PMA诱导K562细胞向巨核系分化过程中线粒体功能的变化及机制研究

发布时间：2018-01-28 03:36

  本文关键词： 核蛋白质复合体 CCT 细胞分化 线粒体功能 呼吸链复合体　出处：《中国人民解放军军事医学科学院》2011年博士论文　论文类型：学位论文

【摘要】：蛋白质复合体是蛋白质在体内发挥功能的重要形式之一。细胞核是真核细胞内最大、最重要的细胞器,是细胞遗传与代谢的调控中心,包含了大量的重要蛋白质复合体如剪接体复合体、DNA聚合酶复合体、核孔复合体等。线粒体是细胞能量代谢的重要场所,通过氧化磷酸化作用,进行能量转换,为细胞进行各项生命活动提供能量。此外,线粒体还与细胞中氧自由基的生成、细胞凋亡、物质代谢等密切相关。有关核蛋白质复合体特别是各复合体间的相互关联、分化过程中线粒体能量代谢复合体的变化及对线粒体功能的影响等目前尚较少报道。 在本论文研究中,我们建立并优化了蓝色温和电泳(Blue-Native Electrophoresis, BN-PAGE)联合质谱分离鉴定蛋白质复合体的技术,并对人白血病细胞系K562细胞核蛋白质复合体进行了分离鉴定,共鉴定了12个蛋白质复合体的成分。对复合体1的深入分析发现,该复合体由40种蛋白质组成,包含了CCT复合体的8个亚基、RNA剪接复合体多个成分及细胞生长、凋亡调控相关蛋白质等。其中87.5%(35/40)的蛋白质组分已被报道可形成蛋白质间的两两相互作用,TUBA4A和TUBA8等是已报道的CCT复合体作用底物,可与CCT复合体的不同亚基有直接的相互作用。采用BN-PAGE联合蛋白质印记技术证明5种蛋白质组分(CCT8、EFTUD2、RPS16、SFPQ和GAPDH)确可与CCT复合体共处同一蛋白质复合体中。CCT复合体的主要功能是负责细胞质中新合成蛋白质的正确折叠,我们的发现提示CCT复合体在细胞核中可能具有协助多种蛋白质复合体聚集,并参与RNA剪切等过程调控的功能,为深入认识CCT复合体的功能提供了重要线索。 进一步,我们以PMA诱导K562细胞向巨核细胞系分化为模型,采用BN-PAGE联合酶活性检测技术分析了分化过程中线粒体呼吸链复合体的动态变化及其与线粒体功能的关系。研究发现:1)在PMA诱导K562细胞向巨核细胞系分化过程中,伴随着显著的线粒体功能受损;2)用环胞菌素A稳定线粒体膜电位,可促进PMA诱导的细胞分化;3)分化过程中线粒体呼吸链复合体Ⅳ活性发生显著变化。在分化诱导早期其活性明显下降,诱导72小时后恢复并略有增加。4)线粒体呼吸链复合体Ⅳ特异抑制剂处理可导致线粒体膜电位的显著下降,并可抑制PMA诱导的细胞分化;环胞菌素A处理细胞稳定线粒体膜电位不能改善PMA诱导的线粒体呼吸链复合体Ⅳ的活性下降;5)PMA诱导K562细胞分化过程中复合体Ⅳ核心亚基COX3、线粒体膜蛋白转运相关蛋白质Tim9和Timl0表达下调。这些结果表明线粒体功能的变化参与了PMA诱导K562细胞分化过程的调节, PMA诱导线粒体呼吸链复合体Ⅳ活性下降,进而引起线粒体膜电位的下调是导致线粒体功能变化的关键因素,维持线粒体功能的稳态是调节细胞分化的重要途径。
[Abstract]:Protein complex is one of the important forms of protein function in vivo. The nucleus is the largest and most important organelle in eukaryotic cells and the regulatory center of cell heredity and metabolism. It contains a large number of important protein complexes such as splicing complex DNA polymerase complex nuclear pore complex and so on. Mitochondria is an important site of cell energy metabolism through oxidative phosphorylation. In addition, mitochondria are associated with the production of oxygen free radicals and apoptosis of cells. Substance metabolism is closely related to the interrelation of nucleoprotein complexes, especially the complexes. The changes of mitochondrial energy metabolism complex and its effect on mitochondrial function during differentiation are seldom reported. In this thesis, we have established and optimized the blue mild electrophoretic Blue-Native Electrophoresis. The protein complex was isolated and identified by BN-PAGE and mass spectrometry, and the nuclear protein complex of human leukemia cell line K562 was isolated and identified. A total of 12 protein complexes were identified. Further analysis of complex 1 revealed that the complex consists of 40 proteins and contains 8 subunits of the CCT complex. Many components of RNA splicing complex and cell growth, apoptosis regulation related proteins, among which 87.5% ~ 35 / 40) protein components have been reported to form pairwise interactions between proteins. TUBA4A and TUBA8 are reported substrates of CCT complex. BN-PAGE combined with protein imprinting technique was used to prove that five protein components, CCT8 / EFTUD2 / RPS16, could interact directly with different subunits of CCT complex. SFPQ and GAPDH) can co-exist with CCT complex in the same protein complex. The main function of CCT complex is responsible for the correct folding of newly synthesized proteins in the cytoplasm. Our findings suggest that the CCT complex may play a role in facilitating the aggregation of multiple protein complexes in the nucleus and participating in the regulation of processes such as RNA shearing. It provides an important clue for further understanding the function of CCT complex. Furthermore, we used PMA to induce K562 cells to differentiate into megakaryocyte cells. The dynamic changes of mitochondrial respiratory chain complex during differentiation and the relationship between mitochondrial respiratory chain complex and mitochondrial function were analyzed by BN-PAGE combined with enzyme activity assay. During the differentiation of K562 cells into megakaryocyte cells induced by PMA. Accompanied by significant mitochondrial dysfunction; 2) cyclosporine A was used to stabilize mitochondrial membrane potential, which could promote cell differentiation induced by PMA. 3) the activity of mitochondrial respiratory chain complex 鈪，

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