Ubiquitination is catalyzed by a cascade of enzymes consisting of E1, E2, and E3. We report here the identification of an E1-like protein, termed E1-L2, that activates both ubiquitin and another ubiquitin-like protein, FAT10. Interestingly, E1-L2 can transfer ubiquitin to Ubc5 and Ubc13, but not Ubc3 and E2-25K, suggesting that E1-L2 may be specialized in a subset of ubiquitination reactions. E1-L2 forms a thioester with FAT10 in vitro, and this reaction requires the active-site cysteine of E1-L2 and the C-terminal diglycine motif of FAT10. Furthermore, endogenous FAT10 forms a thioester with E1-L2 in cells stimulated with tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma), which induce FAT10 expression. Silencing of E1-L2 expression by RNAi blocks the formation of FAT10 conjugates in cells. Deletion of E1-L2 in mice caused embryonic lethality, suggesting that E1-L2 plays an important role in embryogenesis.
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"Similar to ubiquitin, FAT10 contains a C-terminal di-glycine motif which is important for conjugation to different substrates including p53 (Li et al., 2011; Raasi et al., 2001). FAT10 was reported to be activated by the E1-enzyme UBA6/E1-L2 (Chiu et al., 2007) and USE1 (Aichem et al., 2010). Silibinin, a flavanone in milk thistle (Silybum marianum L.), is widely used to treat a range of liver and gallbladder disorders, including hepatitis, cirrhosis, and as a hepatoprotectant against poisoning from wild mushroom, alcohol, chemical, and environmental toxins (Loguercio and Festi, 2011; Rainone, 2005). "
"The covalent modification with the cytokine-inducible ULM HLA-F adjacent transcript 10 (FAT10) targets proteins in a ubiquitinindependent manner for proteasomal degradation (Raasi et al., 2001; Hipp et al., 2005; Schmidtke et al., 2009). As other ULMs, FAT10 is conjugated to its substrates via isopeptide linkage mediated by an E1, E2, and possibly E3 enzyme cascade, where UBA6 (also termed UBE1L2, E1-L2, or MOP-4) and USE1 (UBA6-specific E2 enzyme) serve as E1-type activating and E2-type conjugating enzymes, respectively (Aichem et al., 2010; Pelzer and Groettrup, 2010; Chiu et al., 2007 ). FAT10 has been implicated in multiple cellular processes like apoptosis, spindle check point control during mitotic cell cycle, and NF-B activation. "
" HEK293 cells were transfected with either pcDNA3.1-His-3xFLAG-FAT10 , or with pcDNA3.1-His-3xFLAG-FAT10 ΔGG , by using TransIT-LTI Transfection Reagent (Mirus), or endogenous FAT10 expression was induced as recently described [23,47] . To maintain constantly high levels of FAT10 until harvest after 72 h, cells were transfected a second time or retreated with proinflammatory cytokines after 48 h. Afte"
[Show abstract][Hide abstract]ABSTRACT: The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directly targets its substrates for proteasomal degradation by becoming covalently attached via its C-terminal diglycine motif to internal lysine residues of its substrate proteins. The conjugation machinery consists of the bispecific E1 activating enzyme Ubiquitin-like modifier activating enzyme 6 (UBA6), the likewise bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By mass spectrometry analysis the ubiquitin E1 activating enzyme ubiquitin-activating enzyme 1 (UBE1) was identified as putative substrate of FAT10. Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines. FAT10ylation of UBE1 depends on the diglycine motif of FAT10. By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway. Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway.