Reexamination of the role of ubiquitin-like modifier ISG15 in the phenotype of UBP43-deficient mice - PubMed (original) (raw)

Reexamination of the role of ubiquitin-like modifier ISG15 in the phenotype of UBP43-deficient mice

Klaus-Peter Knobeloch et al. Mol Cell Biol. 2005 Dec.

Abstract

UBP43/USP18 was described as a specific protease that removes conjugated ubiquitin-like modifier ISG15 from target proteins. The severe phenotype of UBP43(-/-) mice characterized by premature death, brain cell injury, and deregulated STAT1 signaling was ascribed to an enhanced conjugation of ISG15. In contrast, no phenotypic changes were detected in ISG15(-/-) mice. To verify the role of ISG15 in the phenotype of UBP43(-/-) mice, we employed mice deficient for both ISG15 and UBP43. Here, we show that the phenotype of UBP43(-/-) mice was not rescued by the absence of ISG15, as evident from unchanged mortality, neurological symptoms, and occurrence of hydrocephalus. Also, the reported hypersensitivity of UBP43(-/-) mice to an interferon inducer, poly(I . C), was ISG15 independent. Furthermore, no evidence for a role of ISG15 in the modulation of STAT1 signaling or in the resistance against lymphocytic choriomeningitis virus and vesicular stomatitis virus was found. Presented results clearly demonstrate that the phenotypic alterations of UBP43(-/-) mice are not caused by the lack of ISG15 deconjugation and must be due to another, non-ISG15-mediated molecular mechanism.

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Figures

FIG. 1.

FIG. 1.

Mortality of ISG15−/− UBP43−/− and UBP43−/− mice. Survival of wild-type (n = 25), ISG15−/− (n = 25), UBP43−/− (n = 36), and ISG15−/− UBP43−/− (n = 24) mice was monitored for a time period of 8 weeks. wt, wild type.

FIG. 2.

FIG. 2.

Hydrocephalus in UBP43−/− and UBP43−/− ISG15−/− mice. Macroscopic images of sculls (A) and hematoxylin-eosin stained coronal sections (B) of brains derived from 8-week-old mice of the indicated genotypes. wt, wild type.

FIG. 3.

FIG. 3.

Hypersensitivity to poly(I · C). Sensitivity of wild-type, UBP43, ISG15−/−, and ISG15−/− UBP43−/− mice to poly(I · C) treatment was assessed by intraperitoneal injection of mice with poly(I · C) (50 mg/g of body weight).

FIG. 4.

FIG. 4.

STAT1 responses of wild-type, UBP43, ISG15−/−, and ISG15−/− UBP43−/− MEF. (A) MEF were stimulated with 100 U/ml of IFN-β for the times indicated. Western blots were incubated with anti-pTyr701 antibody to detect STAT1 phosphorylation and with anti-STAT1 and β-actin as loading controls. (B) MEF were stimulated with 100 U/ml IFN-β for the times indicated. Upon RNA isolation, Northern blots were hybridized with probes specific for the STAT-1 target genes IRF7, 2′,5′ oligoadenylate synthetase (OAS), guanylate-binding protein-1 (GBP1), IRF1, and Mx.1, with β-actin as a loading control. wt, wild type.

FIG. 5.

FIG. 5.

Viral infections. (A) Wild-type (n = 6), UBP43−/− (n = 4), ISG15−/− (n = 5), and ISG15−/− UBP43−/− (n = 6) mice were infected intracranially with 105 IU of LCMV, and survival was monitored daily. (B) RNA from MEF of the indicated genotypes pretreated with the indicated amounts of IFN-β was isolated 1, 2, or 3 days postinfection (d1 p.i., d2 p.i., and d3 p.i., respectively) with LCMV, as described in Materials and Methods. RNA was separated, and Northern blots were hybridized using a probe specific for 5S LCMV and for actin as a loading control, respectively. (C) Virus replication in wild-type, UBP43−/−, ISG15−/−, and ISG15−/− UBP43−/− MEF either untreated (open bars) or treated with 100 U/ml IFN-β (filled bars) 24 h prior to infection with LCMV was assessed by measuring viral titers in the supernatant 1, 2, or 3 days after infection as described in Materials and Methods. wt, wild type.

FIG. 6.

FIG. 6.

Antiviral response of wild-type, UBP43, ISG15−/−, and ISG15−/− UBP43−/− cells upon VSV infection. MEF were incubated with serial dilutions of recombinant murine IFN-β as indicated. After 24 h, cell cultures were infected with VSV and cell viability was monitored 24 h later. wt, wild type.

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