本文選題：去泛素化酶　切入點：Ubp2　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：泛素—蛋白酶體系統(tǒng)(ubiquitin-proteasome system,UPS)是真核細胞特異性降解蛋白質(zhì)的主要場所,實現(xiàn)蛋白質(zhì)質(zhì)、量的精準調(diào)控,影響甚至決定了細胞周期、免疫應(yīng)答和信號傳遞等幾乎所有的生命活動過程。蛋白的泛素化需要E1,E2和E3級聯(lián)反應(yīng),將泛素分子(Ub)轉(zhuǎn)移到底物蛋白的不同賴氨酸上造成翻譯后修飾的宏觀不均一性。底物蛋白上的泛素分子又可繼續(xù)被泛素修飾進而形成不同的泛素鏈修飾。而不同的泛素鏈發(fā)揮不同的生物學(xué)功能。去泛素化酶(DUBs)可將底物蛋白上的泛素鏈或泛素分子特異地水解下來,逆轉(zhuǎn)泛素化過程,以維持泛素—蛋白酶體系統(tǒng)的平衡。其失調(diào)使UPS系統(tǒng)紊亂,從而導(dǎo)致人類諸多疾病。研究發(fā)現(xiàn)DUBs與泛素鏈之間存在特異性對應(yīng)關(guān)系,但由于技術(shù)的限制,特定的DUBs調(diào)控其特異性底物的泛素鏈并參與到生物學(xué)過程的機制尚屬未知,有待我們深入研究。在之前的研究中,我們以芽殖酵母為研究對象,系統(tǒng)評價了酵母所有DUB的泛素鏈的特異性,發(fā)現(xiàn)USP家族的Ubp2對K63鏈有偏好性。本課題以Ubp2的潛在底物L(fēng)sb1為研究對象,通過在其C端加上6×his和biotin雙標簽的策略來系統(tǒng)描述Lsb1。經(jīng)過串聯(lián)純化和SDS-PAGE驗證,我們得到了高純度泛素化的Lsb1,證明我們的研究策略有效。純化的樣品經(jīng)過LC-MS/MS高覆蓋蛋白質(zhì)組分析,我們證實Lsb1的K41和K79位點被泛素化,驗證了以往的研究結(jié)果;與此同時我們還發(fā)現(xiàn)了K37、K85、K98和K108這4個新的泛素化位點。分別將主要的修飾殘基的賴氨酸突變成精氨酸,并做SILAC-IP-MS精準定量蛋白質(zhì)組分析,發(fā)現(xiàn)Lsb1蛋白上的K41和K79位點都含有K48和K63泛素鏈的修飾,其中K41位點主要為K63鏈修飾,而K79位點主要發(fā)生K48修飾,它們受到Ubp2,Ubp3和Ubp14調(diào)控。為闡明Lsb1受Ubp2調(diào)控的分子機制,我們開展了Lsb1特定泛素鏈的SILAC-Protein-Protein Interacting研究。經(jīng)過SILAC定量和嚴格的篩選,我們鑒定了151個高可信的Lsb1相互作用蛋白,這些蛋白主要參與氨基酸代謝、蛋白折疊、細胞離子平衡、內(nèi)吞作用等重要的生物學(xué)過程。在泛素鏈特異性結(jié)合的底物研究中,我們用相同的篩選條件得到了共45個與泛素鏈結(jié)合的蛋白,這群蛋白主要與支鏈氨基酸合成、蛋白折疊、鋅離子跨膜轉(zhuǎn)運等生物學(xué)過程。在隨后的表型實驗中也證明泛素化的Lsb1參與酵母支鏈氨基酸的合成,這與之前的功能分析吻合,也為我們深入研究Lsb1及其泛素鏈的生物學(xué)功能提供了參考。
[Abstract]:Ubiquitin proteasome system system (UPS) is the main site where eukaryotic cells specifically degrade proteins. The precise regulation of protein quality and quantity affects and even determines cell cycle. Almost all biological processes, such as immune response and signal transduction, involve the cascade of E _ 1E _ 2 and E _ 3, and the protein ubiquification requires a cascade of E _ 1E _ 2 and E _ 3. The transfer of ubiquitin molecules onto different lysines of proteins leads to the macroscopic heterogeneity of posttranslational modification. The ubiquitin molecules on the substrate protein can continue to be modified by ubiquitin to form different ubiquitin chains. The ubiquitin chain plays a different biological function. DUBs) can specifically hydrolyze the ubiquitin chain or ubiquitin molecule on the substrate protein. In order to maintain the balance of the ubiquitin proteasome system, the imbalance of Ubiquitin proteasome system leads to the disorder of the UPS system, which leads to many human diseases. It has been found that there is a specific correspondence between the DUBs and the ubiquitin chain, but due to the limitation of technology, The mechanism by which specific DUBs regulates the ubiquitin chain of its specific substrate and participates in biological processes is unknown and remains to be further studied. The specificity of ubiquitin chain of all DUB in yeast was systematically evaluated, and the preference of Ubp2 of USP family to K63 chain was found. In this study, the potential substrate Lsb1 of Ubp2 was studied. Lsb1was systematically described by adding 6 脳 his and biotin double tags to its C-terminal. After tandem purification and SDS-PAGE verification, we obtained a highly purified Ubiquidized Lsb1.It proved that our research strategy was effective. The purified samples were analyzed by LC-MS/MS high covering proteome. We confirmed that the K41 and K79 sites of Lsb1 were ubiquitized, and confirmed the results of previous studies. At the same time, we also found four new ubiquitin sites, K37, K85, K98 and K108, which mutated lysine from the major modified residues to arginine, respectively. SILAC-IP-MS accurate quantitative proteome analysis showed that K41 and K79 sites on Lsb1 protein contained K48 and K63 ubiquitin chains, K41 sites were mainly K63 chain modifications, while K79 sites were mainly K48 modifications. They are regulated by Ubp2Ubp3 and Ubp14. In order to elucidate the molecular mechanism that Lsb1 is regulated by Ubp2, we have carried out the SILAC-Protein-Protein Interacting study on the specific ubiquitin chain of Lsb1. By SILAC quantitative and strict screening, we have identified 151 highly reliable Lsb1 interacting proteins. These proteins are involved in amino acid metabolism, protein folding, cell ion balance, endocytosis and other important biological processes. We used the same screening conditions to get a total of 45 proteins that bind to the ubiquitin chain, which are mainly synthesized with branched amino acids and folded. In subsequent phenotypic experiments, it was also demonstrated that ubiquitin Lsb1 was involved in the biosynthesis of branched amino acids in yeast, which was consistent with previous functional analysis. It also provides a reference for us to study the biological function of Lsb1 and its ubiquitin chain.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q51

【參考文獻】

相關(guān)期刊論文 前1條

1 劉偉;賀福初;姜穎;;蛋白質(zhì)組體內(nèi)標記技術(shù)——SILAC技術(shù)[J];生命的化學(xué);2009年03期

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本文編號：1567290