Keun Il Kim

UBP43 (USP18) specifically removes ISG15 from conjugated proteins.

UBP43 shows significant homology to well characterized ubiquitin-specific proteases and previously was shown to hydrolyze ubiquitin-β-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 andUBP43 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.

UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity

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 ubp43−/− 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.

Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection

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.

Interferon-Inducible Ubiquitin E2, Ubc8, Is a Conjugating Enzyme for Protein ISGylation

ABSTRACT Protein ISGylation is unique among ubiquitin-like conjugation systems in that the expression and conjugation processes are induced by specific stimuli, mainly via the alpha/beta interferon signaling pathway. It has been suggested that protein ISGylation plays a special role in the immune response, because of its interferon-signal dependency and its appearance only in higher eukaryotic organisms. Here, we report the identification of an ISG15-conjugating enzyme, Ubc8. Like other components of the protein ISGylation system (ISG15, UBE1L, and UBP43), Ubc8 is an interferon-inducible protein. Ubc8 clearly mediates protein ISGylation in transfection assays. The reduction of Ubc8 expression by small interfering RNA causes a decrease in protein ISGylation in HeLa cells upon interferon treatment. Neither UbcH7/UbcM4, the closest homologue of Ubc8 among known ubiquitin E2s, nor the small ubiquitin-like modifier E2 Ubc9 supports protein ISGylation. These findings strongly suggest that Ubc8 is a major ISG15-conjugating enzyme responsible for protein ISGylation upon interferon stimulation. Furthermore, we established an assay system to detect ISGylated target proteins by cotransfection of ISG15, UBE1L, and Ubc8 together with a target protein to be analyzed. This method provides an easy and effective way to identify new targets for the ISGylation system and will facilitate related studies.

Ube1L and protein ISGylation are not essential for alpha/beta interferon signaling.

ABSTRACT 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-α/β 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.

Enhanced Antibacterial Potential in UBP43-Deficient Mice against Salmonella typhimurium Infection by Up-Regulating Type I IFN Signaling

ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are dramatically induced upon viral or bacterial infection. We have generated a UBP43 knockout mouse model that is lacking an ISG15-specific isopeptidase to study the biological role of the protein ISGylation system. We report that UBP43-deficient mice are hypersensitive to LPS-induced lethality and that TIR domain-containing adapter inducing IFN-β → IFN regulatory factor 3 → type I IFN is the major axis to induce protein ISGylation and UBP43 expression in macrophages upon LPS treatment. In ubp43−/− macrophages, upon LPS treatment we detected increased expression of IFN-stimulated genes, including genes for several cytokines and chemokines involved in the innate immune response. The ubp43−/− mice were able to restrict the growth of Salmonella typhimurium more efficiently than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but a detriment to the body without strict control.

ISG15, not just another ubiquitin-like protein.

ISG15 is a ubiquitin-like protein containing two ubiquitin homology domains and becomes conjugated to a variety of proteins when cells are treated with type I interferon or lipopolysaccharide. Although ISG15 shares several common properties with those of other ubiquitin-like molecules, it is a unique member, whose expression and conjugation to target proteins are tightly regulated by specific signaling pathways, indicating it may be associated with specialized functions in innate immune system. Loss of UBP43 (USP18), a protease that specifically removes ISG15 from ISG15-modified proteins, in mice leads to decreased life span, brain cell injury, and hypersensitivity to interferon stimulation. In UBP43 deficient cells, interferon induces a prolonged Stat1 tyrosine phosphorylation and DNA binding, which result in a prolonged and enhanced activation of interferon-stimulated genes.

UBP43, an ISG15-specific deconjugating enzyme: expression, purification, and enzymatic assays.

UBP43 is the only deconjugating enzyme for the protein ISGylation system thus far identified. UBP43 activity is not critical for precursor processing, as UBP43-deficent cells can generate ISG15 conjugates upon type I interferon treatment. However, UBP43 deficiency caused a defect in the negative regulation of type I interferon signaling, resulting in enhanced and prolonged activation of Jak/Stat upon type I interferon treatment. This chapter describes the expression, purification, and enzymatic assays for UBP43.

Statistical optimization of culture medium to produce recombinant viral protein by Escherichia coli host for diagnostic kit to detect human immunodeficiency virus (HIV) infection

The maximal production of recombinant HIV1 gp41 by E. coli was examined in optimal culture condition and medium compositions. The culture condition such as growth, initial medium pHs, IPTG concentrations, induction times, temperature (0.5 OD, 7.6, 0.75 mM, 4.6 h, 32 °C respectively), and yeast extract (7.51 g/l), tryptone (7.26 g/l), glucose (2.45 g/l), NaCl (20.40 g/l), betaine (10.41 mM) and ampicillin (71.23 μg/ml) was optimized using statistical experimental design and response surface method (RSM). One of the main popular methods to attain high cell density in fed-batch culture is by controlling the nutrient feeding, which is often necessary for high yield in protein (0.63-0.72 mg/l) and cell (1.7-2 g/l) of the desired product in four litter fermentations.