丙二酸钠诱导应激颗粒形成的分子机制

发布时间：2018-06-10 17:36

本文选题：应激颗粒 + 丙二酸钠　；参考：《天津医科大学》2016年博士论文

【摘要】：目的:生物体生活在一个复杂的、多变的环境中,外界环境中也存在许多有害刺激,如:热休克、冷休克、氧化应激、病毒感染和UV照射等,真核细胞为了适应这些外界刺激,进化出了一系列复杂的应激系统,包括:应激颗粒(Stress Granules,SGs),加工体(Processing Bodies,PBs),线粒体应激,内质网应激(Endoplasmic reticulum Stress,ERS)等。然而在应激情况下去探讨细胞防御机制之间的功能联系是非常有意义的,有研究表明应激颗粒与内质网应激、线粒体应激之间有一定的联系。同时也有研究证明,在一种内质网钙泵的抑制剂毒胡萝卜素的刺激下,TAR DNA结合蛋白43(TAR DNA binding protein 43,TDP-43)和staufen 1蛋白被招募到应激颗粒中,而内质网应激诱导应激颗粒形成的机制已有文献报导。一些线粒体呼吸链抑制剂或解偶联剂等可以引起线粒体应激,同时有研究证明FCCP与百枯草还能诱导应激颗粒的形成,然而线粒体应激与应激颗粒形成之间的相关性,却没有明确的文献报导,因此本课题旨在探讨线粒体应激与应激颗粒形成之间的分子机制。方法:本研究分为三大部分,第一部分:(1)通过细胞增殖实验和细胞免疫荧光(Immunofluorescence,IF)确定丙二酸钠引起SGs形成的最适浓度;(2)通过细胞免疫荧光分析SGs标志性蛋白的共定位情况;(3)通过检测线粒体三磷酸腺苷(adenosine triphosphate,ATP)、活性氧(Reactive oxygen species,ROS)、膜电位(mitochonrial membrane potential,MMP)以及形态的改变,证明丙二酸钠对线粒体功能的影响;(4)利用细胞免疫荧光、活细胞工作系统等技术,综合分析SGs与线粒体的定位现象。第二部分:(1)利用多聚核糖体蔗糖密度梯度离心分析SGs形成与翻译抑制的相关性;(2)通过蛋白质印迹法,小干扰RNA(small interfering RNA,siRNA)转染,细胞免疫荧光等综合分析真核翻译起始因子2α(eukaryotic Initiation Factor2α,eIF2α)与SGs形成的相关性;(3)通过蛋白印迹,结合si RNA干扰、细胞免疫荧光分析真核起始因子4E结合蛋白(eIF4E binding protein,4EBP1)与SGs形成的相关性;(4)通过蛋白印迹,结合激酶抑制剂、细胞免疫荧光分析丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)途径与SGs形成间的相关性。第三部分:(1)通过Cell Titer-Glo?萤光细胞活性,蛋白印迹,免疫荧光,凋亡及细胞增殖的检测来探讨ATP与SGs形成之间的关系;(2)通过ROS检测,蛋白印迹,免疫荧光,细胞凋亡及增殖的检测来探讨ROS与SGs形成之间的关系。结果:第一部分:(1)当丙二酸钠的刺激浓度为0~200 mM时,细胞的增殖活性并未受到明显影响。在丙二酸钠浓度为50 mM时,胞浆内开始出现细小的SGs,当药物浓度增大到100 mM时,出现的SGs最为明显,浓度继续增大后SGs逐渐消失。(2)浓度为100 mM的丙二酸钠刺激后,HeLa细胞内源性的SGs标志性蛋白Ras-GTP酶激活蛋白SH3结构域结合蛋白(Ras-GTPase-activating protein SH3domain-binding protein,G3BP)分别和T细胞胞内抗原相关蛋白(TIA-1-related protein,TIAR)、T细胞胞内抗原1(T cell intracellular antigen 1,TIA-1)、人类抗原R(human antigen R,HuR)蛋白有共定位的现象。(3)与未处理组相比,100 mM丙二酸钠导致活性氧增多、膜电位降低、ATP减少。在未刺激时,线粒体呈现长条网络状结构,刺激后皱缩成小的团块。(4)在丙二酸钠刺激后,线粒体聚缩成为团块状,并在胞核周围聚集,形成的SGs聚集在线粒体的周围。第二部分:(1)与对照组相比,亚砷酸钠与丙二酸钠刺激时,多聚核糖体都出现了解聚的现象。(2)在丙二酸钠处理后,显著提高了eIF2α蛋白的磷酸化水平。利用RNA干扰技术,下调细胞内源性的蛋白激酶GCN2(general control nonderepressible 2,GCN2)或血红素调节的起始因子2α(heme-regulated initiation factor 2αkinase,HRI)的含量后,降低了eIF2α的磷酸化水平,蛋白激酶R(protein kinase R,PKR)和类蛋白激酶R的内质网激酶(PKR-like endoplasmic reticulum kinase,PERK)对其影响不大,但应激时GCN2或HRI这两个蛋白的敲低并未影响SGs的形成。(3)应激状态下,4EBP1的磷酸化被抑制,当下调细胞内4EBP1蛋白表达时,形成的SGs颗粒变少,体积减小。(4)应激时,MAPK途径的p38、细胞外调节蛋白激酶1/2(extracellular regulated protein kinases,Erk1/2)和c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)都被激活,当分别使用p38、Erk1/2、JNK蛋白的抑制剂SB203580、PD98059、SP600125处理后,SGs的形成并未发现明显改变。第三部分:(1)在丙二酸钠刺激情况下,提高细胞内ATP的含量时,形成SGs的数目不变,但体积有所增大。丙二酸钠处理后,补充或者不补充ATP都能引起4EBP1磷酸化的降低和eIF2α磷酸化的增强,然而ATP的补充与否并未引起eIF2α和4EBP1磷酸化状态的改变。在应激后,切割的caspase 3增多,凋亡增多,然而ATP的补充并未影响切割的caspase 3的含量以及凋亡情况,也未影响其细胞的增殖情况。(2)在丙二酸钠刺激情况下,当使用N-乙酰半胱氨酸(N-Acetyl Cysteine,NAC)降低ROS的水平后,形成SGs的数目、体积均未发生显著改变,与此同时eIF2α和4EBP1的磷酸化状态也没有明显变化。在应激后,ROS的清除并未影响切割的caspase 3的含量和凋亡情况,细胞的增殖情况也未受到影响。结论:1)在丙二酸钠刺激情况下,SGs标志性蛋白G3BP蛋白和TIAR、TIA-1、HuR三种SGs相关蛋白完全共定位,细胞胞浆中形成SGs。同时线粒体的相关功能指标,如:ATP、ROS、MMP、形态等都发生了明显改变,证明线粒体发生了功能紊乱。2)应激时,发生eIF2α、4EBP1和MAPK磷酸化的变化,其中4EBP1磷酸化的改变是诱导SGs形成的关键因素,eIF2α与MAPK的磷酸化并不是起主导作用。3)在刺激时,ATP的补充能促进SGs体积增大,此改变并不是通过eIF2α、4EBP1起作用的,ATP水平的改变对细胞的增殖和凋亡没有显著影响。而清除ROS后对SGs的体积和数目,细胞的增殖和凋亡也没有明显影响。
[Abstract]:Objective: living organisms live in a complex and changeable environment, and there are many harmful stimuli in the external environment, such as heat shock, cold shock, oxidative stress, virus infection and UV irradiation. In order to adapt to these external stimuli, eukaryotic cells have evolved a series of complex stress systems, including stress particles (Stress Granules, SGs). Processing Bodies (PBs), mitochondrial stress, endoplasmic reticulum stress (Endoplasmic reticulum Stress, ERS) and so on. However, it is very meaningful to explore the functional connections between cell defense mechanisms under stress. Some studies have shown that stress particles are excited by endoplasmic reticulum, and there is a certain relationship between mitochondrial stress. The TAR DNA binding protein 43 (TAR DNA binding protein 43, TDP-43) and Staufen 1 protein were recruited to stress particles under the stimulation of a kind of endoplasmic reticulum calcium pump inhibitor. The mechanism of endoplasmic reticulum stress induced stress particles formation has been reported in the literature. Some mitochondrial respiratory chain inhibitors or uncoupling agents are available. In order to induce mitochondrial stress, some studies have shown that FCCP and 100 withered grass can also induce the formation of stress particles. However, there is no clear literature on the correlation between mitochondrial stress and the formation of stress particles. Therefore, this study aims to explore the molecular mechanism between mitochondrial stress and stress particles formation. Methods: This study is divided into three Most, the first part: (1) the optimum concentration of SGs formation caused by sodium malonate was determined by cell proliferation experiment and cell immunofluorescence (Immunofluorescence, IF); (2) the co localization of SGs markers by cell immunofluorescence; (3) through the detection of mitochondrial ATP (adenosine triphosphate, ATP), reactive oxygen species (Reactive). Oxygen species, ROS), membrane potential (mitochonrial membrane potential, MMP) and morphological changes, which prove the effect of sodium malonate on mitochondrial function; (4) using cell immunofluorescence, living cell working system and other techniques to analyze the localization of SGs and mitochondria synthetically. The second part: (1) use polyribosome sucrose density gradient centrifugation The correlation between SGs formation and translation inhibition was analyzed. (2) the correlation between eukaryotic translation initiation factor 2 alpha (eukaryotic Initiation Factor2 a, eIF2 a) and SGs formation was analyzed by Western blot, transfection of small interference RNA (small interfering RNA, siRNA), cell immunofluorescence and so on. (3) immunoblotting, combined with Si interference, cell immunity The correlation between the fluorescence analysis of the eukaryotic initiation factor 4E binding protein (eIF4E binding protein, 4EBP1) and the formation of SGs; (4) the correlation between mitogen activated protein kinase (mitogen-activated protein kinase, MAPK) pathway and SGs formation through Western blot and kinase inhibitor, cell immunofluorescence analysis (mitogen-activated protein kinase, MAPK). The third part: (1) through Cell Glo? Fluorescence cell activity, Western blot, immunofluorescence, apoptosis and cell proliferation detection to explore the relationship between ATP and SGs formation; (2) the relationship between ROS and SGs formation through detection of ROS, Western blot, immunofluorescence, apoptosis and proliferation. Results: (1) the stimulation concentration of sodium malonate is 0~200 mM When the concentration of sodium malonate was 50 mM, a small SGs began to appear in the cytoplasm. When the concentration of the drug increased to 100 mM, the SGs was most obvious. The concentration continued to increase and SGs gradually disappeared. (2) the endogenous SGs marker protein Ras in HeLa cells was stimulated by the concentration of 100 mM. The -GTP protein SH3domain-binding protein (G3BP) and the intracellular antigen related proteins (TIA-1-related protein, TIAR) of the T cells (TIA-1-related protein, TIAR), and the protein of the intracellular antigen 1 of the T cells (3). Compared with the untreated group, 100 mM sodium malonate leads to the increase of reactive oxygen species, the decrease of membrane potential and the decrease of ATP. In the absence of stimulation, the mitochondria present a long strip network structure and then shrink into small lumps. (4) after the stimulation of sodium malonate, the mitochondria are condensed into mass, and the formation of SGs is gathered around the mitochondria. The two part: (1) compared with the control group, when sodium arsenite and sodium malonate were stimulated by sodium arsenite and sodium malonate, the accumulation of polyribosomes all appeared. (2) after the treatment of sodium malonate, the phosphorylation level of eIF2 alpha protein was significantly improved. RNA interference technique was used to reduce the endogenous egg white kinase GCN2 (general control nonderepressible 2, GCN2) or blood red. After the content of the starting factor 2 alpha (heme-regulated initiation factor 2 alpha kinase, HRI), the level of phosphorylation of eIF2 alpha is reduced, and the protein kinase R (protein kinase R, PKR) and the protein kinase R like endoplasmic reticulum kinase have little effect on it, but the two proteins are stressed during stress. The knock down did not affect the formation of SGs. (3) the phosphorylation of 4EBP1 was inhibited under stress. When the expression of 4EBP1 protein was downregulated, the formation of SGs particles became less and the volume decreased. (4) when stress was stressed, the p38 of the MAPK pathway, the extracellular regulated protein kinases, Erk1/2) and the terminal amino terminal kinase Rminal kinase, JNK) were activated, and when the inhibitors SB203580, PD98059, and SP600125 were treated with p38, Erk1/2, and JNK protein, the formation of SGs was not obviously changed. Third: (1) when the sodium propanate was stimulated, the number of SGs was increased, but the volume increased. After sodium malonate treatment, Supplementation or without supplementation of ATP could cause the decrease of phosphorylation of 4EBP1 and the enhancement of eIF2 alpha phosphorylation, however, the supplementation of ATP did not cause changes in the state of phosphorylation of eIF2 A and 4EBP1. After stress, the number of caspase 3 increased and apoptosis increased. However, ATP supplementation did not affect the content of caspase 3 and apoptosis, and no shadow of ATP. (2) under the stimulation of sodium malonate, when N- acetylcysteine (N-Acetyl Cysteine, NAC) was used to reduce the level of ROS, the number of SGs was formed, and the volume of SGs did not change significantly. At the same time, there was no significant change in the phosphorylation of eIF2 A and 4EBP1. After stress, the clearance of ROS did not affect the cutting C. The content and apoptosis of Aspase 3 and the cell proliferation were also not affected. Conclusion: 1) under the condition of sodium malonate, the SGs marker protein G3BP protein and the three SGs related proteins of TIAR, TIA-1, HuR are completely Co located, and the cellular cytoplasm forms SGs. and the mitochondrial function indexes, such as ATP, ROS, MMP, morphology, etc. The change of eIF2 alpha, 4EBP1 and MAPK phosphorylation, in which the phosphorylation of 4EBP1 is the key factor in the induction of SGs formation, the phosphorylation of 4EBP1 is not the dominant function of.3) the phosphorylation of eIF2 A and MAPK does not play a dominant role in the stimulation of the 4EBP1. The ATP supplement can increase the SGs volume, and this change does not pass through eIF2 alpha. The effects of 4EBP1 on the proliferation and apoptosis of cells were not significantly affected by the changes of ATP level, but the clearance of ROS had no significant effect on the volume and number of SGs, the proliferation and apoptosis of the cells.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R329.2

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