本文選題：BMAL1　切入點：TRAF2　出處：《蘇州大學》2016年碩士論文　論文類型：學位論文

【摘要】：研究背景和目的:生物節(jié)律是指生物的生理和行為活動都存在與晝夜環(huán)境保持同步的周期性節(jié)律。這一生物節(jié)律幾乎存在于世界上所有的生物體內(nèi)。生物節(jié)律與人類的健康息息相關(guān)。生物節(jié)律的紊亂嚴重影響生物體的生理和行為,導致內(nèi)分泌失調(diào)、內(nèi)環(huán)境紊亂等癥狀。在哺乳動物小鼠體內(nèi)已克隆鑒定了七種生物鐘相關(guān)基因,分別為Per1、Per2、Per3、Cry1、Cry2、Clock和Bmal1。這些基因和它們的蛋白質(zhì)產(chǎn)物構(gòu)成的自主調(diào)節(jié)的轉(zhuǎn)錄和翻譯負反饋環(huán),是生物鐘運轉(zhuǎn)的分子機制。本論文一方面通過蛋白質(zhì)相互作用網(wǎng)絡(luò)平臺篩選出與生物鐘核心蛋白BMAL1相互作用的E3泛素連接酶,探究其調(diào)控生物節(jié)律的分子機制;另一方面通過非標記譜圖計數(shù)定量蛋白質(zhì)組學鑒定BMAL1的相互作用蛋白以及它們對BMAL1翻譯后修飾和代謝的影響,進一步探究其對生物節(jié)律的調(diào)控研究方法:一方面通過蛋白質(zhì)相互作用網(wǎng)絡(luò)平臺篩選出與BMAL1存在潛在相互作用的E3泛素連接酶,通過免疫沉淀和免疫印跡驗證它們與BMAL1的相互作用及其對BMAL1的調(diào)控機制,利用qPCR檢測在N2a細胞中敲低相關(guān)基因后的節(jié)律變化;另一方面過表達生物鐘核心蛋白BMAL1,通過免疫共沉淀獲取BMAL1相互作用蛋白,胰酶酶解后得到肽段進行質(zhì)譜檢測,經(jīng)過數(shù)據(jù)庫搜索和相互作用蛋白的生物信息學分析,發(fā)現(xiàn)BMAL1相關(guān)的生物學功能并通過免疫印跡法驗證蛋白之間的相互作用,通過一系列的生物化學方法驗證它們對BMAL1的調(diào)控機制。研究結(jié)果:我們通過蛋白質(zhì)相互作用網(wǎng)絡(luò)平臺篩選出的E3泛素連接酶TRAF2對生物節(jié)律有著調(diào)控作用。免疫沉淀和免疫印跡實驗發(fā)現(xiàn)TRAF2與BMAL1存在相互作用。穩(wěn)定性實驗發(fā)現(xiàn)TRAF2下調(diào)BMAL1的表達。泛素化檢測實驗發(fā)現(xiàn)TRAF2可促進BMAL1的泛素化修飾。功能上的初步研究顯示TRAF2缺失會改變細胞生物節(jié)律,節(jié)律周期增長、振幅增加。我們還通過非標記譜圖計數(shù)定量蛋白質(zhì)組學方法分析鑒定得到216個BMAL1相互作用蛋白,通過生物信息學分析發(fā)現(xiàn)它們在蛋白酶體泛素依賴的蛋白分解代謝過程、蛋白輸入、細胞核轉(zhuǎn)位等方面有較為重要的生物學功能,通過生化實驗驗證了BMAL1與其中一些蛋白的相互作用。篩選出的泛素蛋白酶體系統(tǒng)相關(guān)蛋白調(diào)控著BMAL1的穩(wěn)定性和泛素化:過表達E3泛素連接酶UBR5和STUB1促進BMAL1的泛素化并下調(diào)BMAL1的表達,而在細胞內(nèi)敲低這兩個基因后BMAL1的穩(wěn)定性增加。敲低去泛素化酶USP9X可以降低BMAL1蛋白的表達。研究結(jié)論:我們發(fā)現(xiàn)由PINA篩選獲得的E3泛素連接酶TRAF2通過調(diào)節(jié)BMAL1的泛素化和穩(wěn)定性來調(diào)控細胞的生物節(jié)律。通過定量蛋白質(zhì)組學、生物信息學和生化實驗發(fā)現(xiàn)了若干個泛素蛋白酶體系統(tǒng)相關(guān)蛋白通過泛素-蛋白酶體途徑來介導BMAL1的泛素化降解從而調(diào)控BMAL1的穩(wěn)定性。它們對生物節(jié)律的影響有待進一步探究。這些發(fā)現(xiàn)為泛素蛋白酶體系統(tǒng)調(diào)控生物節(jié)律的分子機制研究提供了必要的基礎(chǔ)。
[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.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R96

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