Ubp2特异性调控Lsb1泛素链状态及其功能的定量蛋白质组学研究

发布时间：2018-03-04 21:04

本文选题：去泛素化酶　切入点：Ubp2　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：泛素—蛋白酶体系统(ubiquitin-proteasome system,UPS)是真核细胞特异性降解蛋白质的主要场所,实现蛋白质质、量的精准调控,影响甚至决定了细胞周期、免疫应答和信号传递等几乎所有的生命活动过程。蛋白的泛素化需要E1,E2和E3级联反应,将泛素分子(Ub)转移到底物蛋白的不同赖氨酸上造成翻译后修饰的宏观不均一性。底物蛋白上的泛素分子又可继续被泛素修饰进而形成不同的泛素链修饰。而不同的泛素链发挥不同的生物学功能。去泛素化酶(DUBs)可将底物蛋白上的泛素链或泛素分子特异地水解下来,逆转泛素化过程,以维持泛素—蛋白酶体系统的平衡。其失调使UPS系统紊乱,从而导致人类诸多疾病。研究发现DUBs与泛素链之间存在特异性对应关系,但由于技术的限制,特定的DUBs调控其特异性底物的泛素链并参与到生物学过程的机制尚属未知,有待我们深入研究。在之前的研究中,我们以芽殖酵母为研究对象,系统评价了酵母所有DUB的泛素链的特异性,发现USP家族的Ubp2对K63链有偏好性。本课题以Ubp2的潜在底物Lsb1为研究对象,通过在其C端加上6×his和biotin双标签的策略来系统描述Lsb1。经过串联纯化和SDS-PAGE验证,我们得到了高纯度泛素化的Lsb1,证明我们的研究策略有效。纯化的样品经过LC-MS/MS高覆盖蛋白质组分析,我们证实Lsb1的K41和K79位点被泛素化,验证了以往的研究结果;与此同时我们还发现了K37、K85、K98和K108这4个新的泛素化位点。分别将主要的修饰残基的赖氨酸突变成精氨酸,并做SILAC-IP-MS精准定量蛋白质组分析,发现Lsb1蛋白上的K41和K79位点都含有K48和K63泛素链的修饰,其中K41位点主要为K63链修饰,而K79位点主要发生K48修饰,它们受到Ubp2,Ubp3和Ubp14调控。为阐明Lsb1受Ubp2调控的分子机制,我们开展了Lsb1特定泛素链的SILAC-Protein-Protein Interacting研究。经过SILAC定量和严格的筛选,我们鉴定了151个高可信的Lsb1相互作用蛋白,这些蛋白主要参与氨基酸代谢、蛋白折叠、细胞离子平衡、内吞作用等重要的生物学过程。在泛素链特异性结合的底物研究中,我们用相同的筛选条件得到了共45个与泛素链结合的蛋白,这群蛋白主要与支链氨基酸合成、蛋白折叠、锌离子跨膜转运等生物学过程。在随后的表型实验中也证明泛素化的Lsb1参与酵母支链氨基酸的合成,这与之前的功能分析吻合,也为我们深入研究Lsb1及其泛素链的生物学功能提供了参考。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51

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