自噬相关蛋白ATG4B的线粒体定位及其促HCC细胞生长的作用和机制研究

发布时间：2018-03-27 09:19

本文选题：自噬相关蛋白4B　切入点：蛋白激酶B　出处：《第三军医大学》2016年博士论文

【摘要】：自噬相关蛋白4B(autophagy related 4B,ATG4B)是C54肽酶家族的一个成员,其在调控哺乳类细胞自噬过程中发挥着关键作用。在自噬发生过程中,细胞通过形成自噬小体包裹受损和衰老的细胞器,并进一步与溶酶体融合,在溶酶体酸性水解酶的作用下将内容物降解成氨基酸等小分子物质,从而为细胞内大分子的重新合成提供原料。在哺乳动物细胞中,微管相关蛋白1轻链3(MAP1LC3/LC3)在自噬小体的形成过程中发挥着十分重要的作用。前体LC3可以通过剪切修饰加工等步骤转化为酯化形式的LC3,从而成为自噬小体的关键组分。研究表明ATG4B可通过对微管相关蛋白轻链3(LC3)系统的调控显著影响自噬小体的组装,进而调控细胞自噬水平,并在细胞代谢,感染与炎症,神经系统疾病以及肿瘤等多种生理和病理过程中发挥着重要作用。近年来,有关ATG4B调控肿瘤细胞增殖的研究开始受到关注。研究表明,ATG4B可通过调控自噬促进骨肉瘤细胞的生长,缺乏ATG4B的骨肉瘤Saos-2细胞不能启动自噬反应,该细胞系在小鼠模型中不能成瘤;使用ATG4B抑制剂能有效抑制骨肉瘤细胞的生长和成瘤。此外,还有研究表明ATG4B可以通过自噬非依赖的方式促进结肠癌细胞的恶性生长。我们的前期研究结果提示ATG4B可能在细胞线粒体中存在定位,但是否具有功能意义尚不清楚。线粒体作为细胞内的能量中心,产生了大多数的ATP以满足细胞能量需求,线粒体功能紊乱可以导致细胞病理性的能量代谢障碍。此外,线粒体产生了细胞内绝大多数的活性氧簇(Reactive Oxygen Species,ROS),成为了调控细胞生命活动的重要信号传导者。目前,已有大量研究表明线粒体的功能稳态与肿瘤细胞的恶性进展密切相关,但有关ATG4B是否能直接调控线粒体功能继而影响肿瘤细胞的表型还未见报道。因此,结合文献报道和课题组的前期研究线索,本课题拟通过相关实验研究验证ATG4B的线粒体定位,解析其在促HCC细胞生长中的作用和机制,以期拓展ATG4B的功能研究意义,为基于ATG4B的抗HCC治疗提高新的思路和科学依据。目的:本研究以肝癌细胞为模型,探讨ATG4B的线粒体定位及其促HCC生长的作用及分子机制。方法:1.从不同细胞系和手术切除的组织中分离线粒体,通过westernblot实验检测atg4b在细胞线粒体的表达;2.构建带有gfp标记的重组atg4b质粒,转染细胞后,对细胞进行荧光线粒体标记,并在荧光共聚焦显微镜下观察atg4b与线粒体的共定位;3.通过免疫胶体金标记电镜、线粒体亚成分分离及蛋白酶k保护实验等解析atg4b在线粒体的具体定位;4.使用免疫沉淀联合质谱的方法,筛选与atg4b相互作用的蛋白(含高评分的线粒体蛋白);5.应用免疫共沉淀等技术鉴定atg4b与线粒体线粒体atp合酶亚单位的互作蛋白(atp5a1,atp5b和atp5c1,三者均为线粒体atp合酶f1部分的亚单位);6.使用crispr/cas9基因编辑技术建立杂合敲除的hcc细胞系(atg4b+/-),采用基因过表达等方法,检测atg4b对线粒体atp合酶的活性影响;7.应用westernblot,探索过表达atg4b对细胞生长信号通路的作用及其激活的信号通路对细胞生长的影响;8.使用westernblot,免疫共沉淀及荧光成像等技术,探索akt对atg4b线粒体转位的影响;9.运用生物信息学方法及westernblot,免疫共沉淀及基因克隆等技术,解析akt促atg4b线粒体转位的可能机制(akt磷酸化atg4b第34位的丝氨酸);10.使用akt质粒,atg4b野生型质粒及第34位丝氨酸突变的质粒转染细胞,通过westernblot和cck-8实验等方法,验证akt介导的atg4b第34位磷酸化在促细胞增殖中的重要性;11.使用akt质粒,atg4b野生型质粒及第34位丝氨酸突变的质粒转染细胞,通过westernblot证实atg4b第34位丝氨酸磷酸化对hcc细胞自噬的作用;12.建立atg4细胞内活性检测方法,测量atg4b第34位丝氨酸磷酸化对atg4b切割自噬底物的影响。结果:1.atg4b在哺乳动物细胞的线粒体(尤其是线粒体内部)存在明显定位;2.线粒体的atg4b可以与atp合酶亚单位相互作用而抑制atp合酶的活性;3.蛋白激酶akt/pkb可促进atg4b的线粒体转位;4.atg4b是akt的新的靶蛋白;5.akt通过磷酸化atg4b第34位的丝氨酸而促进atg4b向线粒体转位;6.ATG4B第34位丝氨酸的磷酸化对HCC细胞的生长发挥着重要作用,此种作用可不依赖于ATG4B对细胞自噬的调控。结论:HCC细胞中,激活的AKT会介导ATG4B第34位的丝氨酸发生磷酸化,促进ATG4B的线粒体转位,增加其与线粒体F0F1-ATP合酶亚单位的结合,从而抑制该合酶的活性,继而使细胞代谢发生改变,最终促进了HCC细胞的恶性生长。
[Abstract]:Autophagy related protein 4B (autophagy related 4B, ATG4B) is a member of the C54 peptide family of enzymes, which play a key role in the regulation of mammalian autophagy process. In the process of autophagy, cell organelle formation of autophagosomes damaged and aging through the package, and further the lysosome fusion with lysosomes. Under the action of acid hydrolase content degraded into small molecular substances such as amino acids, and large molecules in cells to provide raw materials. The synthesis in mammalian cells, microtubule associated protein 1 light chain 3 (MAP1LC3/LC3) plays a very important role in the formation of autophagy. Small body precursor LC3 by shear modified processing steps into esterified forms of LC3, which has become a key group of autophagosome. Studies show that ATG4B can be based on microtubule associated protein light chain 3 (LC3) system control effect of autophagy in small The assembly and regulation of cell autophagy, and in cell metabolism, infection and inflammation, plays an important role in many physiological and pathological processes of the nervous system disease and tumor. In recent years, the study on ATG4B regulation of tumor cell proliferation is beginning to receive attention. Studies show that ATG4B can promote the regulation of autophagy in human osteosarcoma cell line growth, lack of human osteosarcoma cell line Saos-2 ATG4B cannot start autophagy reaction, not tumor formation in a mouse model of this cell line; using ATG4B inhibitors can effectively inhibit the growth of osteosarcoma cells and tumor formation. In addition, another study showed that ATG4B can promote autophagy dependent mechanism of malignant growth of colon cancer cells. Previous research results we suggested that ATG4B may be located in mitochondria, but whether it has functional significance is not clear. Mitochondria as an intracellular energy center, the Most of the ATP cells to meet the energy demand, mitochondrial dysfunction can lead to pathological cell energy metabolism. In addition, mitochondrial ROS produced most of the cells (Reactive Oxygen Species, ROS), become the most important signal transduction to regulate cellular activities. At present, there are a large number of studies show that malignant progression the function of steady state and tumor cell mitochondria are closely related, but about whether ATG4B can directly regulate mitochondrial function and affect the phenotype of tumor cells has not been reported. Therefore, according to research reported in the literature and early clues to the project group, this paper through the experimental study verified mitochondrial localization of ATG4B, the promoting effect and mechanism analysis HCC cell growth, to expand the function of the significance of the research of ATG4B, anti HCC therapy for ATG4B based on improved new ideas and objective: the scientific basis. Study on the hepatic cancer cells as a model to investigate the mitochondrial localization of ATG4B and its promoting effect on HCC growth and its molecular mechanism. Methods: isolated mitochondria from resection in 1. different cell lines and operation organization, through the Westernblot experiment to detect the expression of atg4b in mitochondria; 2. construction of recombinant plasmid atg4b with GFP markers, after the transfection, fluorescence mitochondrial markers on the cell, and to observe the co localization of atg4b and mitochondria in fluorescence confocal microscope; 3. by immunogold labeling electron microscopy, mitochondrial sub component separation and protection experiment analysis of atg4b protease K in the specific location of mitochondria; 4. methods using immunoprecipitation combined with mass spectrometry, screening and atg4b interaction protein (mitochondrial protein containing high score); interacting proteins of 5. co immunoprecipitation techniques such as identification of atg4b and mitochondrial ATP synthase subunit (atp5a1, atp5b And atp5c1, subunit three are part of the mitochondrial ATP synthase F1); 6. crispr/cas9 gene editing technology to establish a hybrid cell line on HCC (atg4b+/-), in addition to using gene expression method, to study the effect of atg4b on mitochondrial ATP synthase activity; 7. by Westernblot, explored the effects of expression. The growth of atg4b signaling pathway in cells and its signaling pathway on cell growth; 8. Westernblot, CO immunoprecipitation and fluorescence imaging technology, explore the effect of Akt on atg4b mitochondrial translocation; 9. by bioinformatics methods and Westernblot, CO immunoprecipitation and gene cloning technology, the possible mechanism of Akt induced mitochondrial atg4b analysis translocation (Akt phosphorylation of atg4b serine thirty-fourth); 10. using the Akt plasmid, atg4b plasmid and wild type 34 serine mutant plasmid transfected cells by Westernblot and CCK-8 experiments. Method validation of Akt mediated atg4b thirty-fourth phosphorylation of importance in promoting cell proliferation in 11.; the use of Akt plasmid, atg4b plasmid and wild type 34 serine mutant plasmid transfected cells confirmed by Westernblot serine thirty-fourth phosphorylation of atg4b on autophagy in HCC cells; 12. set up a method for the detection of activated atg4 cells. Measurement of serine thirty-fourth phosphorylation of atg4b effect on atg4b cutting autophagy substrate. Results: the 1.atg4b in mammalian cell mitochondria (especially mitochondrial inner) has obvious orientation; 2. mitochondrial atg4b and ATP synthase subunit interactions and inhibit the activity of ATP synthase; 3. protein kinase akt/pkb can promote mitochondrial translocation of atg4b 4.atg4b is a new target protein; Akt; 5.akt and atg4b to promote mitochondrial translocation through phosphorylation of atg4b serine thirty-fourth phosphorylation of serine thirty-fourth; 6.ATG4B of HCC fine Cell growth plays an important role, this action does not depend on the regulation of ATG4B on autophagy. Conclusion: HCC cells, AKT activation mediated by ATG4B thirty-fourth serine phosphorylation, promote mitochondrial translocation of ATG4B, increase the F0F1-ATP level and mitochondrial enzyme combined sub, thereby inhibiting activity the synthase, and then make the cell metabolism change, and ultimately promote the malignant growth of HCC cells.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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