Herc5, an interferon-induced HECT E3 enzyme, is required for conjugation of ISG15 in human cells
ABSTRACT ISG15 is an interferon (IFN)-alpha/beta-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections. A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-beta, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.
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ABSTRACT: Protein modification by the ubiquitin-like protein ISG15 is an interferon (IFN) effector system, which plays a major role in antiviral defense. ISG15 modification is counteracted by the isopeptidase USP18, a major negative regulator of IFN signaling, which was also shown to exert its regulatory function in an isopeptidase-indepen-dent manner. To dissect enzymatic and nonenzymatic functions of USP18 in vivo, we generated knock-in mice (USP18 C61A/C61AProceedings of the National Academy of Sciences 01/2015; 112(5). DOI:10.1073/pnas.1412881112 · 9.81 Impact Factor
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ABSTRACT: The interferon-stimulated gene 15 (Isg15) encodes a ubiquitin-like protein that is induced in the endometrium by pregnancy in mice, humans and ruminants. Because ISG15 is a component of the innate immune system, we hypothesized that development of the embryo, fetus and postnatal pup may be impaired in mice lacking Isg15 (Isg15(-/-)) and that this development would be further impaired in response to environmental insults such as hypoxia. The number of implantation sites, resorption sites, dead embryos and the changes in overall gross morphology of the uterus were evaluated in Isg15(-/-) mice on Days 7.5 and 12.5 postcoitum (dpc). Postnatal development also was monitored from birth to 12 wk of age. On 7.5 dpc, the number of implantation sites and serum progesterone concentrations were similar. However, embryo mortality increased (P < 0.05) in Isg15(-/-) dams by 12.5 dpc, resulting in smaller litter sizes (4.2 ± 0.24 embryos; n = 83 litters) compared to Isg15(+/+) females (7.94 ± 0.32 pups; n = 47 litters). Embryo mortality in Isg15(-/-) mice was further exacerbated to 70% when dams were stressed through housing under hypoxic conditions (PB = 445 mmHg; 6.5-12.5 dpc). Transmission electron microscopy revealed lesions in antimesometrial decidua as well as trophobast cells adjacent to decidual cells on 7.5 dpc. ISG15 was localized to mesometrial decidua on 7.5 dpc. By 12.5 dpc, ISG15 was intensely localized to the labyrinth of the placenta. By 7.5 dpc, uterine natural killer cell (uNK) migration into the mesometrial pole was diminished by 65% and was less prevalent in Isg15(-/-) compared to Isg15(+/+) deciduum. Postnatal growth rate of offspring that survived to birth from Isg15(-/-) and Isg15(+/+) dams was not different. Embryo mortality occurs in pregnant Isg15(-/-) mice, is exacerbated by environmental insults like maternal hypoxia and might result from impaired early decidualization, vascular development and formation of the labyrinth. Copyright 2014 by The Society for the Study of Reproduction.Biology of Reproduction 12/2014; 92(2). DOI:10.1095/biolreprod.114.122002 · 3.45 Impact Factor
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ABSTRACT: The ubiquitin-like protein FAT10 and the homeobox protein HOXB9 each promote metastatic progression in hepatocellular carcinoma (HCC). In this study, we investigated the clinicopathologic significance of FAT10 and HOXB9 in HCC and investigated a mechanistic role for FAT10 in HOXB9-mediated invasiveness and metastasis. Relative to adjacent normal tissues, FAT10 and HOXB9 were markedly overexpressed in HCC, where a positive correlation in their expression and associated malignant characteristics were found. RNAi-mediated silencing of FAT10 decreased HOXB9 expression and inhibited HCC invasion and metastasis in vitro and in vivo. The effects of FAT10 silencing were reversed by HOXB9 overexpression, whereas RNAi-mediated silencing of HOXB9 decreased HCC invasion and metastasis driven by FAT10 overexpression. Mechanistically, FAT10 regulated HOXB9 expression by modulating the β-catenin/TCF4 pathway, directly binding to β-catenin and preventing its ubiquitination and degradation. Together, our results identiﬁed a novel HCC regulatory circuit involving FAT10, β-catenin/TCF4 and HOXB9, the dysfunction of which drives invasive and metastatic character in HCC.Cancer Research 07/2014; 74(18). DOI:10.1158/0008-5472.CAN-14-0284 · 9.28 Impact Factor