生物钟核心蛋白BMAL1相互作用泛素连接酶的鉴定及其对BMAL1代谢的调控研究

发布时间：2018-03-12 11:51

本文选题：BMAL1　切入点：TRAF2　出处：《苏州大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的:生物节律是指生物的生理和行为活动都存在与昼夜环境保持同步的周期性节律。这一生物节律几乎存在于世界上所有的生物体内。生物节律与人类的健康息息相关。生物节律的紊乱严重影响生物体的生理和行为,导致内分泌失调、内环境紊乱等症状。在哺乳动物小鼠体内已克隆鉴定了七种生物钟相关基因,分别为Per1、Per2、Per3、Cry1、Cry2、Clock和Bmal1。这些基因和它们的蛋白质产物构成的自主调节的转录和翻译负反馈环,是生物钟运转的分子机制。本论文一方面通过蛋白质相互作用网络平台筛选出与生物钟核心蛋白BMAL1相互作用的E3泛素连接酶,探究其调控生物节律的分子机制;另一方面通过非标记谱图计数定量蛋白质组学鉴定BMAL1的相互作用蛋白以及它们对BMAL1翻译后修饰和代谢的影响,进一步探究其对生物节律的调控研究方法:一方面通过蛋白质相互作用网络平台筛选出与BMAL1存在潜在相互作用的E3泛素连接酶,通过免疫沉淀和免疫印迹验证它们与BMAL1的相互作用及其对BMAL1的调控机制,利用qPCR检测在N2a细胞中敲低相关基因后的节律变化;另一方面过表达生物钟核心蛋白BMAL1,通过免疫共沉淀获取BMAL1相互作用蛋白,胰酶酶解后得到肽段进行质谱检测,经过数据库搜索和相互作用蛋白的生物信息学分析,发现BMAL1相关的生物学功能并通过免疫印迹法验证蛋白之间的相互作用,通过一系列的生物化学方法验证它们对BMAL1的调控机制。研究结果:我们通过蛋白质相互作用网络平台筛选出的E3泛素连接酶TRAF2对生物节律有着调控作用。免疫沉淀和免疫印迹实验发现TRAF2与BMAL1存在相互作用。稳定性实验发现TRAF2下调BMAL1的表达。泛素化检测实验发现TRAF2可促进BMAL1的泛素化修饰。功能上的初步研究显示TRAF2缺失会改变细胞生物节律,节律周期增长、振幅增加。我们还通过非标记谱图计数定量蛋白质组学方法分析鉴定得到216个BMAL1相互作用蛋白,通过生物信息学分析发现它们在蛋白酶体泛素依赖的蛋白分解代谢过程、蛋白输入、细胞核转位等方面有较为重要的生物学功能,通过生化实验验证了BMAL1与其中一些蛋白的相互作用。筛选出的泛素蛋白酶体系统相关蛋白调控着BMAL1的稳定性和泛素化:过表达E3泛素连接酶UBR5和STUB1促进BMAL1的泛素化并下调BMAL1的表达,而在细胞内敲低这两个基因后BMAL1的稳定性增加。敲低去泛素化酶USP9X可以降低BMAL1蛋白的表达。研究结论:我们发现由PINA筛选获得的E3泛素连接酶TRAF2通过调节BMAL1的泛素化和稳定性来调控细胞的生物节律。通过定量蛋白质组学、生物信息学和生化实验发现了若干个泛素蛋白酶体系统相关蛋白通过泛素-蛋白酶体途径来介导BMAL1的泛素化降解从而调控BMAL1的稳定性。它们对生物节律的影响有待进一步探究。这些发现为泛素蛋白酶体系统调控生物节律的分子机制研究提供了必要的基础。
[Abstract]:Background and objective: biological rhythm refers to the physiology and behavior of biological activities are kept in sync with the circadian rhythm. The circadian rhythm exists in almost all the world's biology. Biological rhythms and human health are closely related. The biological rhythm disorders seriously affect the organism's physiology and behavior, leading to endocrine disorders in the environment, disorders and other symptoms. In mammals, mice that have been cloned and identified seven kinds of clock genes, respectively Per1, Per2, Per3, Cry1, Cry2, Clock and Bmal1. genes and their protein products constitute the autonomic regulation of the transcription and translation of the negative feedback loop, is the molecular mechanism of biological clock operation on the one hand. Through protein-protein interaction network platform screening and biological clock core protein BMAL1 interacting E3 ubiquitin ligase, explore the regulation of circadian rhythm of Sub mechanism; on the other hand, the interaction of protein by unlabeled spectrum counting quantitative proteomic identification of BMAL1 and their influence on the metabolism of BMAL1 and modification after translation, further explore the research methods of regulation of biological rhythms: on the one hand through the protein interaction network platform were screened for the presence of potential interaction with BMAL1 ubiquitin E3 ligase, by immunoprecipitation and Western blotting were performed to verify their interaction with BMAL1 and its regulation mechanism of BMAL1, on rhythm changes after low related genes in N2a cells was detected by qPCR; on the other hand, over expression of clock core protein BMAL1, obtain BMAL1 interacting protein by immunoprecipitation, trypsin enzymolysis get peptide mass spectrometry detection, through the analysis of biological information database search and interacting protein, found that the biological function of BMAL1 and the related immune The interaction between protein blot verification, through a series of biochemical methods to verify their regulatory mechanisms of BMAL1. Results: We screened through protein-protein interaction network platform E3 ubiquitin ligase TRAF2 has a role in the regulation of biological rhythms. The immune precipitation and Western blot showed that TRAF2 interacts with BMAL1. Stability experiments showed that down-regulation of TRAF2 expression. BMAL1 ubiquitination assay demonstrated that TRAF2 could promote the ubiquitination of BMAL1. A preliminary study on the function of the TRAF2 deletion will change the cell biological rhythm, rhythm of growth period, we also increased the amplitude. By unlabeled spectrum counting quantitative proteomic analysis of protein interaction was 216 BMAL1 the identification methodology, through bioinformatics analysis revealed that the metabolic process, decomposition of their dependence on the ubiquitin proteasome protein input cell There is an important biological function of nuclear translocation etc., through biochemical experiments and BMAL1 interacting proteins. Some proteins related to the ubiquitin proteasome system screened regulates BMAL1 stability and ubiquitination: over expression of E3 ubiquitin ligase UBR5 and STUB1 promote BMAL1 ubiquitination and down-regulation of BMAL1 expression, and knock in cells increase the stability of BMAL1 is low. After the two gene knockdown deubiquitinase USP9X can decrease the expression of BMAL1. Conclusion: we found that PINA obtained by screening E3 ubiquitin ligase TRAF2 by regulating BMAL1 ubiquitination and stability to the regulation of cell biological rhythms. Through quantitative proteomics. Bioinformatics and biochemical experiments revealed several proteins associated with the ubiquitin proteasome system to BMAL1 mediated by the ubiquitin proteasome pathway to regulate the ubiquitination of BMAL1 The effect of their effects on circadian rhythm needs further exploration. These findings provide a necessary basis for studying the molecular mechanism of ubiquitin proteasome system in regulating biological rhythms.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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