Regulation of Rap2A by the ubiquitin ligase Nedd4-1 controls neurite development.

Nedd4-1 is a "neuronal precursor cell expressed and developmentally downregulated protein" and among the most abundant E3 ubiquitin ligases in mammalian neurons. In analyses of conventional and conditional Nedd4-1-deficient mice, we found that Nedd4-1 plays a critical role in dendrite formation. Nedd4-1, the serine/threonine kinase TNIK, and Rap2A form a complex that controls Nedd4-1-mediated ubiquitination of Rap2A. Ubiquitination by Nedd4-1 inhibits Rap2A function, which reduces the activity of Rap2 effector kinases of the TNIK family and promotes dendrite growth. We conclude that a Nedd4-1/Rap2A/TNIK signaling pathway regulates neurite growth and arborization in mammalian neurons.

Disruption of the NHR4 domain structure in AML1-ETO abrogates SON binding and promotes leukemogenesis.

AML1-ETO is generated from t(8;21)(q22;q22), which is a common form of chromosomal translocation associated with development of acute myeloid leukemia (AML). Although full-length AML1-ETO alone fails to promote leukemia because of its detrimental effects on cell proliferation, an alternatively spliced isoform, AML1-ETO9a, without its C-terminal NHR3/NHR4 domains, strongly induces leukemia. However, full-length AML1-ETO is a major form of fusion product in many t(8;21) AML patients, suggesting additional molecular mechanisms of t(8;21)-related leukemogenesis. Here, we report that disruption of the zinc-chelating structure in the NHR4 domain of AML1-ETO by replacing only one critical amino acid leads to rapid onset of leukemia, demonstrating that the NHR4 domain with the intact structure generates inhibitory effects on leukemogenesis. Furthermore, we identified SON, a DNA/RNA-binding domain containing protein, as a novel NHR4-interacting protein. Knock-down of SON by siRNA resulted in significant growth arrest, and disruption of the interaction between AML1-ETO and endogenous SON rescued cells from AML1-ETO-induced growth arrest, suggesting that SON is an indispensable factor for cell growth, and AML1-ETO binding to SON may trigger signals inhibiting leukemogenesis. In t(8;21) AML patient-derived primary leukemic cells and cell lines, abnormal cytoplasmic localization of SON was detected, which may keep cells proliferating in the presence of full-length AML1-ETO. These results uncovered the crucial role of the NHR4 domain in determination of cellular fate during AML1-ETO-associated leukemogenesis.

ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response.

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral 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 anti-viral 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.

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.

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-beta --> 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.

SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins.

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.

High-throughput immunoblotting. Ubiquitiin-like protein ISG15 modifies key regulators of signal transduction.

ISG15 is a ubiquitin-like protein that conjugates to numerous proteins in cells treated with interferon or lipopolysaccharide. Dysregulation of protein ISG15 modification (ISGylation) in mice leads to decreased life expectancy, brain cell injury, and hypersensitivity to interferon. Although ISG15 was identified more than two decades ago, the exact biochemical and physiological functions of ISG15-modification remain unknown, and the proteins targeted by ISG15 have not been identified. The major purpose of this work was to identify ISG15 targets among well characterized proteins that could be used as models for biological studies. We purified ISGylated proteins from human thymus by immunoaffinity chromatography and analyzed ISG15 conjugates by a high-throughput Western blot screen (PowerBlot). We found that three key regulators of signal transduction, phospholipase Cgamma1, Jak1, and ERK1 are modified by ISG15. In addition to that, we demonstrate that transcription factor Stat1, an immediate substrate of Jak1 kinase, is also ISGylated. Using whole cell protein extracts and phospholipase Cgamma1 as an example we demonstrate that ISG15 conjugates are not accumulated in cells treated with specific inhibitors of proteasomes. Our work suggests a role for ISG15 in the regulation of multiple signal transduction pathways and offers attractive models to further elucidate the biochemical function of ISGylation.

Protein ISGylation modulates the JAK-STAT signaling pathway.

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-beta 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.

Dysregulation of protein modification by ISG15 results in brain cell injury.

UBP43 (USP18) is a protease that removes the ubiquitin-like 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.

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-beta-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.