高尔基体蛋白STK16和G蛋白GNG7在有丝分裂和膜转运中的功能研究

发布时间：2018-04-18 05:39

本文选题：G蛋白 + GNG7　；参考：《中国科学技术大学》2017年博士论文

【摘要】：有丝分裂和膜转运异常是导致恶性肿瘤等疾病发生和发展的重要因素。近年来的证据表明,这两个看似相对独立的关键生命过程之间存在着某种紧密联系。然而至今,在有丝分裂和膜转运中起双重调控功能的蛋白及其调控机制研究相对匮乏,因此发现更多的、在两过程中均起关键调控功能的蛋白对于阐明肿瘤等疾病的发病机理有重要的指导意义。本文分别从高尔基体蛋白STK16激酶和Gγ蛋白GNG7入手,联合细胞生物学、生物化学和分子生物学等手段,深入探究两者在有丝分裂和膜转运中的功能及调控机制,获得的主要研究结果如下:1.高尔基体蛋白STK16在有丝分裂和膜转运中的功能研究STK16激酶主要定位于高尔基体和细胞膜上。高尔基体部分的STK16在间期时与高尔基体的中间膜囊标记蛋白Giantin共定位,而有丝分裂期时定位发生变化。STK16干扰或活性抑制后不仅推迟细胞进入分裂期,导致G2期细胞比例增高,而且细胞在分裂期的持续时间延长,前中期细胞和胞质分裂期细胞也大幅度增多。并且,STK16干扰或活性抑制后影响高尔基体在G2期和分裂前期的片段化,以及分裂后期高尔基体的重组装。进一步研究发现,STK16激酶可以与肌动蛋白actin直接结合,并以浓度依赖型和激酶活性依赖型形式调控actin的聚合和解聚动力学。因此,STK16激酶能直接通过调节actin的动态平衡影响高尔基体形态并调控细胞的有丝分裂进程。VSVG-GFP质粒转染显示,STK16并不影响内质网到高尔基体以及高尔基体到细胞质膜之间的顺式转运。然而,STK16却参与了经典的胞吞、铁转运蛋白的胞内循环以及表皮生长因子受体的降解途径;并且,STK16在细胞质膜和早期内体、以及高尔基体和内体之间的膜转运以及自噬中发挥功能。定点突变结果表明,肉豆蔻酰化和棕榈酰化修饰是STK16定位于细胞质膜和高尔基体的前提;两种修饰均可作用于STK16对actin的聚合动力学的调控、高尔基体形态和蛋白转运抑制剂刺激下的核质穿梭,提示STK16可能作为转录因子调控其他基因的表达和功能。2. G蛋白GNG7在有丝分裂和自噬中的功能研究报道显示,Gy亚基成员GNG7在几种肿瘤细胞中低表达;通过半定量RT-PCR检测,我们也发现GNG7在多种肿瘤细胞中的转录本水平显著低于正常细胞,表明GNG7可能是一个抑癌蛋白。因此,在GNG7低表达的骨肉瘤和宫颈癌细胞中过表达GNG7蛋白,并比较生长曲线的变化,结果表明GNG7过表达后不仅细胞生长被抑制,而且细胞大量死亡。进一步结合流式细胞术、细胞周期同步化以及RNA干扰等技术,探讨GNG7抑制细胞生长的机制。结果显示,GNG7过表达和干扰均使细胞阻滞于分裂期,导致大量双核/多核细胞的产生/出现;并且,GNG7在分裂期特别是分裂后期高表达,这些均提示GNG7可能主要作用于胞质分裂期。在此基础上,分析GNG7对细胞骨架微丝的影响,结果表明GNG7可通过调控体内actin的动态平衡导致胞质分裂异常。通过流式细胞术、细胞染色和免疫印迹等技术检测了 GNG7促进细胞死亡的途径。结果显示,GNG7过表达会促进细胞凋亡和自噬。通过免疫共沉淀,筛选获得了自噬过程中与GNG7存在相互作用的蛋白,哺乳动物雷帕霉素靶蛋白mTOR。进一步实验结果表明,GNG7还可作用于mTOR信号通路进而诱导自噬,其干扰会明显抑制mTOR抑制剂和EBSS饥饿引起的自噬潮变化。因此,GNG7对细胞生长的抑制和对自噬性死亡的诱导效应使其呈现出良好的抑癌效果。一方面,本研究首次发现定位于高尔基体上的丝氨酸/苏氨酸激酶可与actin直接相互作用,并能通过调控高尔基体的形态和功能影响整个有丝分裂期,为丝氨酸/苏氨酸激酶家族在细胞分裂中的调控模式提供了借鉴;并且,STK16参与多条膜转运途径;另一方面,GNG7作为第一个被发现的参与细胞自噬的Gγ亚基,可与mTOR相互作用并正调控自噬,并可通过影响actin作用于有丝分裂。因此,STK16和GNG7作为调控有丝分裂和膜转运的枢纽蛋白,其作用机制研究为两大生命过程的紧密联系提供了有力的证据,也为下一步更深入地探讨奠定了基础。
[Abstract]:Mitosis and membrane transport abnormality is the leading cause of cancer occurrence and development of important factors. In recent years, the evidence shows that there is a close relationship between these two seemingly independent key life process. However, in mitosis and membrane transport of protein and study of its regulation mechanism of dual control function relatively scarce, therefore found more, in the two plays key regulatory proteins have important guiding significance to clarify the pathogenesis of tumor and other diseases. This paper from the Golgi protein STK16 kinase and G protein of GNG7 gamma, combined with cell biology, biochemistry and molecular biology methods, exploring both in have the function of mitosis and membrane transport and regulation mechanism, the main results are as follows: 1. Golgi protein STK16 in mitosis and membrane transport in function research The STK16 kinase is localized in the Golgi and cell membrane. STK16 Golgi part in interphase Golgi membrane vesicles and intermediate marker protein Giantin co localization, and mitosis orientation changes.STK16 interference or inhibition not only delayed cell mitosis, resulting in increased the percentage of cells in G2 phase, and in the duration of mitotic cell elongation, prometaphase cells and cytokinesis are increased greatly. And the inhibition of STK16 activity after the effect of interference or the Golgi apparatus in G2 phase and prophase of fragmentation and re assembly after the split phase Golgi. Further studies showed that STK16 kinase can directly bind with actin actin kinetics of polymerization and depolymerization, and in a concentration dependent and kinase activity dependent regulation of actin form. Therefore, STK16 kinase can directly regulate actin through the dynamic balance. Ring Golgi morphology and cell mitosis process.VSVG-GFP plasmid transfection showed that STK16 does not affect the CIS transport between the endoplasmic reticulum to the Golgi and Golgi to plasma membrane. However, STK16 is involved in the classical endocytosis, iron transport protein intracellular loop and the degradation pathway of epidermal growth factor receptor STK16; and, in early endosomes and plasma membrane, and membrane transport between the Golgi and endosome and autophagy in function. The results showed that point mutation, myristoylation and palmitoylation modification is the premise of STK16 localized in the cell membrane and the Golgi apparatus; regulation of two kinds of modified STK16 can effect on polymerization kinetics on actin, Golgi morphology and protein transport inhibitor stimulated nucleocytoplasmic shuttling, suggesting that STK16 may act as a transcription factor regulating other gene expression and function of.2. G protein GNG7 The function study reported in mitosis and autophagy, Gy subunit members of low expression of GNG7 in several tumor cells; by semi quantitative RT-PCR assay, we also found that the transcription of GNG7 in tumor cells in the level was significantly lower than that of normal cells, suggesting that GNG7 may be a tumor suppressor protein. Therefore, overexpression of GNG7 protein in osteosarcoma and cervical cancer cells with low expression of GNG7, and compare the changes of the growth curve, the results show that overexpression of GNG7 only after the cell growth was inhibited, and cell death. Further combined with flow cytometry, cell cycle synchronization and RNA interference technology, to explore the mechanism of GNG7 inhibition of cell growth. Showed that GNG7 overexpression and interference are the cell cycle arrest in mitosis, resulting in a large number of binuclear / coenocyte production /; and GNG7 in mitotic anaphase especially high expression, which suggested that GNG7 May be a major role in cytokinesis. On this basis, analyze the influence of GNG7 on microfilament cytoskeleton. The results show that the GNG7 can control the body dynamic balance by abnormal cytokinesis in actin. By flow cytometry, cell staining and Western blot of GNG7 promotes cell death pathway. The results showed that GNG7 overexpression promote cell apoptosis and autophagy. By immunoprecipitation, the proteins interacting with GNG7 in the process of autophagy screening, mammalian target of rapamycin mTOR. further experimental results show that GNG7 can also act on mTOR signaling pathway and induce autophagy, which can inhibit the interference caused by mTOR and EBSS inhibitors of autophagy changes. So the tide of hunger and the inhibition of GNG7 on cell growth and induced autophagic cell death of the cancer showed a good inhibitory effect. On the one hand, this is the first study That is localized in the Golgi serine / threonine kinase can directly interact with actin, and through the morphological and functional regulation of Golgi affects mitosis, as a serine / threonine kinase family in the regulation of cell division provides reference; and STK16, involved in a number of other membrane transport pathway; GNG7, as the first to be found in autophagy G gamma subunit interacts with mTOR and positive regulation of autophagy, and can affect the actin effect of Yu Yousi split. Therefore, STK16 and GNG7 as the control terminal protein fiber splitting and membrane transport, to study its mechanism for two life process the close contact provides strong evidence also laid a foundation for more in-depth discussion.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.2

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