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: 14.46). 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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    • "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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    • "Thus FAT10, the FAT10-binding adapter molecule Nub1 and its recently reported E1 enzyme UBE1L2 that also activates Ub belong to the few genes found to be induced under all four stimulation strategies used in this study (Figs. 2 and 4). In agreement with studies reporting the rapid proteasome dependent degradation of FAT10 conjugates in target cells (Chiu et al., 2007), accumulated FAT10-conjugates were stabilized by proteasome inhibition (Fig. 4). No obvious histological differences were found in FAT10 deficient mice (Hipp et al., 2004, 2005). "
    Dataset: Ebstein2009
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    • "EVI2A is an uncharacterized factor not included in the IPA network with blood-specific expression [42]. Cluster 7 members regulate a range of cellular mechanisms, including protein recycling [94], signal transduction [95], immuno-modulation [92], and transcript maturation [96]. "
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    ABSTRACT: As part of the civil aviation safety program to define the adverse effects of ethanol on flying performance, we performed a DNA microarray analysis of human whole blood samples from a five-time point study of subjects administered ethanol orally, followed by breathalyzer analysis, to monitor blood alcohol concentration (BAC) to discover significant gene expression changes in response to the ethanol exposure. Subjects were administered either orange juice or orange juice with ethanol. Blood samples were taken based on BAC and total RNA was isolated from PaxGeneTM blood tubes. The amplified cDNA was used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses to evaluate differential gene expression. Microarray data was analyzed in a pipeline fashion to summarize and normalize and the results evaluated for relative expression across time points with multiple methods. Candidate genes showing distinctive expression patterns in response to ethanol were clustered by pattern and further analyzed for related function, pathway membership and common transcription factor binding within and across clusters. RT-qPCR was used with representative genes to confirm relative transcript levels across time to those detected in microarrays. Microarray analysis of samples representing 0%, 0.04%, 0.08%, return to 0.04%, and 0.02% wt/vol BAC showed that changes in gene expression could be detected across the time course. The expression changes were verified by qRT-PCR.The candidate genes of interest (GOI) identified from the microarray analysis and clustered by expression pattern across the five BAC points showed seven coordinately expressed groups. Analysis showed function-based networks, shared transcription factor binding sites and signaling pathways for members of the clusters. These include hematological functions, innate immunity and inflammation functions, metabolic functions expected of ethanol metabolism, and pancreatic and hepatic function. Five of the seven clusters showed links to the p38 MAPK pathway. The results of this study provide a first look at changing gene expression patterns in human blood during an acute rise in blood ethanol concentration and its depletion because of metabolism and excretion, and demonstrate that it is possible to detect changes in gene expression using total RNA isolated from whole blood. The analysis approach for this study serves as a workflow to investigate the biology linked to expression changes across a time course and from these changes, to identify target genes that could serve as biomarkers linked to pilot performance.
    BMC Medical Genomics 07/2013; 6(1):26. DOI:10.1186/1755-8794-6-26 · 3.91 Impact Factor
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