Jin, J., Li, X., Gygi, S.P. & Harper, J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature 447, 1135-1138

Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 41.46). 07/2007; 447(7148):1135-8. DOI: 10.1038/nature05902
Source: PubMed


Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1-E2-E3 cascade controls many signalling networks. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction. Charged E2s function with E3s to ubiquitinate substrates. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s.

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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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    • "Our study suggests that DC-UbP binds to USP5 on the tandem UBA domains, implying that binding of DC-UbP to USP5 potentially regulates the deubiquitination process. On the other hand, the UbE1 enzyme utilizes ATP to activate the terminal glycine residue of Ub generating a covalent thioester linkage between ‘activated’ Ub and the UbE1 enzyme itself [44]. The UbE1 protein is mainly comprised of three domains, the adenylation domain that binds ATP and Ub, the catalytic cysteine domain that binds activated Ub, and the C-terminal Ub-fold domain (UFD) that recruits specific E2 conjugating enzymes [27]. "
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    ABSTRACT: The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.
    PLoS ONE 09/2014; 9(9):e107509. DOI:10.1371/journal.pone.0107509 · 3.23 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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