UBP43 shows significant homology to well characterized ubiquitin-specific proteases and previously was shown to hydrolyze ubiquitin-␤-galactosidase fusions in Escherichia coli. In our assays, the activity of UBP43 toward Ub fusions was undetectable in vitro directing us to investigate the possibility of Ub-like proteins such as SUMO, Nedd8, and ISG15 as probable substrates. We consequently demonstrate that UBP43 can efficiently cleave only ISG15 fusions including native ISG15 conjugates linked via isopeptide bonds. In addition to commonly used methods we introduce a new experimental design featuring ISG15-UBP43 fusion self-processing. Deletion of the UBP43 gene in mouse leads to a massive increase of ISG15 conjugates in tissues indicating that UBP43 is a major ISG15-specific protease. UBP43 is the first bona fide ISG15-specific protease reported. Both ISG15 and UBP43 genes are known to be strongly induced by interferon, genotoxic stress, and viral infection. We postulate that UBP43 is necessary to maintain a critical cellular balance of ISG15-conjugated proteins in both healthy and stressed organisms.ISG15 is one of the most strongly induced genes after interferon (IFN) 1 treatment (1, 2) and is also significantly induced by influenza B virus (3), lipopolysaccharide (4), and genotoxic stress (5). ISG15 was originally identified by Farrell et al. (1) and later characterized by Knight and co-workers (6, 7). Subsequently, the sequence of ISG15 protein was noted to possess significant homology to a diubiquitin sequence, accounting for its cross-reactivity with affinity purified anti-ubiquitin antibodies (8). Several reports demonstrate that ISG15 is released by various cell types and can act as cytokine leading to proliferation of NK cells (9 -11). Most remarkably, ISG15 was found to be conjugated to intracellular proteins via an isopeptide bond in a manner similar to ubiquitin (Ub), SUMO, and Nedd8 (12). Conjugation of ubiquitin-like proteins (Ubls) involves a three-step mechanism whereby specific enzymes (or enzyme complexes) activate and covalently link Ubls to their substrates (13,14). Narasimhan et al. (15) demonstrated that ISG15 conjugation occurs via a similar but distinct pathway compared with Ub conjugation. Yuan and Krug (3) discovered that an activating enzyme for ISG15 is UBE1L. Although the role of Ub, Nedd8, and SUMO conjugation has been assessed in numerous studies (16 -20), the biological significance of ISG15 modification remains unknown and the proteins that are targeted by ISG15 have not been identified. It is unknown whether ISG15 conjugates can be targeted to proteasomes in a way similar to Ub conjugates. Alternatively, ISG15 conjugation might antagonize binding of Ub and save proteins from degradation or modify biological activities of targeted proteins as is the case with Nedd8 and SUMO modification (16,18). Loeb and Haas (21) demonstrated that a substantial amount of ISG15 conjugates are co-localized with intermediate filaments of the cytoskeleton. It is therefore possible that one of th...

ISG15 is one of the most strongly induced genes upon viral infection, type I interferon (IFN) stimulation, and lipopolysaccharide (LPS) stimulation. Here we report that mice lacking UBP43, a protease that removes ISG15 from ISGylated proteins, are hypersensitive to type I IFN. Most importantly, in UBP43-deficient cells, IFN-β induces a prolonged Stat1 tyrosine phosphorylation, DNA binding, and IFN-mediated gene activation. Furthermore, restoration of ISG15 conjugation in protein ISGylation-defective K562 cells increases IFN-stimulated promoter activity. These findings identify UBP43 as a novel negative regulator of IFN signaling and suggest the involvement of protein ISGylation in the regulation of the JAK-STAT pathway.

Innate immune responses provide the host with an early protection barrier against infectious agents, including viruses, and help shape the nature and quality of the subsequent adaptive immune responses of the host. Expression of ISG15 (UCRP), a ubiquitin-like protein, and protein ISGylation are highly increased upon viral infection. We have identified UBP43 (USP18) as an ISG15 deconjugating protease. Protein ISGylation is enhanced in cells deficient in UBP43 (ref. 6). Here we have examined the role of UBP43, encoded by the gene Usp18, in innate immunity to virus infection. Usp18(-/-) mice were resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that developed in wild-type mice after intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV), respectively. Survival of Usp18(-/-) mice after intracerebral LCMV infection correlated with a severe inhibition of LCMV RNA replication and antigen expression in the brain and increased levels of protein ISGylation. Consistent with these findings, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages from Usp18(-/-) mice showed restricted LCMV replication. Moreover, MEF from Usp18(-/-) mice showed enhanced interferon-mediated resistance to the cytopathic effect caused by VSV and Sindbis virus (SNV). This report provides the first direct evidence that the ISG15 protease UBP43 and possibly protein ISGylation have a role in innate immunity against viral infection.

UBP43 (USP18) is a protease that removes the ubiquitinlike modifier ISG15 from conjugated proteins. Here we present the first report of dysregulation of protein ISG15 modification by the generation of UBP43 knockout mice. In the absence of UBP43, brain tissue showed an elevated level of ISG15 conjugates, and cellular necrosis was evident in the ependyma. Such disruption of the blood-brain barrier resulted in severe neurologic disorders. These results demonstrate that UBP43 plays a critical role in maintaining the homeostatic balance of ISG15-conjugated protein, and that regulation of cellular levels of ISG15 protein modification is essential for brain cell function.

No abstract

Human cancer is controlled by a complex interaction between genetic and environmental factors. Such environmental factors are well defined for smoking-induced lung cancer; however, the roles of specific genes have still to be elucidated. Glutathione transferase P (GSTP) catalyzes the detoxification of electrophilic diol epoxides produced by the metabolism of polycyclic aromatic hydrocarbons such as benzo [a]pyrene (BaP), a common constituent of tobacco smoke. Activityaltering polymorphisms in Gstp have therefore been speculated to be potential risk modifiers in lung cancer development. To clearly establish a role for GSTP in lung tumorigenesis, we investigated whether deletion of the murine Gstp genes (Gstp1 and Gstp2) alters susceptibility to chemically induced lung tumors following exposure to BaP, 3-methylcholanthrene (3-MC), and urethane. Gstp-null mice were found to have substantially increased numbers of adenomas relative to wildtype mice following exposure to all three compounds (8.3-, 4.3-, and 8.7-fold increase for BaP, 3-MC, and urethane, respectively). In Gstp-null mice, the capacity of pulmonary cytosol to catalyze conjugation of the BaP diol epoxide was significantly reduced. Concomitant with this, a significant increase in the level of BaP DNA adducts was measured in the lungs of null animals; however, no increase in DNA adducts was measured in the case of 3-MC exposure, suggesting that an alternative protective pathway exists. Indeed, significant differences in pulmonary gene expression profiles were also noted between wild-type and null mice. This is the first report to establish a clear correlation between Gstp status and lung cancer in vivo.

Glutathione transferases are a multigene family of proteins that catalyze the conjugation of toxic electrophiles and carcinogens to glutathione. Glutathione transferase Pi (GSTP) is commonly overexpressed in human tumors and there is emerging evidence that the enzyme has additional cellular functions in addition to its role in drug and carcinogen detoxification. To investigate the unique functions of this enzyme, we have crossed Gstp null mice with an initiated model of colon cancer, the Apc Min mouse. In contrast to the Apc Min/؉ Gstp1/p2 ؉/؉ (Gstp-wt Apc Min ) mice, which rarely develop colonic tumours, Apc Min/؉ Gstp1/p2 ؊/؊ (Gstp-null Apc Min ) mice had a 6-fold increase in colon adenoma incidence, and a 50-fold increase in colorectal adenoma multiplicity, relative to Gstp-wt Apc Min . This increase was associated with early tumor onset and decreased survival. Analysis of the biochemical changes in the colon tissue of Gstp-null Apc Min mice demonstrated a marked induction of many inflammatory genes, including IL-6, IL-4, IFN-␥, and inducible nitric oxide synthase. In support of the induction of inducible nitric oxide synthase, a profound induction of nitrotyrosine adducts was observed. Gstp therefore appears to play a role in controlling inflammatory responses in the colon, which would explain the change in tumor incidence observed. These data also suggest that individual variation in GSTP levels may be a factor in colon cancer susceptibility.cancer ͉ colorectal ͉ inflammation G lutathione S-transferases play a key role in chemical detoxification by catalyzing the conjugation of reduced glutathione to reactive electrophiles (1). In a genetic approach to study GST functions, we have generated mice nulled at the glutathione transferase Pi (GSTP) Gstp gene locus (2). These mice develop normally, are fertile, and show no obvious abnormalities. Topical application of the tumor initiator 7,12-dimethylbenz[a]anthracene, followed by the promoting agent 12-0-tetradecanoylphorbol-13-acetate, resulted in a significant increase in the number of papillomas in null animals (2). Similarly, increased adenoma formation in the lungs of Gstp-null mice relative to wild-type mice was also observed following dosing with benzo[a]pyrene, 3-methylcholanthene and urethane (3). In recent studies we have obtained evidence that this protein can also modulate toxicological or carcinogenic response in a manner distinct from its role in chemical detoxification (4). Further evidence of unique GSTP function is demonstrated by its ability to form protein-protein interactions and regulate the activities of several cellular proteins (JNK and TRAF2) independently of its catalytic function (5, 6). GSTP has also been shown to potentiate S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo (7).To further evaluate the role of GSTP in disease processes, we have investigated whether GSTP can alter colon cancer susceptibility in the Apc Min mouse. Mutations in the Apc gene is a major initiating factor in the aet...

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, ...