Reexamination of the Role of Ubiquitin-Like Modifier ISG15 in the Phenotype of UBP43-Deficient Mice

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.Postranslational modification of proteins by covalently linked ubiquitin and ubiquitin-like modifiers (UBLs) is a rapid and versatile regulatory mechanism involved in many cellular processes (5,14). ISG15 is a 15-kDa interferon (IFN)-induced UBL which bears two ubiquitin-like domains, each about 30% homologous to ubiquitin (1,3,6,12). Analogous to ubiquitin and other UBLs, ISG15 is conjugated by an isopeptide bond to target proteins (7). Correspondingly, an ISG15-specific enzymatic cascade that consists of an activating UBE1L (E1) and a conjugating UbcH8 (E2) enzyme was recently identified (18,19). Several ISG15-modified substrates such as serpin 2a (4), JAK1, STAT1, phospholipase C␥1, and ERK1 (8) were reported, but rigorous evidence for an ISG15 covalently linked to these proteins, with the exception of serpin 2a, has not yet been found. Most recently, a large number (158) of ISG15 target proteins that function in diverse cellular pathways were identified by mass spectroscopy (20).Previous work from several laboratories implicated ISG15 and ISGylation in diverse biological activities, ranging from a role in pregnancy to antiviral and antitumor defense (11,17). Speculations on the biological significance of ISG15 were further amplified by analyses of mice lacking UBP43/USP18 (16), a protease that removes conjugated ISG15 from substrate proteins (9). Mice with a targeted deletion of UBP43 (UBP43 Ϫ/Ϫ ) have elevated levels of ISG15 conjugates and develop brain injury due to necrosis of ependymal cells, accompanied by hydrocephalus and early death (16). Investigators reported a prolonged STAT1 phosphorylation in the absence of UBP43 and claimed that ISG15 modification is an important factor in the regulation of the JAK-STAT pathway and IFN signaling (10, 17). Most recently, some evidence was presented for a role of UBP43 and possibly ISGylation in innate immun...

Microarray analysis reveals that Type I interferon strongly increases the expression of immune-response related genes in Ubp43 (Usp18) deficient macrophages

“…Our data showed that a variety of CXC (CXCL10, CXCL11) and CC chemokines (CCL2, 3, 4 and 5) as well as CC chemokine receptor (CCRL2) are induced by IFN-β in Ubp43 −/− BMMs (Fig. 4 and supplemental data Although there are arguments about the effect of Ubp43 deficiency on the IFN signaling pathway and the expression of IFN stimulated genes [14], our present study using Microarray analysis reveals that Ubp43 deficiency in BMMs generally enhanced the expression of genes that are induced by IFN in wild-type cells. The IFN hypersensitivity of Ubp43 −/− cells and subsequent higher induction of immune response related genes in Ubp43 −/− cells (Fig.…”

“…However, the expression of three other IFN-induced genes, GBP1, IRF1, and Mx1, remained unaffected [14]. All five genes were in our list as ISGs in Ubp43 −/− BMM.…”

“…Comparing IFN treated wild-type murine embryonic fibroblasts (MEFs) with Ubp43 −/− cells , Knobeloch et al detected prolonged expression of two ISGs, IRF7 and 2′,5′ oligoadenylate synthetase [14]. However, the expression of three other IFN-induced genes, GBP1, IRF1, and Mx1, remained unaffected [14].…”

IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses

“…Recent studies have identified Ͼ100 target proteins of ISG15, which encompass diverse cellular pathways (15)(16)(17). However, the consequence of ISGylation of target proteins and thus the cellular function(s) of ISG15 remain unknown (18,19).…”

Fighting mycobacteria through ISGylation

“…Furthermore, ISG15 can inhibit the budding of certain viruses and modify viral proteins, and some viruses have developed strategies to inhibit ISGylation, underscoring the function of ISG15 and ISGylation in the antiviral response [6]. However, other viruses-such as vesicular stomatitis and lymphocytic choriomeningitis virus-have similar effects on ISG15 -/-and wild-type mice [7], suggesting specialized functions of ISGylation after viral infection. In addition, upfront hot of f the press a closer look at the role of ISG15 in regulating human viruses complicates the picture further: ISG15 has been found to stimulate rather than inhibit hepatitis C virus production in vitro, probably by preventing the degradation of viral proteins through competition between ISGylation and ubiquitylation [8].…”