because of their high stability, strong lipophobic and hydrophobic properties. As a result, they have been found to be globally distributed in both environmental and biological media. These compounds have been detected in indoor and outdoor air, house dust, drinking water and food. PFCs have also been found in the liver, fat, and serum of wild animals, as well as in human serum, breast milk and semen. Perfluorooctane sulfonates (PFOS) and perfluorooctanoic acid (PFOA) are the two most commonly present PFCs. In May 2009, PFOS was added to the list of persistent organic pollutants of the Stockholm Convention to reduce and eventually eliminate its production and use. Due to their environmental persistence, bioaccumulation and biomagnification through the food web, the risks of PFCs to human health are of great concern and currently under evaluation. The toxicity of PFCs was investigated in the past decade or so. The liver toxicity and lipid disruption are the most important toxicity induced by PFCs, and reproductive toxicity, endocrine toxicity, and immunotoxicity also have been reported. Some epidemiological studies revealed positive correlation between PFOA and PFOS exposure and disorders of lipid metabolism, diabetes, and some cancers. The toxicology of PFCs were obtained from the physiological index or histopathology in animal studies. However, the mechanism(s) by which PFCs induce their toxicologies is not very clear. Due to their structural similarity with fatty acids, we speculate that the proteins whose endogenous ligands are fatty acids may play an important role in the PFCs-induced toxicology. So, we investigate the interactions between PFCs and three important proteins which can bind FA, including binding protein, nuclear receptor and membrane receptor. The binding affinity with PFCs, protein structure alteration, and subsequent bioactivity of the proteins were investigated. We hope this study would help to improve our understanding on the mechanisms of PFCs toxicology. 1. The binding interactions between PFCs and human liver fatty acid binding protein (hL-FABP) were investigated and the disruption of PFCs on the protein’s physiological function was evaluated. The binding properties between PFCs and hL-FABP were determined using a competitive fluorescence method. The results indicated that the PFCs could directly bind hL-FABP and the binding affinity was dependent on the carbon numbers and function groups of PFCs. The binding affinity of perfluorinated carboxylic acids (PFCAs) increased significantly with their carbon number from 4 to 11, and decreased slightly when the number was over 11. The three perfluorinated sulfonic acids (PFSAs) displayed comparable affinity, but no binding was detected for the fluorotelomer alcohols. The molecular docking revealed that the binding affinity depended on the binding cavity length of hL-FABP and the number of formed hydrogen bounds between PFCs and the protein. Based on the binding constant obtained in this work, the possibility of in vivo competitive displacement of fatty acid from hL-FABP by PFCs was estimated, and the results indicated that the competitive displacement of fatty acid by PFCs would be insignificant for the general population, but there were as much as ~38% and ~20% fatty acid would be displaced by PFOS and PFOA, respectively, for occupationally exposed workers. 2. The direct interaction between PFCs and human peroxisome proliferatoractivated receptor-γ (hPPARγ) was investigated and subsequent alteration of PPARγ pathway activity was studied. A structure-activity relationship between hPPARγ binding affinity and fluorinated carbon chain and functional group was evident, with medium-sized PFCs having the highest binding affinity. Circular dichroism (CD)results showed that PFCs binding induced conformational changes of the receptor. PPRE-luciferase reporter gene assay showed that PFCs were capable of activating hPPARγ in transiently transfected Hep G2 cells, and the activation potencies were in line with their binding affinities. Based on molecular docking analyses, we concluded that PFCs with different chain length were positioned into different pocket space of hPPARγ, and the number of hydrogen bonds formed with key residues (Tyr-473, His-323 and His-449) might be an important factor for the ligand binding and receptor activation. 3. The G-protein coupled receptor 40 (GPR40)-binding and activation properties of PFCs were investigated. The direct binding affinity between PFCs and GPR40 was obtained using flow cytomety (FCM) and found that the PFOA, PFDA and PFDoA, with a medium chain length, could bind to GPR40. The PFDA has the highest binding affinity with a Kd of 29.4 μM, which is about 1/14 of the oleic acid. PFDA could induce the rise of intracellular calcium ([Ca2+]i), and further study proved that the [Ca2+]i rise induced by PFDA was GPR40-dependent. The PFDA also could activate the ERK1/2 pathway, and the ERK1/2 activation was partly GPR40-mediated. This study proved that the PFCs can generate toxicology via binding membrane receptor and opening the second messenger. Furthermore, the disruption of GPR40 signaling pathway may be an important reason for the diabetes mellitus observed in the epidemiological studies. |
| 公开日期 | 2015-06-16 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/13483]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 张连营. 全氟化合物与脂肪酸靶蛋白的相互作用及干扰效应研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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