本文選題：自噬相關蛋白4B　切入點：蛋白激酶B　出處：《第三軍醫(yī)大學》2016年博士論文

【摘要】：自噬相關蛋白4B(autophagy related 4B,ATG4B)是C54肽酶家族的一個成員,其在調控哺乳類細胞自噬過程中發(fā)揮著關鍵作用。在自噬發(fā)生過程中,細胞通過形成自噬小體包裹受損和衰老的細胞器,并進一步與溶酶體融合,在溶酶體酸性水解酶的作用下將內容物降解成氨基酸等小分子物質,從而為細胞內大分子的重新合成提供原料。在哺乳動物細胞中,微管相關蛋白1輕鏈3(MAP1LC3/LC3)在自噬小體的形成過程中發(fā)揮著十分重要的作用。前體LC3可以通過剪切修飾加工等步驟轉化為酯化形式的LC3,從而成為自噬小體的關鍵組分。研究表明ATG4B可通過對微管相關蛋白輕鏈3(LC3)系統(tǒng)的調控顯著影響自噬小體的組裝,進而調控細胞自噬水平,并在細胞代謝,感染與炎癥,神經系統(tǒng)疾病以及腫瘤等多種生理和病理過程中發(fā)揮著重要作用。近年來,有關ATG4B調控腫瘤細胞增殖的研究開始受到關注。研究表明,ATG4B可通過調控自噬促進骨肉瘤細胞的生長,缺乏ATG4B的骨肉瘤Saos-2細胞不能啟動自噬反應,該細胞系在小鼠模型中不能成瘤;使用ATG4B抑制劑能有效抑制骨肉瘤細胞的生長和成瘤。此外,還有研究表明ATG4B可以通過自噬非依賴的方式促進結腸癌細胞的惡性生長。我們的前期研究結果提示ATG4B可能在細胞線粒體中存在定位,但是否具有功能意義尚不清楚。線粒體作為細胞內的能量中心,產生了大多數(shù)的ATP以滿足細胞能量需求,線粒體功能紊亂可以導致細胞病理性的能量代謝障礙。此外,線粒體產生了細胞內絕大多數(shù)的活性氧簇(Reactive Oxygen Species,ROS),成為了調控細胞生命活動的重要信號傳導者。目前,已有大量研究表明線粒體的功能穩(wěn)態(tài)與腫瘤細胞的惡性進展密切相關,但有關ATG4B是否能直接調控線粒體功能繼而影響腫瘤細胞的表型還未見報道。因此,結合文獻報道和課題組的前期研究線索,本課題擬通過相關實驗研究驗證ATG4B的線粒體定位,解析其在促HCC細胞生長中的作用和機制,以期拓展ATG4B的功能研究意義,為基于ATG4B的抗HCC治療提高新的思路和科學依據。目的:本研究以肝癌細胞為模型,探討ATG4B的線粒體定位及其促HCC生長的作用及分子機制。方法:1.從不同細胞系和手術切除的組織中分離線粒體,通過westernblot實驗檢測atg4b在細胞線粒體的表達;2.構建帶有gfp標記的重組atg4b質粒,轉染細胞后,對細胞進行熒光線粒體標記,并在熒光共聚焦顯微鏡下觀察atg4b與線粒體的共定位;3.通過免疫膠體金標記電鏡、線粒體亞成分分離及蛋白酶k保護實驗等解析atg4b在線粒體的具體定位;4.使用免疫沉淀聯(lián)合質譜的方法,篩選與atg4b相互作用的蛋白(含高評分的線粒體蛋白);5.應用免疫共沉淀等技術鑒定atg4b與線粒體線粒體atp合酶亞單位的互作蛋白(atp5a1,atp5b和atp5c1,三者均為線粒體atp合酶f1部分的亞單位);6.使用crispr/cas9基因編輯技術建立雜合敲除的hcc細胞系(atg4b+/-),采用基因過表達等方法,檢測atg4b對線粒體atp合酶的活性影響;7.應用westernblot,探索過表達atg4b對細胞生長信號通路的作用及其激活的信號通路對細胞生長的影響;8.使用westernblot,免疫共沉淀及熒光成像等技術,探索akt對atg4b線粒體轉位的影響;9.運用生物信息學方法及westernblot,免疫共沉淀及基因克隆等技術,解析akt促atg4b線粒體轉位的可能機制(akt磷酸化atg4b第34位的絲氨酸);10.使用akt質粒,atg4b野生型質粒及第34位絲氨酸突變的質粒轉染細胞,通過westernblot和cck-8實驗等方法,驗證akt介導的atg4b第34位磷酸化在促細胞增殖中的重要性;11.使用akt質粒,atg4b野生型質粒及第34位絲氨酸突變的質粒轉染細胞,通過westernblot證實atg4b第34位絲氨酸磷酸化對hcc細胞自噬的作用;12.建立atg4細胞內活性檢測方法,測量atg4b第34位絲氨酸磷酸化對atg4b切割自噬底物的影響。結果:1.atg4b在哺乳動物細胞的線粒體(尤其是線粒體內部)存在明顯定位;2.線粒體的atg4b可以與atp合酶亞單位相互作用而抑制atp合酶的活性;3.蛋白激酶akt/pkb可促進atg4b的線粒體轉位;4.atg4b是akt的新的靶蛋白;5.akt通過磷酸化atg4b第34位的絲氨酸而促進atg4b向線粒體轉位;6.ATG4B第34位絲氨酸的磷酸化對HCC細胞的生長發(fā)揮著重要作用,此種作用可不依賴于ATG4B對細胞自噬的調控。結論:HCC細胞中,激活的AKT會介導ATG4B第34位的絲氨酸發(fā)生磷酸化,促進ATG4B的線粒體轉位,增加其與線粒體F0F1-ATP合酶亞單位的結合,從而抑制該合酶的活性,繼而使細胞代謝發(fā)生改變,最終促進了HCC細胞的惡性生長。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.7

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