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2018年有机化学:化学工具来探测蛋白质泛素化——6月阴

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泛素(乌兰巴托)是通过一个E1-E2-E3转移酶级联的基质蛋白调节细胞的稳定性和生物功能。人类基因组编码2 e1, 45 e2和e3 600多。他们一起组装复杂网络的乌兰巴托转移细胞蛋白的改性。目前,关键问题是解决如何识别重要的e3泛素化目标映射在细胞信号网络,以及如何乌兰巴托的具体联系组装链编码细胞中的独特信号。我们已经开发出一种方法,我们称之为¢€Aoeorthogonal乌兰巴托transferA¢a€•(出)理清蛋白质泛素化网络的复杂性。的关键是工程师一连串的工程E1, E2和E3酶(xE1、xE2 xE3),只传输一个工程乌兰巴托(xUB) xE3的基质。我们表达xUB和级联的细胞,净化xUB-conjugated蛋白质,蛋白质组学和披露他们的身份。屏幕是潜在的基质的蛋白E3的级联。我们开发了级联与HECT E3 E6AP U-box E3 E4B和芯片和确定新细胞电路由这些E3。调查E2-catalyzed乌兰巴托链合成的机理,我们生成linkage-specific di-UB轭合物的非自然氨基酸结合,表示蛋白质结扎。 The di-UB conjugates mimic the binding modes of donor and acceptor UBs at the E2 active site for UB chain synthesis. By characterizing the structure of E2-diUB conjugates, we are to reveal how E2 regulates the synthesis of UB chains of different linkages. Ubiquitin (Ub) may be a small (8.6 kDa) regulatory protein of 76 amino acids that adopts a β-grasp fold. Ub is very conserved in eukaryotic organisms. The conjugation of ubiquitin to a target protein is named ubiquitination or ubiquitylation.1 Typically, Ub is attached to proteins through an isopeptide linkage between the C-terminal carboxylate of ubiquitin (glycine 76) and an ε-amino group of a lysine residue within the acceptor proteins. Ubiquitination is a crucial , reversible post-translational modification (PTM) in eukaryotic cells. Since its discovery within the late 1970s and early 1980s, the modification by ubiquitin has emerged as an important regulatory mechanism in most cellular processes in eukaryotes. Ubiquitination affects substrate proteins in many various ways including signaling, proteasomal degradation, altering cellular localization, modulating catalytic activity, and promoting or preventing protein interactions.2, 3 The cellular processes regulated by ubiquitination include cell cycle, transcription, trafficking, inflammation and DNA repair. Notably, many of the processes are independent of proteasome-mediated protein degradation. Ubiquitination involves three main enzymatic steps catalyzed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s) (see Fig. 1 ).5 First, ubiquitin is activated during a two-step reaction by an E1 (ubiquitin-activating enzyme) with the consumption of ATP, forming a ubiquitin adenylate intermediate and subsequently a thioester bond between the C-terminal carboxyl of ubiquitin and therefore the site cysteine of E1.The human genome contains two E1s, i.e. UBA1 and UBA6.8 E2 catalyzes the transfer of Ub from the Ub-E1 conjugate to the site cysteine of E2 and forms the Ub-E2 conjugate through a transthioesterification reaction. The human genome possesses quite 30 different E2 enzymes. The E3 ubiquitin ligase catalyzes the ultimate step of the ubiquitination cascade by transferring Ub from the Ub-E2 conjugate to the substrate protein. E3s have substrate specificity for the E2 enzymes. The cullin-RING ligases, which constitute the most important group of E3s (around 600 members), don't form a chemical bond with Ub. Two smaller groups of E3s, the HECT ligases (around 30 members) and RBR ligases (around 12 members), form a Ub-thioester intermediate with the E3 site cystein

6月阴

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