SUMO (small ubiquitin-related modifier) modulates protein structure and function by covalently binding to the lysine side chains of the target proteins. Yeast cells contain two SUMO proteases, Ulp1 and Ulp2, that cleave sumoylated proteins in the cell. Ulp1 (SUMO protease 1) processes the SUMO precursor to its mature form and also de-conjugates SUMO from side chain lysines of target proteins. Here we demonstrate that attachment of SUMO to the N-terminus of under-expressed proteins dramatically enhances their expression in E. coli. SUMO protease 1 was able to cleave a variety of SUMO fusions robustly and with impeccable specificity. Purified recombinant SUMO-GFPs were efficiently cleaved when any amino acid, except proline, was in the+1 position of the cleavage site. The enzyme was active over a broad range of buffer and temperature conditions. Purification of certain recombinant proteins is accomplished by production of Ub-fusions from which Ub can be subsequently removed by de-ubiquitinating enzymes (DUBs). However, DUBs are unstable enzymes that are difficult to produce and inexpensive DUBs are not available commercially. Our findings demonstrate that SUMO protease 1/SUMO-fusion system may be preferable to DUB/Ub-fusion. Enhanced expression and solubility of proteins fused to SUMO combined with broad specificity and highly efficient cleavage properties of the SUMO protease 1 indicates that SUMO-fusion technology will become a useful tool in purification of proteins and peptides.

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.

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.

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in antiviral responses. The interferon-inducible ubiquitin-like protein ISG15 is expressed in cells in response to a variety of stress conditions like viral or bacterial infection and is present in its free form or is conjugated to cellular proteins. In addition, protein ubiquitination plays a regulatory role in the immune system. Many viruses modulate the ubiquitin (Ub) pathway to alter cellular signaling and the antiviral response. Ubiquitination of retroviral group-specific antigen precursors and matrix proteins of the Ebola, vesicular stomatitis, and rabies viruses by Nedd4 family HECT domain E3 ligases is an important step in facilitating viral release. We found that Nedd4 is negatively regulated by ISG15. Free ISG15 specifically bound to Nedd4 and blocked its interaction with Ub-E2 molecules, thus preventing further Ub transfer from E2 to E3. Furthermore, overexpression of ISG15 diminished the ability of Nedd4 to ubiquitinate viral matrix proteins and led to a decrease in the release of Ebola VP40 virus-like particles from the cells. These results point to a mechanistically novel function of ISG15 in the enhancement of the innate antiviral response through specific inhibition of Nedd4 Ub-E3 activity. To our knowledge, this is the first example of a Ub-like protein with the ability to interfere with Ub-E2 and E3 interaction to inhibit protein ubiquitination. IFNs2 are secreted pleiotropic cytokines that mediate diverse biological functions, including induction of the antiviral response and immunomodulatory activities (1-3). IFNs activate the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway, which leads to the induction of hundreds of ISGs. Along with other ISGs, IFN stimulation leads to the up-regulation of ISG15, a Ub-like protein that is present in cells either in the free form or conjugated to various cellular substrates (4, 5). Recently, ISG15 was found to function as an antiviral protein against influenza, herpes, Sindbis, and human immunodeficiency virus type 1 (6 -8), although the molecular mechanism remains largely unknown.Ubiquitination and Ub-mediated proteolysis play a regulatory role in many biological systems. In general, polyubiquitination targets proteins to degradation by the 26 S proteasome, whereas monoubiquitination signals for internalization and vesicle sorting (9, 10). The mammalian Nedd4 protein, originally identified as a gene developmentally down-regulated in the early embryonic mouse central nervous system (11), is a membrane-localized, HECT class Ub-E3 ligase (10). Nedd4 and Nedd4-like proteins are found in eukaryotes from yeast to mammals and are defined by a similar domain organization (12, 13). All contain a catalytic HECT domain at the C terminus and an N-terminal region involved in substrate recognition that includes a C2 domain and a series of WW domains. The WW domains of Nedd4 bind to the viral PPXY motifs, and its Ub-...