蛋白激酶NEK6调控细胞抗病毒信号转导机制及DNAzyme抗病毒活性研究

发布时间：2018-06-16 07:53

本文选题：Nek6 + Ⅰ型干扰素　；参考：《武汉大学》2012年博士论文

【摘要】：抗病毒天然免疫系统是宿主防御病毒感染的第一道防线。病毒感染机体后,机体的免疫系统很快启动免疫级联反应激活工型干扰素和多种细胞因子的表达。病毒感染细胞分泌的I型干扰素通过JAK-STAT信号通路诱导周围细胞表达多种抗病毒基因,这些抗病毒蛋白抑制病毒繁殖,促进感染细胞凋亡。Ⅰ型干扰素还可以活化适应性免疫系统,从而建立起长期的病毒防御机制。抗病毒天然免疫系统主要通过胚系表达的模式识别受体(pattern-recogniton receptors, PRR)识别病毒复制过程产生的病原体相关分子模式(pathogen-associated molecular patterns, PAMPs)。参与病毒PAMP识别的PRR主要包括Toll样受体(Toll-like receptor, TLR)、RIG-I样受体(RIG-I-like receptor, RLR)和DNA识别分子。定位在膜结构上的TLR识别病毒成份,通过接头分子MyD88或者TRIF激活下游信号通路,诱导Ⅰ型干扰素和炎症因子的表达。胞质中RIG-I样受体识别病毒复制产物后,迅速招募组装VISA(virus-induced signaling adaptor) TRAFs/TANK/NAP1/SINTBAD复合物活化蛋白激酶TBK1和IKK蛋白激酶复合物。随后转录因子IRF3和NF-κB活化入核诱导Ⅰ型干扰素的表达。蛋白质翻译后修饰如磷酸化和泛素化等在细胞生命活动过程扮演着重要的角色。为寻找调控细胞抗病毒信号转导新的蛋白激酶,我们通过克隆表达实验对蛋白激酶表达库进行了系统筛选并发现一个影响病毒诱导Ⅰ型干扰素表达的新蛋白激酶——Nek6。表达外源Nek6能抑制病毒诱导的ISRE,IFNβ启动子的激活并抑制IFNB1及受其诱导细胞因子RANTES的表达。通过siRNA抑制内源Nek6的表达不仅可以协同仙台病毒对ISRE的激活,而且能显著抑制水泡口炎病毒的复制。我们的生化实验进一步表明Nek6能够以泛素化依赖方式促进活化IRF3的降解。我们的研究工作充分说明了Nek6参与了细胞抗病毒信号转导的负调控过程。在过表达实验中我们发现Nek6激酶失活突变体失去了抑制活性,这表明Nek6对细胞抗病毒信号传导的调控是依赖其激酶活性的。过表达条件下Nek6能够对IRF3173位丝氨基酸进行磷酸化修饰。我们拟在后期通过磷酸位点特异抗体进一步证实内源性IRF3173位丝氨酸磷酸化在病毒感染过程中的动态变化。因为Nek6所属NIMA蛋白激酶家族主要参与了细胞M期的起始,我们拟在后期开展实验检测IRF3173位丝氨酸磷酸化的水平是否随细胞周期变化。以上研究工作能帮助我们更全面阐明Nek6调控细胞抗病毒信号转导的分子机制。 2003年爆发的重症呼吸衰竭综合症(severe acute respiratory syndrome, SARS)是由冠状病毒家族一个新的成员SARS-CoV引起的。SARS病毒为正义链单链包膜RNA病毒,基因组大约由30Kb碱基组成,编码14个开发阅读框,两端为高度保守的非编码区(untranslated region, UTR)。目前,我们仍然缺乏对SARS-CoV有很好疗效的药物。DNA酶是一类人工合成对RNA具有切割活性的单链脱氧核酸,根据其催化结构域可以分为10-23和8-17两种模式。人们已经设计了多个有效抑制HIV和流感病毒的DNA酶,我们试图设计并筛选能够特异抑制SARS-CoV的DNA酶。SARS5'UTR大约有265个核苷酸组成,在分子进化上高度保守,在病毒的生活周期有重要的功能,因此我们以SARS5'UTR RNA为靶标设计了DNA酶。体外切割实验表明,我们设计的DNA酶Dz-104,而不是其突变体能特异切割来源SARS5'UTR的RNA。我们将SARS5'UTR和绿色荧光蛋白基因融合在一起,成功构建了靶向SARS5'UTR的DNA酶细胞筛选系统。通过共转染实验,我们发现Dz-104在胞内能特异切割SARS5'UTR,并抑制绿色荧光蛋白的表达。我们的研究工作充分说明了DNA酶能有效识别并切割SARS病毒来源的5’非编码序列RNA。我们拟在后期利用SARS亚基因组复制子检测Dz-104对SARS病毒的抑制活性并以SARS病毒全基因组为靶向设计筛选更多的DNA酶。我们也将尝试各种化学修饰提高DNA酶的活性和稳定性。以上两项工作取得的进展将为开发新型抗SARS药物奠定基础。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R373

【共引文献】