Interferons (IFNs) regulate diverse cellular functions through activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Lack of Ubp43, an IFN-inducible ISG15 deconjugating enzyme, leads to IFN hypersensitivity in ubp43À/À mice, suggesting an important function of Ubp43 in downregulation of IFN responses. Here, we show that Ubp43 negatively regulates IFN signaling independent of its isopeptidase activity towards ISG15. Ubp43 functions specifically for type I IFN signaling by downregulating the JAK-STAT pathway at the level of the IFN receptor. Using molecular, biochemical, and genetic approaches, we demonstrate that Ubp43 specifically binds to the IFNAR2 receptor subunit and inhibits the activity of receptor-associated JAK1 by blocking the interaction between JAK and the IFN receptor. These data implicate Ubp43 as a novel in vivo inhibitor of signal transduction pathways that are specifically triggered by type I IFN.

The expression of ubiquitin-like modifier ISG15 and its conjugation to target proteins are highly induced by interferon (IFN) stimulation and during viral and bacterial infections. However, the biological significance of this modification has not been clearly understood. To investigate the function of protein modification by ISG15, we generated a mouse model deficient in UBE1L, an ISG15-activating enzyme. Ube1L ؊/؊ mice did not produce ISG15 conjugates but expressed free ISG15 normally. ISGylation has been implicated in the reproduction and innate immunity. However, Ube1L؊/؊ mice were fertile and exhibited normal antiviral responses against vesicular stomatitis virus and lymphocytic choriomeningitis virus infection. Our results indicate that UBE1L and protein ISGylation are not critical for IFN-␣/␤ signaling via JAK/STAT activation. Moreover, using Ube1L/Ubp43 double-deficient mice, we showed that lack of UBP43, but not the increase of protein ISGylation, is related to the increased IFN signaling in Ubp43-deficient mice.

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.

Hepatitis B virus (HBV) causes both acute and chronic infection of the human liver and is associated with the development of liver cirrhosis and hepatocellular carcinoma. UBP43 (USP18) is known as an ISG15-deconjugating enzyme and an inhibitor of type I IFN signaling independent of its enzyme activity. In this study, we examined the role of these two previously identified functions of UBP43 in the innate immune response to HBV viral infection. As an in vivo HBV replication model system, a replication-competent DNA construct was injected hydrodynamically into the tail veins of mice. Although the lack of ISG15 conjugation in the absence of ISG15-activating enzyme UBE1L (UBA7) did not affect the level of HBV replication, the steady-state level of HBV DNA was substantially reduced in the UBP43-deficient mice in comparison to the wild-type controls. In addition, introduction of short hairpin RNA against UBP43 resulted in substantially lower levels of HBV DNA at day 4 postinjection and higher levels of ISG mRNAs. These results suggest that HBV infection is more rapidly cleared if UBP43 expression is reduced. Furthermore, these results illustrate the therapeutic potential of modulating UBP43 levels in treating viral infection, especially for viruses sensitive to IFN signaling.

Interferon (IFN) signaling induces the expression of interferon-responsive genes and leads to the activation of pathways that are involved in the innate immune response. Ubp43 is an ISG15-specific isopeptidase, the expression of which is activated by IFN. Ubp43 knock-out mice are hypersensitive to IFN-␣/␤ and have enhanced resistance to lethal viral and bacterial infections. Here we show that in addition to protection against foreign pathogens, Ubp43 deficiency increases the resistance to oncogenic transformation by BCR-ABL. BCR-ABL viral transduction/transplantation of wild-type bone marrow cells results in the rapid development of a chronic myeloid leukemia (CML)-like myeloproliferative disease; in contrast, a significantly increased latency of disease development is observed following BCR-ABL viral transduction/transplantation of Ubp43-deficient bone marrow cells. 4,5 These kinases then phosphorylate STAT1 and STAT2, leading to the activation of downstream signal transduction pathways. 6-9 Furthermore, a family of suppressors of cytokine signaling (SOCS) and several protein tyrosine phosphatases negatively regulate the STAT signaling pathway. [10][11][12] Defects in such regulators may result either in the loss of response or a hyperresponse to IFN stimulation.Type 1 IFN signaling triggers the expression of hundreds of IFN-stimulated genes (ISGs). 13,14 Among these is the ISG15 deconjugating enzyme Ubp43 (Usp18). [15][16][17][18][19] ISG15 is a ubiquitinlike modifier whose expression and conjugation to other proteins (ISGylation) is strongly increased upon type 1 IFN stimulation. 20,21 Ubp43-deficient cells accumulate higher levels of ISGylated proteins and are hypersensitive to type 1 IFN treatment, as evidenced by the enhanced and prolonged activation of STAT phosphorylation in these cells. 22,23 Furthermore, Ubp43 knock-out mice show a higher resistance to viral and bacterial infection, 24,25 indicating an important role for Ubp43 in the regulation of IFN signal transduction. Recently, using cells with different levels of protein ISGylation and Ubp43 expression, we demonstrated that UBP43 is a novel negative regulator of type 1 interferon signaling and this function is independent of Ubp43 isopeptidase activity against ISG15 conjugates. 26,27 Type 1 IFNs suppress cell proliferation and promote apoptosis, 28 as such they have been used in the clinical treatment of several cancers, including leukemia. 29 A specific example is in the treatment of chronic myeloid leukemia (CML), where IFN was the primary choice before imatinib mesylate became available. [30][31][32] In nearly all cases of CML, patients carry a reciprocal translocation between chromosomes 9 and 22. 33,34 This results in a fusion protein consisting of the N-terminal portion of BCR joined to most of the ABL tyrosine kinase. The chimeric BCR-ABL tyrosine kinase is constitutively activated as a result of the oligomerization domain provided by BCR. The tyrosine kinase activity of BCR-ABL activates several signaling intermediates, such as Ras, Akt, ...