Browsing by Subject "Ubiquitin-Activating Enzymes"
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Item Identification and Characterization of a New E1 That Activates Ubiquitin and FAT10(2008-05-13) Chiu, Yu-Hsin; Chen, Zhijian J.Ubiquitination is one of many post-translational modifications in eukaryotes. Three enzymes (E1, E2, and E3) are involved in conjugating ubiquitin to protein substrates. I identified a novel E1-like protein, E1-L2, which is homologous to the ubiquitin E1 and another E1 involved in the activation of the ubiquitin-like protein ISG-15 (E1-L1). E1-L2 activates both ubiquitin and FAT10, a ubiquitin-like protein. Interestingly, E1-L2 can transfer ubiquitin to only a subset of E2 enzymes, Ubc5 and Ubc13, but not Ubc3 and E2-25K, suggesting that E1-L2 may function in certain ubiquitination reactions. E1-L2, but not E1 or E1-L1, forms a thioester with FAT10 in vitro. The formation of the thioester bond requires the active site cysteine residue of E1-L2 and the C-terminal diglycine motif of FAT10. In addition, endogenous FAT10 forms a thioester with E1-L2 in cells stimulated with tumor necrosis factor-alpha and interferon-gamma, which induce FAT10 expression. Silencing of E1-L2 expression by RNAi impaired the formation of FAT10 conjugates in cells. Furthermore, E1-L2 deficient embryos died before embryonic day 13.5, suggesting that E1-L2 is essential for early embryonic development. Since the FAT10-deficient mice develop normally, it is likely that specific ubiquitination reactions catalyzed by E1-L2 play an important role in animal development.Item Mechanistic Studies of the Activation of Ubiquitin-Conjugating Enzymes by Ring-Type Ubiquitin Ligases(2006-05-15) Özkan, Engin; Deisenhofer, JohannUbiquitination, modification with ubiquitin, is a post-translational regulation of proteins in eukaryotes. Ubiquitin-activating enzymes (E1) activate ubiquitin and form thioester linkages with ubiquitin, which are then transferred onto ubiquitin-conjugating enzymes (E2). Ubiquitin-conjugating enzymes, with the help of ubiquitin ligases (E3) transfer ubiquitin onto ubiquitination targets to form isopeptide linkages between the targets and ubiquitin. The function of ubiquitin ligases in the ubiquitination process has not been clearly understood. Recently, it has been shown that ubiquitin ligases are modular proteins or protein complexes that bring together the ubiquitination targets and the E2 enzyme. In this study, we show that E3 enzymes without their target-binding domains are effective activators of the ubiquitination reaction. We demonstrate that E3 binding to E2 enzymes increases the rate of the breakdown of the E2-ubiquitin thioester bond. Using the Statistical Coupling Analysis, a pairwise residue covariation analysis, we observe that E2 enzymes have a set of residues that vary as a group throughout evolution. Extensive mutagenesis of the E2 enzyme UbcH5b, including its covarying residues, yield mutants that can accept ubiquitin, break the thioester linkage, bind E3 enzymes, but cannot be stimulated to release their ubiquitin thioesters in the presence of an E3 enzyme. We also show that thioester release measurements agree with polyubiquitination of physiological targets. We have structurally characterized our model E2 enzyme UbcH5b, it mutants, and its interaction with E3 enzymes. Taken together, our data strongly demonstrate that E3 enzymes are, apart from their scaffolding function, activators of E2 enzymes to ubiquitinate targets. We discuss possible physical mechanisms of this activation, and hypothesize a long-distance communication pathway between the E3-binding surface and the thioester-forming active site of E2 enzymes. We also report attempts to crystallize several E2 and E3 enzymes and their complexes.