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


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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    ABSTRACT: Pleiotropic pro-inflammatory cytokines, TNF-α and IFN-γ (TI), play important yet diverse roles in cell survival, proliferation, and death. Recent evidence highlights FAT10 as a downstream molecule in the pathway of inflammation-induced tumorigenesis through mediating the effect of cytokines in causing numerical CIN and protecting cells from cytokines-induced cell death. cDNA microarray analysis of cells treated with TI revealed 493 deregulated genes with FAT10 being the most up-regulated (85.7-fold) gene and NF-κB being the key nodal hub of TI-response genes. Silibinin is reported to be a powerful antioxidant and has anti-C effects against various carcinomas by affecting various signaling molecules/pathways including MAPK, NF-κB and STATs. As NF-κB signaling pathway is a major mediator of the tumor-promoting activities of TI, we thus examine the effects of silibinin on TI-induced FAT10 expression and CIN. Our data showed that silibinin inhibited expression of FAT10, TI-induced chromosome instability (CIN) as well as sensitizes cells to TI-induced apoptosis. Significantly, silibinin suppressed intra-tumorally injected TNF-α-induced tumor growth. This represents the first report associating silibinin with FAT10 and demonstrating that silibinin can modulate TI-induced CIN, apoptosis sensitivity and suppressing TNF-α-induced tumor growth. © 2015. Published by The Company of Biologists Ltd.
    Biology Open 07/2015; 4(8). DOI:10.1242/bio.011189 · 2.42 Impact Factor
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    • "In most other tissues, FAT10 is not expressed unless the pro-inflammatory cytokines IFNc and TNFa are present (Liu et al., 1999; Raasi et al., 1999). FAT10 needs no processing, but is activated and conjugated by the constitutively expressed E1 and E2 enzymes UBA6 and USE1, respectively (Aichem et al., 2010; Chiu et al., 2007; Jin et al., 2007; Pelzer et al., 2007). Potential E3 ligases and deconjugating enzymes have not yet been identified. "
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    ABSTRACT: Bacterial invasion of eukaryotic cells is counteracted by cell-autonomous innate immune mechanisms including xenophagy. The identification of cytosolic bacteria by ubiquitylation and Galectin-8 leads to recruitment of autophagy adaptors like p62, NDP52, and optineurin which initiate the destruction of bacteria by xenophagy. Here we show that the functionally barely characterized IFNγ- and TNFα-inducible ubiquitin-like modifier FAT10, which binds to the autophagy adaptor p62 but has not been shown to associate with pathogens before, is recruited to cytosolic Salmonella Typhimurium in human cells. FAT10 decorated S. Typhimurium were simultaneously decorated with ubiquitin, p62, NDP52, and the autophagy marker LC3. FAT10 co-localized with p62 positive microdomains on S. Typhimurium whereas co-localization with NDP52 was only partial. A kinetic analysis revealed an early but only transient decoration of bacteria by FAT10 which resembled that of p62. While bacterial replication was not detectably altered in FAT10-depleted or overexpressing cells in vitro, survival experiments revealed a higher susceptibility of FAT10-deficient than wild type NRAMP1-transgenic mice to orally inoculated S. Typhimurium. Taken together, our data suggest a role for FAT10 in the intracellular defense against bacteria.
    Journal of Cell Science 09/2014; 127(22). DOI:10.1242/jcs.152371 · 5.43 Impact Factor
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    • "To avoid confusion in terminology, we respectively refer to these two isoforms as Uba1A, defined here as the predominantly nuclear form of Uba1, and Uba1B, defined here as the cytoplasmic form of Uba1, instead of E1a and E1b. Uba6 is required to activate the E2 Use1 (Uba6-specific E2) both in vitro and in vivo [6] and can also activate another ubiquitin-like modifier, FAT10 [7]. "
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    ABSTRACT: Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus.
    PLoS ONE 05/2014; 9(5):e96666. DOI:10.1371/journal.pone.0096666 · 3.23 Impact Factor
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