發(fā)布時(shí)間：2018-01-09 20:22

  本文關(guān)鍵詞：核仁蛋白Def與半胱氨酸蛋白酶CAPN3的互作研究及其在核仁中的靶蛋白鑒定　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： CAPN3/Capn3b Def p53 核仁 斑馬魚 肝臟 細(xì)胞周期 蛋白降解 蛋白質(zhì)組學(xué)

【摘要】：核仁不僅是核糖體合成與加工的場(chǎng)所,也與細(xì)胞周期調(diào)控、DNA損傷修復(fù)、壓力信號(hào)應(yīng)答等生化過程有關(guān)。核仁作為細(xì)胞核內(nèi)致密的非膜細(xì)胞器,富集了超過4,500種不同蛋白質(zhì)。然而,人們對(duì)于這些蛋白質(zhì)在核仁中的功能以及這些蛋白在核仁中是如何被調(diào)控的所知甚少。本實(shí)驗(yàn)室之前的研究發(fā)現(xiàn)核仁蛋白Def(Digestive-organexpansion factor)能夠介導(dǎo)半胱氨酸蛋白酶 CAPN3(Calpain3)降解p53。但是我們并不清楚Def如何幫助CAPN3降解p53,也不知Def-CAPN3在核仁內(nèi)是否還存在其他靶蛋白。本研究首先發(fā)現(xiàn)人CAPN3能夠降解p53A138V、p53M2371、p53R248W和p53R273P,而不能降解p53R175H。p53R175H作為癌癥樣品中出現(xiàn)頻率最高的p53點(diǎn)突變,提示了 CAPN3在腫瘤形成中的意義。通過免疫共沉淀實(shí)驗(yàn),我們證明Def與人CAPN3能夠直接互作,并在核仁內(nèi)形成蛋白復(fù)合體。我們發(fā)現(xiàn)這種互作在斑馬魚上也具有保守性,斑馬魚Def能夠與Capn3b(人CAPN3在斑馬魚上的同源蛋白)在核仁內(nèi)形成蛋白復(fù)合體。進(jìn)一步的研究發(fā)現(xiàn),Def能夠通過磷酸化修飾調(diào)控其與CAPN3的結(jié)合能力,招募CAPN3進(jìn)入核仁降解p53。此外,Def-CAPN3對(duì)細(xì)胞周期及斑馬魚器官發(fā)育的調(diào)控也是部分通過p53實(shí)現(xiàn)的。在兩個(gè)不同的Caapn3b基因敲除品系斑馬魚的肝臟細(xì)胞中,p53都大量積累于核仁。雖然該斑馬魚純合突變體可正常繁育,但是成年魚表現(xiàn)出體長(zhǎng)變短、軀體易側(cè)彎的癥狀,與capn3基因突變引起的先天性肢帶型肌肉營(yíng)養(yǎng)不良(LGMD2A)癥狀類似。為了尋找Def-CAPN3在核仁內(nèi)的其他靶蛋白,我們通過蛋白質(zhì)組學(xué)技術(shù)分析了capn3b基因敲除斑馬魚與野生型斑馬魚肝臟細(xì)胞核仁中差異表達(dá)的蛋白,發(fā)現(xiàn)了 119個(gè)在capn3b突變魚中上調(diào)(超過1.45倍)的核蛋白,其中核仁蛋白33個(gè)。對(duì)其中8個(gè)候選蛋白進(jìn)行體內(nèi)、體外的驗(yàn)證實(shí)驗(yàn),證明其中7個(gè)(LmnA、Nkap、Ddx18、Cdk9、Hmgb1、Tra2a、Bbofl)蛋白能夠被CAPN3降解,而且能夠被Def誘導(dǎo)降解。此外,通過對(duì)蛋白質(zhì)組學(xué)數(shù)據(jù)的深入分析,我們發(fā)現(xiàn)Capn3b還參與了免疫系統(tǒng)的調(diào)節(jié),這解釋了為何部分LGMD2A患者在發(fā)病早期出現(xiàn)炎癥反應(yīng)。綜上所述,我們的研究首次闡明了 Def-CAPN3降解途徑能夠在核仁內(nèi)通過對(duì)靶蛋白的降解,調(diào)控細(xì)胞周期進(jìn)程及器官發(fā)育。作為首個(gè)斑馬魚核仁蛋白質(zhì)組學(xué)研究,我們的工作不僅為斑馬魚核仁蛋白功能的研究提供了幫助,也通過這種方法鑒定出了 7個(gè)Def-CAPN3的新底物,為Def-CAPN3的功能研究提供了新的基礎(chǔ)。此外,我們所構(gòu)建的capn3b基因敲除斑馬魚能夠作為L(zhǎng)GMD2A疾病研究模型,將來可用于藥物篩選。
[Abstract]:Nucleoli are not only the sites for ribosome synthesis and processing, but also related to the biochemical processes such as cell cycle regulation, DNA damage repair, pressure signal response, etc. Nucleoli act as dense non-membranous organelles in the nucleus. Enriched more than 4,500 different proteins. Little is known about the function of these proteins in nucleoli and how they are regulated in nucleoli. Digestive-organexpansion factor can mediate cysteine protease CAPN3 and Calpain3). However, we do not know how Def can help CAPN3 to degrade p53. It is also unknown whether there are other target proteins in the nucleoli of Def-CAPN3. In this study, we first found that human CAPN3 can degrade p53A138VP53M2371. P53R248W and p53R273Pcould not degrade p53R175H.p53R175H as the most frequent point mutation of p53 in cancer samples. By immunoprecipitation, we have proved that Def and human CAPN3 can interact directly with each other. We found that this interaction is also conserved in zebrafish. Zebrafish Def can form protein complex in nucleoli with Capn3b (the homologous protein of human CAPN3 on zebrafish). Def can regulate its binding ability to CAPN3 by phosphorylation and enlist CAPN3 into nucleolus to degrade p53. in addition. The regulation of Def-CAPN3 on cell cycle and organ development of zebrafish is also partly mediated by p53. In two different Caapn3b gene knockout strains of zebrafish liver cells. Although the homozygous mutant of zebrafish could be bred normally, adult fish showed the symptoms of shorter body length and easy lateral curvature. Similar to the capn3 gene mutation caused by congenital limb-band muscular dystrophy (LGMD2A). In order to search for other target proteins of Def-CAPN3 in nucleoli. We analyzed the differentially expressed proteins in liver nuclei of capn3b knockout zebrafish and wild type zebrafish by proteomics. One hundred and nineteen nucleoproteins were identified in capn3b mutant fish, of which 33 were nucleolus proteins. Eight candidate proteins were tested in vitro and in vivo. It was proved that seven of them, Ddx18Cdk9, Hmgb1Tra2aBboflon, could be degraded by CAPN3. In addition, through the in-depth analysis of proteomics data, we found that Capn3b is also involved in the regulation of the immune system. This explains why some patients with LGMD2A develop inflammation at the early stage of the disease. Our study demonstrated for the first time that Def-CAPN3 degradation pathway can degrade target proteins in nucleoli. As the first zebrafish nucleolus proteomics research, our work not only provides help for the study of zebrafish nucleolar protein function. Seven new substrates of Def-CAPN3 were also identified by this method, which provided a new basis for the functional study of Def-CAPN3. Our constructed capn3b knockout zebrafish can be used as a LGMD2A disease research model and can be used for drug screening in the future.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q75

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