GA6对自噬的调节作用及其机制

发布时间：2018-03-17 15:05

  本文选题：GA6　切入点：自噬　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:自噬是从真核生物到人都高度保守的一种细胞自我消耗的途径。在饥饿条件下,细胞通过自噬途径降解自身蛋白和细胞器得到核苷酸、氨基酸、脂肪酸、糖类和ATP,以维持细胞代谢的正常进行而得以在恶劣环境下继续存活,自噬还能清除细胞内错误折叠的蛋白和破损的细胞器,维持胞内蛋白和细胞器处于正常水平。自噬的适时发生和适当的自噬水平是机体正常运作的一大保证。自噬异常与炎症、肿瘤和神经退行性疾病等多种疾病有关,目前靶向自噬的药物3-甲基腺嘌呤和氯喹都已经被用于肿瘤的治疗,对自噬的深入了解能够辅助医疗,指导药物的开发和临床用药,促进新的治疗策略的产生。自噬流的主要检测指标包括pro-LC3向LC3-I的转化、LC3-I向LC3-II的转化,P62在自噬溶酶体中的降解。自噬促进时,pro-LC3的切割增加,LC3-I的脂化增加,P62的降解增加。自噬事件包括自噬的起始、成核、自噬前体膜的延长、自噬体的成熟、自噬体与溶酶体的融合、吞噬物的降解再利用。自噬前体膜的延长涉及到pro-LC3在半胱氨酸酶Atg4B的作用下被切割掉C末端的5个氨基酸(MKLSV)变成LC3-I,LC3-I经Atg7、Atg3、Atg10、Atg12-Atg5-Atg16L的共同作用被脂化形成磷脂酰乙醇胺(PE,Phosphatidylethanolamine)修饰形式的LC3-II,并特异性定位于自噬前体膜上,这为自噬体的成熟创造条件。但这一切割和脂化过程被如何抑制还不清楚,因此本研究拟探讨本实验室发现的GA6癌蛋白在这一切割和脂化过程中的作用及GA6对自噬的影响。内容:本研究的研究内容主要包括如下:1)探讨GA6是否与自噬有关。筛选与GA6有相互作用的蛋白,并进行验证。2)GA6对自噬有什么影响。GA6是促进自噬还是抑制自噬,有哪些表现可以证明。3)GA6对LC3切割的影响。GA6对LC3的切割有什么影响,具体影响机制是什么。4)GA6对LC3-I脂化的影响。GA6对LC3-I的脂化有什么影响,该影响是否因为LC3-I的形成变化引起。5)GA6对自噬的影响在C57小鼠上有什么表型。结合(60)Co照射,分析GA6、自噬与放射敏感性的关系。方法:利用CRISPR/Cas9技术构建了GA6-/-的乳腺癌细胞亚株ZR75-1和GA6-/-C57小鼠,并确定了能够有效敲低GA6的siRNA,建立了敲低GA6的宫颈癌细胞亚株Hela。利用免疫共沉淀结合质谱法和快速蛋白液相色谱法筛选与GA6有关的自噬蛋白,并与GA6进行Co-IP,进一步判断其是否与GA6具有相互作用,接着我们用GST pulldown实验最终确定具体是哪个自噬蛋白与GA6有直接相互作用,并确定此自噬蛋白为本研究的中心。应用免疫荧光技术观察点状LC3的形成情况,应用透射电子显微镜技术观察自噬体和自噬溶酶体的形成情况,应用Western blot技术检测LC3-I向LC3-II的转化情况。通过氯喹和雷帕霉素的处理,探究GA6对自噬的影响具体表现在哪个自噬环节,又是否通过mTOR通路。为探讨GA6对LC3的切割和脂化的影响,我们构建了Myc-LC3-PLA2的重组质粒,在体内研究GA6是否影响LC3的切割;构建了GST-LC3-GFP的重组质粒,通过观察GFP荧光的强弱来体外研究GA6是否影响LC3的切割;通过GST pull-down实验探讨GA6、LC3、Atg4B三者之间的定位关系,明确GA6影响LC3的切割的具体机制。为了研究脂化,我们构建了GFP-LC3-Hela的稳定细胞亚株,体内通过流式细胞术检测GFP的平均荧光强度(MFI,Median Fluorescent intensities)来反映LC3-II的形成情况;体外通过LC3-I与PE beads的结合情况来反映GA6对LC3-I的脂化的影响。在动物水平方面,我们将野生型小鼠分为正常饲养组和自噬诱导组,分别进行1000cGy剂量的(60)Co照射,统计生存曲线来研究自噬对小鼠放射敏感性的影响;将GA+/+组和GA6-/-组小鼠分别进行1000 cGy剂量的(60)Co照射,统计生存曲线来研究GA6对小鼠放射敏感性的影响。结果:(1)LC3与GA6有直接相互作用。通过免疫共沉淀结合质谱法、快速蛋白液相色谱法和免疫共沉淀法检测到GA6与LC3有相互作用,通过GST pull-down确定LC3与GA6有直接相互作用,因此将研究中心定为LC3。(2)GA6抑制自噬。免疫荧光显示GA6敲低或者敲除均导致细胞质中点状LC3的形成增加,Western blot显示GA6抑制LC3向LC3-II的转化,透射电子显微镜显示GA6抑制自噬体的形成。因为磷酸氯喹(CQD,Chloroquine Diphosphate Salt)处理后GA6对自噬的抑制作用更明显,所以我们判断GA6对自噬的抑制出现在自噬体与溶酶体融合之前,也就是自噬前体延长成自噬体的过程。并通过雷帕霉素的处理发现GA6抑制自噬不依赖于mTOR通路。(3)GA6抑制Atg4B对LC3的切割。体内实验表明,当GA6过表达时,LC3-PLA2的切割减少;体外实验表明,Atg4B单独存在时,GST-LC3-GFP的绿色荧光变为无色,说明Atg4B可切割LC3;GA6、Atg4B同时存在时,GST-LC3-GFP的绿色荧光变化不大,表明GA6抑制Atg4B对LC3的切割。又因为GA6、Atg4B定位于LC3的同一片段,表明GA6与Atg4B竞争性结合于LC3而导致GA6抑制Atg4B对LC3的切割。(4)GA6抑制LC3-I的脂化。体内实验表明,当GA6过表达时,MFI of GFP-LC3-II减少;体外实验表明,当有GA6存在时,结合在PE beads上的LC3-I减少。(5)小鼠诱导自噬后表现为放射抵抗,GA6-/-小鼠表现为放射敏感。当野生型正常饲养组和自噬诱导组小鼠经过相同剂量的(60)Co照射后,生存曲线分析显示,与正常饲养组相比,自噬诱导组小鼠死亡更慢;当GA6+/+与GA6-/-小鼠经过相同剂量的(60)Co照射后,生存曲线分析显示,与GA6+/+小鼠相比,GA6-/-小鼠死亡更快。结论:发现了一个新的抑制自噬的分子——GA6。GA6通过与Atg4B竞争性结合于LC3抑制Atg4B对pro-LC3的切割,从而抑制LC3-I的形成,GA6还抑制LC3-I的脂化。GA6由于抑制pro-LC3的切割和LC3-I的脂化导致自噬前体延长障碍,从而抑制自噬。
[Abstract]:Objective: autophagy is a cellular pathway from eukaryotes to have highly conserved self consumption. Under conditions of starvation, cells through autophagy pathway to degrade its nucleotides, proteins and organelles of amino acids, fatty acids, carbohydrates and ATP, in order to maintain the normal metabolism of the cells to survive in the harsh environment autophagy, can remove protein misfolding and intracellular organelle damage, maintain cytoplasmic proteins and organelles at normal levels. The timely and appropriate occurrence of autophagy autophagy is a major guarantee the normal operation. Autophagy abnormalities and inflammation, tumor and neurodegenerative diseases and other diseases, the current target to autophagy 3- methyladenine and chloroquine drugs have been used for the treatment of cancer, in-depth understanding of autophagy can help guide the development of medical, medicine and clinical medicine, promote new treatment The strategy is produced. The main indexes of autophagy flow including pro-LC3 conversion to LC3-I, LC3-I to LC3-II conversion, the P62 degradation in autolysosome. Promote autophagy, cutting increased pro-LC3, increased lipid LC3-I, the degradation of P62 increased. The autophagy events include starting autophagy, nucleation and extension of autophagy the precursor film, autophagosome maturation, fusion of autophagosomes and lysosomes, degradation of phagosomes recycling. 5 amino acids involved in caspase pro-LC3 under the action of Atg4B was cut off at the end of the extended C autophagy precursor film (MKLSV) into LC3-I, LC3-I by Atg7, Atg3, Atg10, CO the role of Atg12-Atg5-Atg16L is to form lipid phosphatidylethanolamine (PE, Phosphatidylethanolamine) modified forms of LC3-II, and specifically located in the autophagy precursor film, which create the conditions for autophagosome maturation. But all this process is how to cut and fat suppression Is not clear, so this study intends to explore the effects of GA6 and GA6 in our laboratory found cancer protein in the cutting and fat in the process of autophagy. Content: This study mainly includes the following: 1) to investigate whether GA6 is related to autophagy. Screening the interaction between GA6 and protein. Verify.2 GA6) what is the effect of.GA6 on autophagy promotes autophagy or inhibition of autophagy, which performance can prove the effect of GA6 on LC3.3) cut.GA6 LC3 cleavage of what effect, the influence mechanism is what.4 GA6) effects on LC3-I lipid.GA6 of LC3-I fat what effect whether this effect because.5 caused the change of LC3-I) what is the effect of GA6 on autophagy phenotype in C57 mice. (60) combined with Co irradiation, GA6 analysis, the relationship between autophagy and radiosensitivity. Methods: to construct the GA6- / - breast cancer using CRISPR/Cas9 Technology Cell sublines ZR75-1 and GA6-/-C57 mice, and to determine the effective on siRNA low GA6, established the knockdown of GA6 cervical cancer cell line Hela. by immunization combined with mass spectrometry and fast protein liquid chromatography screening GA6 associated with autophagy protein co precipitation, and Co-IP and GA6, a judge whether it has interaction with GA6, then we use the GST pulldown experiment and ultimately determine which specific autophagy protein GA6 with direct interaction, and to determine the autophagy protein for the research center. The formation of immunofluorescence observation point LC3, the formation of application of transmission electron microscopy observation of autophagosomes and autolysosomes. The application of Western blot technique to detect LC3-I conversion to LC3-II. Through the treatment of chloroquine and rapamycin on autophagy, which link to explore the effects of GA6 on autophagy specific performance, and whether the MTOR pathway. In order to explore the effect of LC3 and GA6 cutting fat, we constructed the recombinant plasmid Myc-LC3-PLA2, in vivo effects of LC3 GA6 is cutting; construction of the recombinant plasmid GST-LC3-GFP, to study the in vitro effect of LC3 GA6 is cutting through the observation of GFP fluorescence intensity by GST pull-down; experimental study of GA6. LC3 Atg4B, relationship between the three, the specific mechanism of clear GA6 effect of LC3 cutting. In order to study the lipid, we construct GFP-LC3-Hela stable cell line in vivo, by the mean fluorescence intensity of GFP by flow cytometry (MFI Median, Fluorescent intensities) to reflect the formation of LC3-II in vitro by combining; LC3-I and PE beads to reflect the effect of GA6 on LC3-I resin. In the animal level, we will be the wild type mice were divided into normal diet group and autophagy induction group, respectively 1000cGy agent The amount of (60) Co irradiation, statistical survival curves to study autophagy of mice Radiosensitivity; GA+/+ group and GA6-/- group were respectively 1000 doses of cGy (60) Co irradiation, the statistical survival curve to study the influence of GA6 on the radiosensitivity of mice. Results: (1) LC3 and GA6 have each other directly the role of the immune co precipitation method combined with mass spectrometry, fast protein liquid chromatography method to detect the GA6 and LC3 interaction and co immunoprecipitation, LC3 and GA6 have a direct interaction with GST pull-down, the Research Center for LC3. (2) GA6 inhibited autophagy. Immunofluorescence showed that GA6 knockdown or knockout were led to the formation of cytoplasmic punctate LC3 increases, Western blot showed that GA6 inhibited LC3 to LC3-II conversion, transmission electron microscopy showed the formation of autophagosomes. Because of the inhibition of GA6 (CQD Chloroquine Diphosphate, chloroquine phosphate Salt) after the treatment of GA6 on autophagy The inhibitory effect is more obvious, so we determine the inhibition of GA6 on autophagy occurs before and lysosome fusion, is also the precursor to extend into the process of autophagy autophagy by rapamycin treatment. It was found that GA6 inhibited autophagy is not dependent on mTOR pathway. (3) GA6 inhibited Atg4B LC3 cleavage of the in vivo experiments show. When, over expression of GA6, LC3-PLA2 cutting decreased; the in vitro experiments showed that Atg4B alone, the green fluorescence of GST-LC3-GFP becomes colorless, Atg4B LC3 GA6, Atg4B cutting; existing at the same time, the green fluorescence of GST-LC3-GFP changed little, suggesting that GA6 inhibits LC3 cleavage of Atg4B. Because GA6, Atg4B positioning the same segment to LC3, GA6 inhibited Atg4B LC3 cleavage of which showed that GA6 and Atg4B competitive binding to LC3. (4) GA6 inhibits LC3-I lipidation. In vivo experiments showed that the overexpression of GA6, MFI of GFP-LC3-II was decreased; in vitro. The results show that, when there is GA6, with PE beads in LC3-I decreased. (5) mice showed resistance to radiation induced autophagy, GA6-/- mice exhibited radiation sensitive. When the wild type normal feeding group and autophagy induced group of mice after the same dose (60 Co) after irradiation, the survival curve analysis showed that compared with the normal diet group, slower death induced autophagy mice; when GA6+/+ and GA6-/- mice after the same dose (60 Co) after irradiation, the survival curve analysis showed that, compared with GA6+/+ mice, GA6-/- mice died faster. Conclusion: the discovery of a new molecular GA6.GA6 inhibition of autophagy by binding to the Atg4B competition in LC3 suppressed Atg4B cleavage of pro-LC3, thus inhibiting the formation of LC3-I, GA6.GA6 LC3-I also inhibited lipid lipid due to inhibition of pro-LC3 cutting and LC3-I lead to autophagy precursor to inhibit autophagy. Extend the obstacles

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q23
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本文编号：1625257