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题名	环境污染物对生物靶蛋白氨基酸脱羧酶的识别及分子毒理机制研究
作者	汪素芳
学位类别	博士
答辩日期	2015-05
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	全氟烷基酸，有机磷酸酯，有机汞，氨基酸脱羧酶，生物靶点，PFAAs, OPEs, Organomercury, Amino acid decarboxylase, Biological target
其他题名	Study on Environmental Pollutants Recognition to Biological Target Amino Acid Decarboxylase and Molecular Mechanism of Toxicity
学位专业	环境科学
中文摘要	全氟烷基酸（Perfluorinated alkyl acids, PFAAs）、有机磷酸酯阻燃剂（Organophosphate esters, OPEs）和有机汞类物质是一类在环境中广泛存在、严重危害人类健康的有毒环境污染物，毒理学研究表明这些污染物能够对生物体造成多种损害，如肝毒性、免疫毒性、神经毒性、生殖发育毒性、内分泌干扰效应和致癌性等。对职业暴露及普通人群的流行病学调查发现血液中这些污染物的浓度与人类的一些疾病（如糖尿病、心血管、神经退行性疾病）及肿瘤（主要为肾癌、前列腺癌、睾丸癌、胰腺癌）的发生呈显著正相关。目前，这些污染物的毒理学研究主要集中在动物实验、细胞水平和亚细胞水平，而对于其产生毒性作用的分子水平的机理研究较少。污染物与生物分子作用是污染物致毒的源头，因此要明确污染物的毒性分子机制，首先需要弄清污染物在生物体内会与哪些靶分子发生相互作用。氨基酸脱羧酶的生物学功能主要是负责多胺的生物合成。多胺在细胞生长和发育、组织修复、雄性雌性生殖器官的发育和功能调节等方面起着非常重要的作用，并且已有研究证明一些疾病甚至癌症的发生都与多胺水平异常有关，如皮肤癌、肺癌、肝癌、结肠癌、前列腺癌等。本论文分别从分子水平和细胞水平两个层面，研究了污染物与氨基酸脱羧酶的相互作用、作用后酶蛋白的构象变化和催化活性变化、细胞暴露后酶蛋白调控基因的表达变化、活性变化及后续生物学效应，由此阐明污染物通过与酶蛋白相互作用引发细胞内酶蛋白功能障碍和毒性效应的分子机制。同时结合分子对接模拟技术，探讨污染物毒性效应与其化学结构之间的关系，发现污染物的毒性效应取决于它们与酶蛋白结合的空间构型，且其效应强度与二者的结合能力基本一致，疏水作用和氢键为主要驱动力。本论文主要包括以下三部分研究内容。     1. PFAAs与赖氨酸脱羧酶的相互作用及毒性分子机制研究。本工作采用简单、快速、免标记的葫芦[7]脲/Dapoxyl（CB7/Dapoxyl）超分子荧光传感体系，利用酶催化产物和荧光探针与大环主体CB7之间的竞争结合，研究了16种PFAAs与赖氨酸脱羧酶（LDC）的相互作用，其中PFAAs包括13种全氟烷基羧酸（PFCAs）和3种全氟烷基磺酸（PFSAs）。结果表明除了短链PFCAs（少于7个碳）外，其它PFAAs对LDC表现出明显的抑制作用，并且随着碳链长度的增加，抑制作用显著增加，其中含18个碳的PFOcDA抑制作用最强，PFCAs抑制常数为2.960~290.8 μM，PFSAs抑制常数为41.2267.44 μM。圆二色光谱表明PFAAs与酶蛋白的结合能够诱导酶蛋白的二级结构发生显著变化。分子对接结果揭示出抑制效应的差异取决于二者的结合模式，主要受PFAAs的尺寸、取代基团和疏水性的影响。在非毒性剂量下，三种代表性PFAAs包括PFOA、PFOS和PFOcDA在细胞水平进一步验证这种抑制作用及后续产生的生物学效应，研究表明三种PFAAs可明显抑制HepG2肝细胞内LDC的活性，并导致细胞内尸胺水平的下降，这些结果表明LDC很有可能是PFAAs细胞内毒性作用的一个新靶标分子，为PFAAs肝毒性分子机理的阐释提供了一个新线索。     2. OPEs与LDC的相互作用及毒性分子机制研究。在筛选污染物与LDC的选择性相互作用时，为了降低酶的使用量，我们对大环主体/荧光染料信号传导单元进行了优化，采用更加经济的CB7/AO超分子荧光传感体系研究了12种OPEs（包括芳香取代、烷基取代和氯代烷基取代）与LDC的相互作用。研究表明除6种烷基取代的OPEs外，芳香取代和氯代烷基取代的OPEs可显著抑制LDC的活性，IC50为1.329.07 μM，其中三甲苯磷酸酯（TCrP）的抑制作用最强，甚至强于生物体内LDC的天然抑制剂ppGpp（1.60 μM）。而且，在非毒性剂量下，这6种芳香取代和氯代烷基取代的OPEs对 PC12神经细胞内LDC的活性也表现出明显的抑制效应，并导致后续的生物学效应即尸胺含量的显著降低。分子对接结果揭示出不同的结合模式和键合残基导致OPEs抑制效应的差异。我们的研究表明，LDC也可能是OPEs在细胞内毒性作用的一个生物靶点，同时也可能是OPEs暴露导致神经毒性的原因之一。      3.有机汞与精氨酸脱羧酶（ADC）的相互作用及毒性分子机制研究。目前对于甲基汞神经毒性的致毒机制尚没有明确的定论，而胍丁胺合成并储存于神经元细胞，是一种具有重要生理功能的神经递质。因此对ADC活性以及基因蛋白表达的影响可能也是甲基汞神经毒性的一种可能机制。本工作采用CB7/AO荧光传感体系研究了3种有机汞（包括甲基汞、乙基汞和苯基汞）与ADC的相互作用。研究表明，甲基汞可以显著抑制ADC的活性，IC50为8.64 nM。我们进一步在细胞水平研究了甲基汞对ADC基因表达、蛋白表达、酶活性的影响以及PC12细胞内胍丁胺水平的变化。本研究将为甲基汞诱发人类神经毒性提供一个可能的机制。
英文摘要	Perfluorinated alkyl acids (PFAAs), organophosphate esters (OPEs) and organomercury are toxic environmental pollutants, which are harmful to human health. Toxicology studies have shown that these pollutants can cause various damage to organisms, such as hepatoxicity, immunotoxicity, neurotoxicity, reproductive and developmental toxicity, endocrine disrupting effects and carcinogenicity. Occupational exposure and general population epidemiological investigation revealed positive correlation between some of these pollutants in the blood and human diseases such as diabetes, cardiovascular and neurodegenerative diseases) and tumor (mainly for kidney cancer, prostate cancer, testicular cancer, pancreatic cancer). At present, the toxicology research of these pollutants mainly concentrated in animal experiments, cellular and subcellular levels, and the research for the toxic mechanism is still less. The interaction between pollutants and biological molecules is the source of their toxicity, therefore, we need find out the molecule targets with which the pollutants in organisms interact, and so to understand the toxic molecular mechanism of these pollutants. Amino acid decarboxylases play dual roles in acid resistance and the synthesis of polyamines. Polyamines have long been associated with cell growth and development, and are known to be involved in the development of several tissue types. In addition, polyamines have also been implicated in the development and function of both male and female reproductive organs. Early studies indicated that some diseases including cancers are closely associated with the abnormal polyamine level, such as skin cancer, lung cancer, liver cancer, colon cancer, prostate cancer, etc. So we investigate the interactions between pollutants and amino acid decarboxylase from cellular and molecular level, including protein structure and catalytic activity alteration upon binding of pollutants, the expression change of enzyme by activity, protein and mRNA level, and subsequent biological effect in exposed cells. We hope this study would help to improving our understanding on the mechanisms of these pollutions toxicology. Combined with molecular docking, we also explore the relationship between pollutant toxicity and its chemical structure, and found that the toxicity of pollutants highly dependent on the binding geometry of a pollutant with proterin at its active site. The structural dependence was rationalized by structure-activity relationship analysis that took into account the dominant hydrophobic forces and hydrogen bonding. This paper mainly include the following three parts.     1. The interactions between PFAAs and lysine decarboxylase (LDC) were investigated, and the molecular mechanism of toxicity was evaluated. The interaction properties of 16 PFAAs, including 13 perfluorinated carboxylic acids (PFCAs) and 3 perfluorinated sulfonic acids (PFSAs), between lysine decarboxylase (LDC) were determined using macrocyclic receptors and fluorescent dyes such as CB7/Dapoxyl. This product-competitive displacement method is simple, convenient and label-free. The results showed that the inhibitory effect of PFCAs increased significantly with carbon chain (7-18 carbons), whereas the short chain PFCAs (less than 7 carbons) did not show any effect. The inhibition constants fall in the range of 2.960 μM to 290.8 μM for targeted PFCAs, and 41.22 μM to 67.44 μM for targeted PFSAs. Circular dichroism results showed that PFAAs binding induced significant protein secondary structural changes. Molecular docking revealed that the inhibitory effect could be rationalized well by the cleft binding mode as well as the size, substituent group and hydrophobic characteristics of the PFAAs. At non-cytotoxic concentrations, three selected PFAAs inhibited LDC activity in HepG2 cells, and subsequently resulted in the decreased cadaverine level in the exposed cells, suggesting that LDC may be a possible target of PFAAs for their in vivo toxic effects.     2. The interactions between OPEs and LDC was investigated and subsequent molecular mechanism of toxicity were studied. In order to reduce the usage of enzyme in the screening of interaciton between pollustants and LDC, we optimized the macrocycle/fluorescent dye reporter pair. In this work, the effect of twelve OPEs with aromatic, alkyl or chlorinated alkyl substituents on the activity of LDC was assessed quantitatively with a more economic CB7/AO supramolecular fluorescence sensing system. The twelve OPEs were found to vary in their capacity to inhibit LDC activity. Alkyl group substituted OPEs had no inhibitory effect. By contrast, six OPEs substituted with aromatic or chlorinated alkyl groups inhibited LDC activity significantly with IC50 ranging from 1.32 μM to 9.07 μM. Among them, the inhibitory effect of tri-m-cresyl phosphate (TCrP) was even more effective as an inhibitor than guanosine 5'-diphosphate-3'-diphosphate (ppGpp) (1.60 μM), an LDC natural inhibitor in vivo. Moreover, at non-cytotoxic concentrations, these six OPEs showed perceptible inhibitory effects on LDC activity in PC12 living cells, and led to a marked loss in the cadaverine content. Molecular docking analysis of the LDC/OPE complexes revealed that different binding modes contribute to the difference in their inhibitory effect. Our finding suggested that LDC, as a new potential biological target of OPEs, might be implicated in toxicological and pathogenic mechanism of OPEs.     3. The interactions between organomercury and arginine decarboxylase (ADC) were investigated, and the molecular mechanism of toxicity was assessed. Now, the toxic mechanism of methyl mercury (MeHg) neurotoxicity is still not clear, and the agmatine as a neuromodulator is synthesized and stored in neuronal cells have many physiological functions. So the effect of MeHg on ADC activity, gene and protein expression may be a possible mechanism of MeHg neurotoxicity. The effect of organomercury on the activity of ADC was also assessed quantitatively with CB7/AO supramolecular fluorescence sensing system. The three organomercury were found to vary in their capacity to inhibit ADC activity. EtHg and PhHg had no inhibitory effect. By contrast, MeHg inhibited ADC activity significantly with IC50 was 8.63 nM. We further studied the effect of MeHg on ADC activity, gene expression, protein expression, and subquent agmatine level in PC12 cells. This study will provide a new possible target of MeHg for its in vivo toxic effects.
内容类型	学位论文
源URL	[http://ir.rcees.ac.cn/handle/311016/34378]
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	汪素芳. 环境污染物对生物靶蛋白氨基酸脱羧酶的识别及分子毒理机制研究[D]. 北京. 中国科学院研究生院. 2015.

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