本文關(guān)鍵詞： 核蛋白質(zhì)復(fù)合體 CCT 細胞分化 線粒體功能 呼吸鏈復(fù)合體　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年博士論文　論文類型：學(xué)位論文

【摘要】：蛋白質(zhì)復(fù)合體是蛋白質(zhì)在體內(nèi)發(fā)揮功能的重要形式之一。細胞核是真核細胞內(nèi)最大、最重要的細胞器,是細胞遺傳與代謝的調(diào)控中心,包含了大量的重要蛋白質(zhì)復(fù)合體如剪接體復(fù)合體、DNA聚合酶復(fù)合體、核孔復(fù)合體等。線粒體是細胞能量代謝的重要場所,通過氧化磷酸化作用,進行能量轉(zhuǎn)換,為細胞進行各項生命活動提供能量。此外,線粒體還與細胞中氧自由基的生成、細胞凋亡、物質(zhì)代謝等密切相關(guān)。有關(guān)核蛋白質(zhì)復(fù)合體特別是各復(fù)合體間的相互關(guān)聯(lián)、分化過程中線粒體能量代謝復(fù)合體的變化及對線粒體功能的影響等目前尚較少報道。 在本論文研究中,我們建立并優(yōu)化了藍色溫和電泳(Blue-Native Electrophoresis, BN-PAGE)聯(lián)合質(zhì)譜分離鑒定蛋白質(zhì)復(fù)合體的技術(shù),并對人白血病細胞系K562細胞核蛋白質(zhì)復(fù)合體進行了分離鑒定,共鑒定了12個蛋白質(zhì)復(fù)合體的成分。對復(fù)合體1的深入分析發(fā)現(xiàn),該復(fù)合體由40種蛋白質(zhì)組成,包含了CCT復(fù)合體的8個亞基、RNA剪接復(fù)合體多個成分及細胞生長、凋亡調(diào)控相關(guān)蛋白質(zhì)等。其中87.5%(35/40)的蛋白質(zhì)組分已被報道可形成蛋白質(zhì)間的兩兩相互作用,TUBA4A和TUBA8等是已報道的CCT復(fù)合體作用底物,可與CCT復(fù)合體的不同亞基有直接的相互作用。采用BN-PAGE聯(lián)合蛋白質(zhì)印記技術(shù)證明5種蛋白質(zhì)組分(CCT8、EFTUD2、RPS16、SFPQ和GAPDH)確可與CCT復(fù)合體共處同一蛋白質(zhì)復(fù)合體中。CCT復(fù)合體的主要功能是負責細胞質(zhì)中新合成蛋白質(zhì)的正確折疊,我們的發(fā)現(xiàn)提示CCT復(fù)合體在細胞核中可能具有協(xié)助多種蛋白質(zhì)復(fù)合體聚集,并參與RNA剪切等過程調(diào)控的功能,為深入認識CCT復(fù)合體的功能提供了重要線索。 進一步,我們以PMA誘導(dǎo)K562細胞向巨核細胞系分化為模型,采用BN-PAGE聯(lián)合酶活性檢測技術(shù)分析了分化過程中線粒體呼吸鏈復(fù)合體的動態(tài)變化及其與線粒體功能的關(guān)系。研究發(fā)現(xiàn):1)在PMA誘導(dǎo)K562細胞向巨核細胞系分化過程中,伴隨著顯著的線粒體功能受損;2)用環(huán)胞菌素A穩(wěn)定線粒體膜電位,可促進PMA誘導(dǎo)的細胞分化;3)分化過程中線粒體呼吸鏈復(fù)合體Ⅳ活性發(fā)生顯著變化。在分化誘導(dǎo)早期其活性明顯下降,誘導(dǎo)72小時后恢復(fù)并略有增加。4)線粒體呼吸鏈復(fù)合體Ⅳ特異抑制劑處理可導(dǎo)致線粒體膜電位的顯著下降,并可抑制PMA誘導(dǎo)的細胞分化;環(huán)胞菌素A處理細胞穩(wěn)定線粒體膜電位不能改善PMA誘導(dǎo)的線粒體呼吸鏈復(fù)合體Ⅳ的活性下降;5)PMA誘導(dǎo)K562細胞分化過程中復(fù)合體Ⅳ核心亞基COX3、線粒體膜蛋白轉(zhuǎn)運相關(guān)蛋白質(zhì)Tim9和Timl0表達下調(diào)。這些結(jié)果表明線粒體功能的變化參與了PMA誘導(dǎo)K562細胞分化過程的調(diào)節(jié), PMA誘導(dǎo)線粒體呼吸鏈復(fù)合體Ⅳ活性下降,進而引起線粒體膜電位的下調(diào)是導(dǎo)致線粒體功能變化的關(guān)鍵因素,維持線粒體功能的穩(wěn)態(tài)是調(diào)節(jié)細胞分化的重要途徑。
[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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