Article

E1-L2 activates both ubiquitin and FAT10

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.
Molecular Cell (Impact Factor: 14.02). 10/2007; 27(6):1014-23. DOI: 10.1016/j.molcel.2007.08.020
Source: PubMed

ABSTRACT

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). "
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    • "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. "
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    • " HEK293 cells were transfected with either pcDNA3.1-His-3xFLAG-FAT10 [19], or with pcDNA3.1-His-3xFLAG-FAT10 ΔGG [23], 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"
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