卷曲螺旋蛋白52与TRAF6及其家族成员相互作用对NF-κB通路的影响研究

发布时间：2018-06-26 09:03

本文选题：NDP52 + TRAF6　；参考：《安徽医科大学》2011年硕士论文

【摘要】：NF-κB是一类具有多向转录调节作用的核蛋白因子,广泛存在于多种组织细胞中,激活后的NF-κB信号途径参与多种基因的转录调控,在免疫、炎症、氧化应激、细胞增殖、细胞凋亡、肿瘤形成等生理病理过程中发挥作用,对于该通路的研究具有十分重要的生物学意义。TRAF6(tumor necrosis factor receptor associated factor 6)的活化是NF-κB激活信号通路中的关键步骤。为进一步研究NF-κB信号通路的精确调控机制,我们以TRAF6为诱饵,用酵母双杂交技术从胎肝文库中分离了与TRAF6相互作用的蛋白质NDP52(nuclear dot protein 52)。 NDP52(nuclear dot protein 52)是核点家族中的一员,定位于胞质。NDP52在Hella细胞中高表达。目前对NDP52的报导主要集中在NDP52可以参与机体对沙门氏菌的自噬过程,现在认为NDP52是可以直接参与自噬过程的蛋白。但目前对其作用机制了解甚少。本论文以NDP52的相互作用蛋白质为切入点,探讨了NDP52可能的作用机制,着重研究了NDP52与TRAF6的相互作用及生物学意义。本文以酵母双杂交筛选成人肝脏cDNA文库得到的未知相互作用对NDP52和TRAF6作为研究对象,通过一系列实验验证了二者的相互作用,并对其功能意义进行了初步探索。首先我们通过外源性的免疫共沉淀实验实验确证了NDP52和TRAF6之间存在生理性相互作用,mating实验表明NDP52与TRAF3、TRAF4、TRAF6存在相互作用,而免疫共沉淀实验表明NDP52与TRAF1、TRAF3、TRAF5、TRAF6存在相互作用,随后我们通过转录活性实验表明,过表达NDP52可以负调控NF-κB信号通路,TNFα刺激后抑制作用更明显,同时NDP52可以明显负调控TRAF2、TRAF5、TRAF6介导的NF-κB信号通路;mating实验表明,TRAF6N3(1-350aa)与NDP52存在相互作用,免疫共沉淀实验表明,NDP52C1(224-446aa)与TRAF6存在相互作用;最后我们研究了NDP52与TRAF6相互作用是如何负调控NF-κB信号通路的,通过体内泛素化实验我们发现多表达NDP52可以抑制TRAF6的泛素化, 总之,我们筛选到的NDP52和TRAF6相互作用能够影响NF-κB信号通路,此研究结果将有助于拓展对NF-κB信号通路调节机制的认识。
[Abstract]:NF- 魏 B is a kind of nuclear protein factor with multidirectional transcriptional regulation. The activated NF- 魏 B signaling pathway is involved in the transcriptional regulation of many genes, such as immunity, inflammation, oxidative stress, cell proliferation and apoptosis. Tumor formation and other physiological and pathological processes play an important role in the study of this pathway. The activation of TRAF6 (tumor necrosis factor receptor associated factor 6 is a key step in the activation of NF- 魏 B signaling pathway. In order to further study the precise regulation mechanism of NF- 魏 B signaling pathway, we isolated the protein NDP52 (nuclear dot protein 52 interacting with TRAF6 from fetal liver library by yeast two-hybrid technique using TRAF6 as bait. NDP52 (nuclear dot protein 52) is a member of nuclear point family. It was located in cytoplasm. NDP52 was highly expressed in Hella cells. At present, NDP52 is mainly reported to be involved in the autophagy process of Salmonella. NDP52 is considered to be a protein directly involved in autophagy. However, little is known about its mechanism at present. Based on the interaction protein of NDP52, the possible mechanism of NDP52 is discussed, and the interaction between NDP52 and TRAF6 and its biological significance are studied. In this paper, the unknown interaction between NDP52 and TRAF6 from adult liver cDNA library screened by yeast two-hybrid was studied. The interaction between NDP52 and TRAF6 was verified by a series of experiments, and its functional significance was preliminarily explored. First of all, we confirmed the physiological interaction between NDP52 and TRAF6 through exogenous immunoprecipitation experiments. The results showed that NDP52 interacted with TRAF3TRAF4TRAF6, while immunoprecipitation showed that NDP52 interacted with TRAF1TRAF3TRAF5TRAF6. Subsequently, our transcriptional activity experiment showed that overexpression of NDP52 could negatively regulate the inhibitory effect of TNF- 伪 on the NF- 魏 B signaling pathway, and NDP52 could significantly negatively regulate the NF- 魏 B signaling pathway mediated by TRAF2TRAF5TRAF6-mediated NF-kappa B signaling pathway. The results showed that there was interaction between TRAF6N3 (1-350aa) and NDP52. Immunoprecipitation assay showed that NDP52C1 (224-446aa) interacted with TRAF6. Finally, we studied how NDP52C1 (224-446aa) negatively regulated NF- 魏 B signaling pathway. In vivo, we found that overexpression of NDP52 can inhibit the ubiquification of TRAF6. In short, the interaction between NDP52 and TRAF6 we screened can affect the NF- 魏 B signaling pathway. This study will be helpful in understanding the regulation mechanism of NF- 魏 B signaling pathway.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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