本文選題：Nek6 + Ⅰ型干擾素��； 參考：《武漢大學(xué)》2012年博士論文

【摘要】：抗病毒天然免疫系統(tǒng)是宿主防御病毒感染的第一道防線。病毒感染機(jī)體后,機(jī)體的免疫系統(tǒng)很快啟動(dòng)免疫級(jí)聯(lián)反應(yīng)激活工型干擾素和多種細(xì)胞因子的表達(dá)。病毒感染細(xì)胞分泌的I型干擾素通過JAK-STAT信號(hào)通路誘導(dǎo)周圍細(xì)胞表達(dá)多種抗病毒基因,這些抗病毒蛋白抑制病毒繁殖,促進(jìn)感染細(xì)胞凋亡。Ⅰ型干擾素還可以活化適應(yīng)性免疫系統(tǒng),從而建立起長(zhǎng)期的病毒防御機(jī)制。 抗病毒天然免疫系統(tǒng)主要通過胚系表達(dá)的模式識(shí)別受體(pattern-recogniton receptors, PRR)識(shí)別病毒復(fù)制過程產(chǎn)生的病原體相關(guān)分子模式(pathogen-associated molecular patterns, PAMPs)。參與病毒PAMP識(shí)別的PRR主要包括Toll樣受體(Toll-like receptor, TLR)、RIG-I樣受體(RIG-I-like receptor, RLR)和DNA識(shí)別分子。定位在膜結(jié)構(gòu)上的TLR識(shí)別病毒成份,通過接頭分子MyD88或者TRIF激活下游信號(hào)通路,誘導(dǎo)Ⅰ型干擾素和炎癥因子的表達(dá)。胞質(zhì)中RIG-I樣受體識(shí)別病毒復(fù)制產(chǎn)物后,迅速招募組裝VISA(virus-induced signaling adaptor) TRAFs/TANK/NAP1/SINTBAD復(fù)合物活化蛋白激酶TBK1和IKK蛋白激酶復(fù)合物。隨后轉(zhuǎn)錄因子IRF3和NF-κB活化入核誘導(dǎo)Ⅰ型干擾素的表達(dá)。 蛋白質(zhì)翻譯后修飾如磷酸化和泛素化等在細(xì)胞生命活動(dòng)過程扮演著重要的角色。為尋找調(diào)控細(xì)胞抗病毒信號(hào)轉(zhuǎn)導(dǎo)新的蛋白激酶,我們通過克隆表達(dá)實(shí)驗(yàn)對(duì)蛋白激酶表達(dá)庫進(jìn)行了系統(tǒng)篩選并發(fā)現(xiàn)一個(gè)影響病毒誘導(dǎo)Ⅰ型干擾素表達(dá)的新蛋白激酶——Nek6。表達(dá)外源Nek6能抑制病毒誘導(dǎo)的ISRE,IFNβ啟動(dòng)子的激活并抑制IFNB1及受其誘導(dǎo)細(xì)胞因子RANTES的表達(dá)。通過siRNA抑制內(nèi)源Nek6的表達(dá)不僅可以協(xié)同仙臺(tái)病毒對(duì)ISRE的激活,而且能顯著抑制水泡口炎病毒的復(fù)制。我們的生化實(shí)驗(yàn)進(jìn)一步表明Nek6能夠以泛素化依賴方式促進(jìn)活化IRF3的降解。我們的研究工作充分說明了Nek6參與了細(xì)胞抗病毒信號(hào)轉(zhuǎn)導(dǎo)的負(fù)調(diào)控過程。在過表達(dá)實(shí)驗(yàn)中我們發(fā)現(xiàn)Nek6激酶失活突變體失去了抑制活性,這表明Nek6對(duì)細(xì)胞抗病毒信號(hào)傳導(dǎo)的調(diào)控是依賴其激酶活性的。過表達(dá)條件下Nek6能夠?qū)RF3173位絲氨基酸進(jìn)行磷酸化修飾。我們擬在后期通過磷酸位點(diǎn)特異抗體進(jìn)一步證實(shí)內(nèi)源性IRF3173位絲氨酸磷酸化在病毒感染過程中的動(dòng)態(tài)變化。因?yàn)镹ek6所屬NIMA蛋白激酶家族主要參與了細(xì)胞M期的起始,我們擬在后期開展實(shí)驗(yàn)檢測(cè)IRF3173位絲氨酸磷酸化的水平是否隨細(xì)胞周期變化。以上研究工作能幫助我們更全面闡明Nek6調(diào)控細(xì)胞抗病毒信號(hào)轉(zhuǎn)導(dǎo)的分子機(jī)制。 2003年爆發(fā)的重癥呼吸衰竭綜合癥(severe acute respiratory syndrome, SARS)是由冠狀病毒家族一個(gè)新的成員SARS-CoV引起的。SARS病毒為正義鏈單鏈包膜RNA病毒,基因組大約由30Kb堿基組成,編碼14個(gè)開發(fā)閱讀框,兩端為高度保守的非編碼區(qū)(untranslated region, UTR)。目前,我們?nèi)匀蝗狈?duì)SARS-CoV有很好療效的藥物。DNA酶是一類人工合成對(duì)RNA具有切割活性的單鏈脫氧核酸,根據(jù)其催化結(jié)構(gòu)域可以分為10-23和8-17兩種模式。人們已經(jīng)設(shè)計(jì)了多個(gè)有效抑制HIV和流感病毒的DNA酶,我們?cè)噲D設(shè)計(jì)并篩選能夠特異抑制SARS-CoV的DNA酶。SARS5'UTR大約有265個(gè)核苷酸組成,在分子進(jìn)化上高度保守,在病毒的生活周期有重要的功能,因此我們以SARS5'UTR RNA為靶標(biāo)設(shè)計(jì)了DNA酶。體外切割實(shí)驗(yàn)表明,我們?cè)O(shè)計(jì)的DNA酶Dz-104,而不是其突變體能特異切割來源SARS5'UTR的RNA。我們將SARS5'UTR和綠色熒光蛋白基因融合在一起,成功構(gòu)建了靶向SARS5'UTR的DNA酶細(xì)胞篩選系統(tǒng)。通過共轉(zhuǎn)染實(shí)驗(yàn),我們發(fā)現(xiàn)Dz-104在胞內(nèi)能特異切割SARS5'UTR,并抑制綠色熒光蛋白的表達(dá)。我們的研究工作充分說明了DNA酶能有效識(shí)別并切割SARS病毒來源的5’非編碼序列RNA。我們擬在后期利用SARS亞基因組復(fù)制子檢測(cè)Dz-104對(duì)SARS病毒的抑制活性并以SARS病毒全基因組為靶向設(shè)計(jì)篩選更多的DNA酶。我們也將嘗試各種化學(xué)修飾提高DNA酶的活性和穩(wěn)定性。以上兩項(xiàng)工作取得的進(jìn)展將為開發(fā)新型抗SARS藥物奠定基礎(chǔ)。
[Abstract]:The antiviral natural immune system is the first line of defense against the virus infection of the host. After the virus infects the body, the immune system of the body quickly starts the expression of the immune cascade activated interferon and a variety of cytokines. The I type interferon secreted by the virus infected cells can induce a variety of resistance to the peripheral cells through the JAK-STAT signaling pathway. Viral genes, these antiviral proteins inhibit the reproduction of the virus and promote the apoptosis of infected cells. Type I interferon can also activate the adaptive immune system, thus establishing a long-term defense mechanism.
The antiviral natural immune system mainly identifies the pathogen associated molecular model (pathogen-associated molecular patterns, PAMPs) produced by the viral replication process (pathogen-associated molecular patterns, PRR), which is mainly expressed by the embryo line (pattern-recogniton, PRR). The PRR involved in the identification of virus PAMP mainly includes Toll like receptor (Toll-like receptor,), RIG-I-like receptor (RLR) and DNA identification molecules. Locate the TLR on the membrane structure to identify the virus components, activate the downstream signaling pathway through the joint molecule MyD88 or TRIF, and induce the expression of type I interferon and inflammatory factors. In cytoplasm, RIG-I like receptors identify the replication products of the virus and quickly recruit VISA (virus-induced signali). Ng adaptor) TRAFs/TANK/NAP1/SINTBAD complex activated protein kinase TBK1 and IKK protein kinase complex. Then the transcription factor IRF3 and NF- kappa B are activated to induce the expression of type I interferon.
Protein translational modification, such as phosphorylation and ubiquitination, plays an important role in the process of cell life activities. In order to find a new protein kinase for regulating cell antiviral signal transduction, we systematically screened the protein kinase expression library by cloning and expression experiments and found a new effect on the expression of virus induced interferon type I. The expression of exogenous Nek6 by protein kinase Nek6. inhibits the activation of ISRE, IFN beta promoter and inhibits the expression of IFNB1 and its induced cytokine RANTES. The inhibition of the expression of endogenous Nek6 by siRNA can not only cooperate with the activation of Sendai virus to ISRE, but also significantly inhibit the replication of the vesicular stomatitis virus. Our biochemical reality The test further indicates that Nek6 can promote the degradation of activated IRF3 in a ubiquitinated dependent manner. Our work fully illustrates that Nek6 has been involved in the negative regulation of cell antiviral signal transduction. In overexpression experiments, we found that the Nek6 kinase inactivation mutant lost its inhibitory activity, which indicates that Nek6 is a signal transduction of the cell antiviral signal. The regulation is dependent on its kinase activity. Under overexpression, Nek6 can phosphorylate the IRF3173 amino acid amino acids. We intend to further confirm the dynamic changes of the endogenous serine phosphorylation in the virus infection process by the specific antibody of the phosphoric acid site at the later stage, because the main participation of the NIMA protein kinase family of the Nek6 belongs to the NIMA protein kinase family. At the beginning of the cell M phase, we intend to test whether the level of IRF3173 serine phosphorylation changes with the cell cycle in the later period. The above work can help us to elucidate the molecular mechanism of Nek6 regulating cell anti-virus signal transduction more comprehensively.
Severe acute respiratory syndrome (SARS), a new member of the coronavirus family, caused by a new member of the coronavirus family, SARS-CoV, is a just chain envelope RNA virus, which is composed of a 30Kb base, encoding 14 development reading frames, and a highly conservative non coding region (untranslated). Region, UTR). At present, we still lack the drug.DNA enzyme that has a good effect on SARS-CoV. It is a class of artificially synthesized single strand deoxynucleic acid that has cleavage activity to RNA. According to its catalytic domain, it can be divided into 10-23 and 8-17 modes. A number of DNA enzymes that effectively inhibit HIV and influenza virus have been designed, and we are trying to design and The DNA enzyme.SARS5'UTR, which can specifically inhibit SARS-CoV, is composed of about 265 nucleotides, highly conserved in the molecular evolution, and has important functions in the life cycle of the virus. Therefore, we designed the DNA enzyme with SARS5'UTR RNA as a target. In vitro cutting experiments showed that we designed DNA enzyme Dz-104, not the specific cut of its mutant energy. RNA., a source of source SARS5'UTR, combined the SARS5'UTR with the green fluorescent protein gene and successfully constructed a DNA enzyme cell screening system targeting SARS5'UTR. Through co transfection experiments, we found that Dz-104 cut SARS5'UTR specifically in the intracellular energy and inhibited the expression of green fluorescent protein. Our work fully explained the enzyme energy of DNA. The 5 'non coding sequence RNA., which effectively identifies and cuts the source of SARS virus, we intend to detect the inhibitory activity of Dz-104 against the SARS virus by using the SARS subgenome replicator in the later period and select more DNA enzymes by targeting the whole genome of SARS virus. We will also try various chemical modifications to improve the activity and stability of the DNA enzyme. The above two items will be improved. The progress achieved will lay the foundation for developing new anti SARS drugs.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373

【共引文獻(xiàn)】

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