p53信号通路调节砷化物诱导的IKKα自噬降解反应和IKKβ转录抑制的信号传递机制研究

发布时间：2018-04-16 23:39

本文选题：砷化物 + p53　；参考：《广西医科大学》2017年硕士论文

【摘要】：研究背景及内容砷是一种广泛存在于自然界的重金属类毒性元素,对人类健康具有极大危害。通常,高剂量砷化物暴露主要引发以“细胞凋亡”等为主要特征的急性毒性损伤效应;而低剂量砷化物长期暴露主要引发以“细胞癌变”等为主要特征的慢性毒性损伤效应。因此,砷化物的健康危害效应机制研究具有重要的基础理论意义和医学应用价值。本课题组长期开展砷化物诱导细胞凋亡反应的急性毒性损伤效应机制研究工作,在以往研究中获得了大量具有价值的研究发现,为砷化物的健康危害评估和损伤防控策略研究提供了重要的理论依据和干预靶标。在近期工作中,我们又发现了砷化物诱导促细胞凋亡反应中伴随有IKK激酶的两个催化亚基——IKKα和IKKβ的表达水平下调现象,并且这一现象的发生是砷化物诱导细胞走向凋亡的重要前提。在对以上现象的分子机制进行深入研究过程中,我们先后发现了砷化物刺激可通过激活p53并进一步促发细胞自噬反应从而诱导IKKα进入自噬途径降解,同时p53也可通过诱导靶基因ETS-1表达而协同介导对IKKβ的转录抑制作用。本课题以上述研究结果为基础,在砷化物处理的HepG2细胞中,探讨了p53信号通路调节砷化物诱导的IKKα自噬降解反应和IKKβ转录抑制的信号传递机制(见前言部分附图)。结果在第一部分工作中,我们首先筛选了能够介导砷化物促发IKKα自噬降解反应的p53下游靶基因。结果发现:砷化物刺激HepG2细胞后能够诱导自噬相关p53靶基因DRAM1、ISG20L1、DAPK1、TIGAR、SESTRIN2表达,其中DRAM1能够介导砷化物诱导的IKKα自噬降解反应;而TIGAR和SESTRIN2与上述反应状态完全无关。ISG20L1和DAPK1能够介导砷化物刺激诱导的细胞自噬反应,但这种自噬反应并不能够介导IKKα降解;而且ISG20L1在砷化物刺激作用下的诱导表达反应也不受控于p53。以上实验结果说明:DRAM1是能够介导砷化物促发IKKα自噬降解反应的p53下游靶基因。在第二部分工作中进一步分析了砷化物刺激反应中负责催化p53/DRAM1信号传递途径诱导活化的上游蛋白激酶。结果发现:ATR、CHK1、LKB1、AMPKα、PERK均能够调节砷化物刺激作用下p53的转录激活活性,然而只有CHK1、LKB1是介导DRAM1诱导表达并进一步促发IKKα自噬降解反应的p53上游蛋白激酶。在第三部分工作中,我们初步分析了砷化物促发IKKα自噬降解反应的分子机制。由于IKKα和IKKβ高度同源,但只有IKKα能够进入自噬途径实现降解;因此我们推测:这种自噬反应的选择特异性一方面可能与IKKα本身的分子结构密切相关,另一方面也可能存在有协同因子参与协助IKKα的降解反应。我们的实验结果显示:砷化物刺激作用下IKKα能够通过其激酶结构域与LC3发生诱导性结合反应从而进入自噬降解途径,而CHK1、LKB1在此过程中可能发挥协同作用。尽管我们在第一部分工作中排除了ISG20L1和DAPK1在介导砷化物促发IKKα自噬降解反应中的作用,但却意外地发现了这两个信号蛋白能够介导IKKβ的转录抑制反应。因此我们在最后一部分工作中分析了ISG20L1和DAPK1调节砷化物刺激诱导IKKβ转录抑制的分子机制。结果发现:DAPK1能够协同ISG20L1作为活化p53-ETS-1-IKKβ转录抑制信号传递途径的上游蛋白,并进而通过调控MDM2依赖的GADD45α诱导表达反应而介导砷化物诱导的促细胞凋亡效应。结论综上所述,本论文研究结果初步揭示了砷化物刺激诱发IKKα选择性自噬降解反应的分子机制;同时也进一步完善了砷化物刺激诱导IKKβ转录抑制的信号传递机制。以上实验结果不仅为IKK激酶在细胞应激反应中的基因表达调控机制研究提供了创新性研究发现,并且为砷化物的毒性效应机制研究提供了崭新内容。
[Abstract]:Research background and contents of heavy metal arsenic is a toxic element widely exists in the nature, which is harmful to human health. Generally, the high dose of arsenic exposure is mainly caused by acute toxicity injury "apoptosis" as the main feature; while low dose arsenic exposure mainly caused by the effects of chronic toxicity "cancer cells" as the main feature. Therefore, health damage mechanism of arsenic has important theoretical significance and application value. The acute toxicity of medicine based the research group to carry out long-term arsenic induced apoptosis reaction damage study of the mechanism of effects in previous studies were obtained with a large number of the value found, provide a theoretical basis and an important target for intervention health hazard assessment and damage prevention and control strategy research for arsenic. In recent work, we also found The arsenic induced Pro apoptotic reactions with the two catalytic subunit of IKK kinase, the expression level of IKK alpha and IKK beta cut phenomenon, and this phenomenon is an important prerequisite for arsenic induced cell apoptosis. Further study in the process of the molecular mechanism of the above phenomenon, we the discovery of arsenic stimulation can activate p53 and further promote autophagy reaction to induce the autophagy pathway into IKK alpha degradation, while p53 may also co inhibition mediated transcription of IKK beta by inducing the expression of ETS-1 gene. This subject is based on the above research results, in the treatment of arsenic in HepG2 cells, discusses the signal of IKK alpha and IKK beta transcription autophagy degradation reaction of p53 signaling pathway in regulation of arsenic induced inhibition of the transfer mechanism (see preface Figure). Results in the first part, we first Screening can be mediated by arsenic on p53 downstream target genes IKK alpha autophagic degradation reaction. Results showed that arsenic stimulated HepG2 cells can induce autophagy related p53 target genes DRAM1, ISG20L1, DAPK1, TIGAR, SESTRIN2 expression, DRAM1 can IKK a autophagy mediated degradation reaction induced by arsenic while TIGAR and SESTRIN2; and the reaction condition has nothing to do.ISG20L1 and DAPK1 can response to autophagy mediated arsenic induced autophagy, but this reaction is not mediated by IKK and ISG20L1 in alpha degradation; arsenic stimulation induced expression of the reaction is not controlled by the above p53. results show that DRAM1 is can mediate arsenic on p53 downstream target genes IKK alpha autophagic degradation reaction. In the second part in the further analysis of arsenic in the stimuli responsible for catalysis of p53/DRAM1 signaling pathway activation on Tour the protein kinase. The results showed that: ATR, CHK1, LKB1, AMPK alpha, PERK can regulate arsenic stimulates transcription activation activity under the action of p53, but only CHK1, LKB1 mediates DRAM1 induced expression and further promote the development of IKK alpha autophagy degradation reaction of p53 upstream kinase. In the third part of this work. We analyzed the molecular mechanism of arsenic in promoting IKK alpha autophagy degradation reaction. Because of IKK alpha and IKK beta are highly homologous, but only IKK alpha can enter the autophagy pathway to achieve degradation; we hypothesize that the choice of a specific autophagic response may be closely related to the molecular structure and IKK alpha itself, another also there may be synergistic factors to participate in the degradation reaction of IKK alpha. Our experimental results showed that arsenic stimulated by IKK alpha through its kinase domain and LC3 induced binding reaction to enter autophagy degradation. Size, while CHK1 and LKB1 may play a synergistic role in the process. Although we exclude ISG20L1 and DAPK1 in arsenic mediated priming IKK alpha autophagic degradation reaction in the first part, but accidentally discovered the two signal protein transcription mediated IKK beta inhibitory response. Therefore we analyzed ISG20L1 and DAPK1 regulate arsenic induced molecular mechanism of IKK beta transcription inhibition in the last part of this work. The results showed that DAPK1 can cooperate with ISG20L1 as activation of p53-ETS-1-IKK beta signaling pathway upstream of the transcriptional repressor protein, and then through the apoptosis promoting effect of regulation of MDM2 dependent GADD45 expression induced by alpha reaction mediated by arsenic induced. In conclusion, the molecular mechanism of the results of this study revealed arsenic induced IKK alpha selective autophagic degradation reaction; at the same time further end The arsenic induced transcription inhibition of IKK beta signal transmission mechanism. The above results not only provide the regulation mechanism of innovative research found that gene IKK kinase in cell stress response expression, and toxicity mechanism of arsenic with new content.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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