Ovarian Tumor Domain Research Articles

Zebrafish transforming growth factor-β-stimulated clone 22 domain 3 (TSC22D3) plays critical roles in Bmp-dependent dorsoventral patterning via two deubiquitylating enzymes Usp15 and Otud4

BackgroundOsmotic stress transcription factor 1/transforming growth factor-β-stimulated clone 22 domain 3 (Ostf1/Tsc22d3) is a transcription factor that plays an osmoregulatory role in euryhaline fishes. Its mRNA and protein levels are up-regulated under hyperosmotic stress. However, its osmoregulatory and developmental functions have not been studied in any stenohaline freshwater fishes. Zebrafish is an excellent model to perform such study to unfold the functional role of Tsc22d3. MethodsWe identified the zebrafish Tsc22d3 and performed knockdown studies using morpholino antisense oligonucleotide (MO). ResultsZebrafish Tsc22d3 did not response to hypertonic stress and ts22d3 knockdown or overexpression by injecting MO or capped RNA did not change the transcriptional levels of any of the known ionocyte markers. To reveal the unknown function of zebrafish Tsc22d3, we performed several in situ molecular marker studies on tsc22d3 morphants and found that Tsc22d3 plays multi-functional roles in dorsoventral (DV) patterning, segmentation, and brain development. We then aimed to identify the mechanism of Tsc22d3 in the earliest stages of DV patterning. Our results demonstrated that tsc22d3 is a ventralizing gene that can stimulate the transcription of bone morphogenetic protein 4 (bmp4) and, thus, has a positive effect on the Bmp signaling pathway. Furthermore, we showed that Tsc22d3 interacts with deubiquitylating enzymes, ubiquitin-specific protease 15 (Usp15) and ovarian tumor domain containing protein 4 (Otud4). In addition, the interruption of Bmp4 signaling by double knockdown of usp15 and otud4 reduced the ventralized effects in tsc22d3-overexpressing embryos. ConclusionsThis is the first study to identify new developmental functions of Tsc22d3 in zebrafish. General significanceZebrafish tsc22d3 is a ventralizing gene and plays a role in early embryogenesis.

Diversity of Ubiquitin and ISG15 Specificity among Nairoviruses' Viral Ovarian Tumor Domain Proteases

Nairoviruses are responsible for numerous diseases that affect both humans and animal. Recent work has implicated the viral ovarian tumor domain (vOTU) as a possible nairovirus virulence factor due to its ability to edit ubiquitin (Ub) bound to cellular proteins and, at least in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), to cleave the Ub-like protein interferon-stimulated gene 15 (ISG15), a protein involved in the regulation of host immunity. The prospective roles of vOTUs in immune evasion have generated several questions concerning whether vOTUs act through a preserved specificity for Ub- and ISG15-conjugated proteins and where that specificity may originate. To gain insight into the substrate specificity of vOTUs, enzymological studies were conducted on vOTUs from Dugbe, CCHFV, and Erve nairoviruses. These studies revealed that vOTUs originating from different nairoviruses display a significant divergence in their preference toward Ub and ISG15. In addition, a recently identified vOTU from turnip yellow mosaic tymovirus was evaluated to elucidate any possible similarities between vOTUs originating from different viral families. Although possessing a similar preference for certain polymeric Ub moieties, its activity toward Ub in general was significantly less then those of nairoviruses. Lastly, the X-ray crystallographic structure of the vOTU from the Dugbe nairovirus was obtained in complex with Ub to reveal structural commonalities of vOTUs originating from nairoviruses. The structure suggests that divergences between nairovirus vOTUs specificity originate at the primary structural level. Comparison of this structure to that originating from CCHFV identified key residues that infer the substrate specificity of vOTUs.

Intron-derived aberrant splicing of A20 transcript in rheumatoid arthritis

Aberrant splicing is one of the most significant components generating functional diversity in many pathological conditions. The objective of this study was to analyse the mutations or aberrant splicing of A20 transcript, the region encompassing the ovarian tumour (OTU) domain [which is functionally important as an inhibitor of nuclear factor (NF)-κB activation] in fibroblast-like synoviocytes (FLSs) from RA patients. Alterations in A20 transcripts were determined through sequence analysis of 10 clones of A20 cDNA in FLSs from each of the five RA patients. The levels of aberrant A20 transcript were measured by quantitative real-time RT-PCR with primers to specifically recognize the inserted introns. The functional role of A20 and its aberrant variants were examined by analysing NF-κB luciferase reporter activity and NF-κB-dependent target gene expression. In RA FLSs, we discovered four novel aberrant A20 transcripts, most of which resulted from insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these FLSs, sequence analysis revealed that these aberrant insertional sequences were flanked by consensus splice donor and acceptor sequences without nucleotide substitution, suggesting alternative splicing as the likely mutational mechanism. These variants elicited a codon frame shift by creating a premature translational stop codon, and eventually, disruption of the OTU domain (which is functionally important as an inhibitor of NF-κB activation) of A20. The expression level of aberrant A20 transcript was correlated well with persisitently enhanced status of NF-κB signalling, as evident by the phosphorylation of inhibitor of NF-κB (IκB)-α and transcription of NF-κB target genes. The results suggest that A20 inactivation by the novel aberrant splicing may contribute to RA progression by inducing persistent NF-κB activation.

Dugbe virus ovarian tumour domain interferes with ubiquitin/ISG15-regulated innate immune cell signalling

The ovarian tumour (OTU) domain of the nairovirus L protein has been shown to remove ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host cell proteins, which is expected to have multiple effects on cell signalling pathways. We have confirmed that the OTU domain from the L protein of the apathogenic nairovirus Dugbe virus has deubiquitinating and deISGylating activity and shown that, when expressed in cells, it is highly effective at blocking the TNF-α/NF-κB and interferon/JAK/STAT signalling pathways even at low doses. Point mutations of the catalytic site of the OTU [C40A, H151A and a double mutant] both abolished the ability of the OTU domain to deubiquitinate and deISGylate proteins and greatly reduced its effect on cell signalling pathways, confirming that it is this enzymic activity that is responsible for blocking the two signalling pathways. Expression of the inactive mutants at high levels could still block signalling, suggesting that the viral OTU can still bind to its substrate even when mutated at its catalytic site. The nairovirus L protein is a very large protein that is normally confined to the cytoplasm, where the virus replicates. When the OTU domain was prevented from entering the nucleus by expressing it as part of the N-terminal 205 kDa of the viral L protein, it continued to block type I interferon signalling, but no longer blocked the TNF-α-induced activation of NF-κB.

A20/TNFAIP3 inhibits NF-κB activation induced by the Kaposi's sarcoma-associated herpesvirus vFLIP oncoprotein.

Kaposi's sarcoma-associated herpesvirus (KSHV) K13/vFLIP (viral Flice-inhibitory protein) induces transcription of numerous genes through NF-κB activation, including pro-inflammatory cytokines, which contribute to the pathogenesis of Kaposi's sarcoma (KS). In this study, we report that KSHV vFLIP induces the expression of the NF-κB regulatory proteins A20, ABIN-1 and ABIN-3 (A20-binding NF-κB inhibitors) in primary human endothelial cells, and that KS spindle cells express A20 in KS tissue. In reporter assays, A20 strongly impaired vFLIP-induced NF-κB activation in 293T cells, but ABIN-1 and ABIN-3 did not. Mutational analysis established that the C-terminal domain (residues 427-790) is critical for A20 modulation of NF-κB, but the ubiquitin-editing OTU (ovarian tumor) domain is not. In functional assays, A20 inhibited vFLIP-induced expression of the chemokine IP-10, reduced vFLIP-induced cell proliferation and increased IKK1 protein levels. Thus, we demonstrate that A20 negatively regulates NF-κB activation directly induced by KSHV vFLIP. By attenuating excessive and prolonged vFLIP-induced NF-κB activation that could be harmful to KSHV-infected cells, A20 likely has an important role in the pathogenesis of KSHV-associated diseases, in which vFLIP is expressed.

An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains

Eight different types of ubiquitin (Ub) linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are largely unknown. We here reveal how the human Ovarian Tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydrolyzes both Lys29- and Lys33-linked diubiquitin (diUb). A crystal structure of the extended catalytic domain reveals an unpredicted Ankyrin repeat (Ank) domain that precedes an A20-like catalytic core. NMR analysis identifies the Ank domain as a new Ub binding fold termed AnkUBD, and DUB assays in vitro and in vivo show that this domain is crucial for TRABID efficiency and linkage-specificity. Our data are consistent with a role of the AnkUBD as an enzymatic S1' Ub binding site, which orients a Ub chain such that Lys29 and Lys33 linkages are cleaved preferentially.

Crystal Structure of Human ISG15 Protein in Complex with Influenza B Virus NS1B

ISG15 (interferon-stimulated gene 15), the first ubiquitin-like protein (UBL) identified, has emerged as an important cellular antiviral factor. It consists of two UBL domains with a short linker between them. The covalent attachment of ISG15 to host and viral proteins to modify their functions, similar to ubiquitylation, is named ISGylation. Influenza B virus NS1B protein antagonizes human but not mouse ISGylation because NS1B exhibits species specificity; it only binds human and non-human primate ISG15. Previous studies have demonstrated that the N-terminal UBL domain and linker of ISG15 are required for the binding by NS1B and that the linker plays a large role in the species specificity, but the structural basis for them has not been elucidated. Here we report the crystal structure of human ISG15 in complex with NS1B at a resolution of 2.0 Å. A loop in the ISG15 N-terminal UBL domain inserts into a pocket in the NS1B dimer, forming a high affinity binding site. The nonspecific van der Waals contacts around the ISG15 linker form a low affinity site for NS1B binding. However, sequence alignment reveals that residues in the high affinity site are highly conserved in primate and non-primate ISG15. We propose that the low affinity binding around the ISG15 linker is important for the initial contact with NS1B and that the stable complex formation is largely contributed by the following high affinity interactions between ISG15 N-terminal UBL domain and NS1B. This provides a structural basis for the species-specific binding of ISG15 by the NS1B protein.

Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb.

Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain ( V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5′- and 3′-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5′-UTR) and -UUUACUACU (in the 3′-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34 nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).

Molecular basis for ubiquitin and ISG15 cross-reactivity in viral ovarian tumor domains

Crimean Congo hemorrhagic fever virus (CCHFV) is a deadly human pathogen that evades innate immune responses by efficiently interfering with antiviral signaling pathways mediated by NF-κB, IRF3, and IFNα/β. These pathways rely on protein ubiquitination for their activation, and one outcome is the modification of proteins with the ubiquitin (Ub)-like modifier interferon-stimulated gene (ISG)15. CCHFV and related viruses encode a deubiquitinase (DUB) of the ovarian tumor (OTU) family, which unlike eukaryotic OTU DUBs also targets ISG15 modifications. Here we characterized the viral OTU domain of CCHFV (vOTU) biochemically and structurally, revealing that it hydrolyzes four out of six tested Ub linkages, but lacks activity against linear and K29-linked Ub chains. vOTU cleaved Ub and ISG15 with similar kinetics, and we were able to understand vOTU cross-reactivity at the molecular level from crystal structures of vOTU in complex with Ub and ISG15. An N-terminal extension in vOTU not present in eukaryotic OTU binds to the hydrophobic Ile44 patch of Ub, which results in a dramatically different Ub orientation compared to a eukaryotic OTU–Ub complex. The C-terminal Ub-like fold of ISG15 (ISG15-C) adopts an equivalent binding orientation. Interestingly, ISG15-C contains an additional second hydrophobic surface that is specifically contacted by vOTU. These subtle differences in Ub/ISG15 binding allowed the design of vOTU variants specific for either Ub or ISG15, which will be useful tools to understand the relative contribution of ubiquitination vs. ISGylation in viral infection. Furthermore, the crystal structures will allow structure-based design of antiviral agents targeting this enzyme.

Structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease

The attachment of ubiquitin (Ub) and the Ub-like (Ubl) molecule interferon-stimulated gene 15 (ISG15) to cellular proteins mediates important innate antiviral responses. Ovarian tumor (OTU) domain proteases from nairoviruses and arteriviruses were recently found to remove these molecules from host proteins, which inhibits Ub and ISG15-dependent antiviral pathways. This contrasts with the Ub-specific activity of known eukaryotic OTU-domain proteases. Here we describe crystal structures of a viral OTU domain from the highly pathogenic Crimean-Congo haemorrhagic fever virus (CCHFV) bound to Ub and to ISG15 at 2.5-Å and 2.3-Å resolution, respectively. The complexes provide a unique structural example of ISG15 bound to another protein and reveal the molecular mechanism of an ISG15 cross-reactive deubiquitinase. To accommodate structural differences between Ub and ISG15, the viral protease binds the β-grasp folds of Ub and C-terminal Ub-like domain of ISG15 in an orientation that is rotated nearly 75° with respect to that observed for Ub bound to a representative eukaryotic OTU domain from yeast. Distinct structural determinants necessary for binding either substrate were identified and allowed the reengineering of the viral OTU protease into enzymes with increased substrate specificity, either for Ub or for ISG15. Our findings now provide the basis to determine in vivo the relative contributions of deubiquitination and deISGylation to viral immune evasion tactics, and a structural template of a promiscuous deubiquitinase from a haemorrhagic fever virus that can be targeted for inhibition using small-molecule-based strategies.

Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin.

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(-)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 Å, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU's preference for Ub and Ub-like substrates.

Epitope-based immunoinformatics and molecular docking studies of nucleocapsid protein and ovarian tumor domain of crimean-congo hemorrhagic Fever virus.

Crimean–Congo hemorrhagic fever virus (CCHFV), the fatal human pathogen is transmitted to humans by tick bite, or exposure to infected blood or tissues of infected livestock. The CCHFV genome consists of three RNA segments namely, S, M, and L. The unusual large viral L protein has an ovarian tumor (OTU) protease domain located in the N terminus. It is likely that the protein may be autoproteolytically cleaved to generate the active virus L polymerase with additional functions. Identification of the epitope regions of the virus is important for the diagnosis, phylogeny studies, and drug discovery. Early diagnosis and treatment of CCHF infection is critical to the survival of patients and the control of the disease. In this study, we undertook different in silico approaches using molecular docking and immunoinformatics tools to predict epitopes which can be helpful for vaccine designing. Small molecule ligands against OTU domain and protein–protein interaction between a viral and a host protein have been studied using docking tools.

Ubiquitin Binding to A20 ZnF4 Is Required for Modulation of NF-κB Signaling

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.

The Cysteine Protease Domain of Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Possesses Deubiquitinating and Interferon Antagonism Functions

Porcine reproductive and respiratory syndrome (PRRS) virus nonstructural protein 2 (nsp2) contains a cysteine protease domain at its N terminus, which belongs to the ovarian tumor (OTU) protease family. In this study, we demonstrated that the PRRSV nsp2 OTU domain antagonizes the type I interferon induction by interfering with the NF-kappaB signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-kappaB activation by interfering with the polyubiquitination process of IkappaBalpha, which subsequently prevents IkappaBalpha degradation. To determine whether the nsp2 protein antagonist function can be ablated from the virus, we introduced point mutations into the OTU domain region by use of reverse genetics. The D458A, S462A, and D465A mutations targeting on a B-cell epitope in the OTU domain region generated the viable recombinant viruses, and the S462A and D465A mutants were attenuated for growth in cell culture. The OTU domain mutants were examined to determine whether mutations in the nsp2 OTU domain region altered virus ability to inhibit NF-kappaB activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-kappaB activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-kappaB activation as effectively as the wild-type virus. This study represents a fundamental step in elucidating the role of nsp2 in PRRS pathogenesis and provides an important insight in future modified live-virus vaccine development.

Ubiquitin-like Sequence in ASK1 Plays Critical Roles in the Recognition and Stabilization by USP9X and Oxidative Stress-Induced Cell Death

Ubiquitination is an important posttranslational modification that regulates various cellular processes, including signal transduction. However, physiological roles of ubiquitination in the regulation of MAPK pathways are poorly understood. Here, we identified the deubiquitinating enzyme USP9X as a binding partner of ASK1 that mediates oxidative stress-induced cell death through activation of the JNK and p38 MAPK pathways. In the recognition of ubiquitin by deubiquitinating enzymes, the importance of a tandem glycine-glycine sequence in the ubiquitin C terminus has been suggested. Interestingly, ASK1 contains six amino acids identical to the ubiquitin C terminus (LRLRGG), and the GG sequence of ASK1 was required for the USP9X-ASK1 interaction. We also found that USP9X interacted with oxidative stress-activated ASK1 and prevented it from undergoing ubiquitin-dependent degradation. In USP9X-deficient cells, oxidative stress-induced JNK activation and subsequent cell death were reduced. These results demonstrate that USP9X-dependent stabilization of activated ASK1 plays a crucial role in oxidative stress-induced cell death.

Crimean-Congo Hemorrhagic Fever Virus-Encoded Ovarian Tumor Protease Activity Is Dispensable for Virus RNA Polymerase Function

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus (genus Nairovirus, family Bunyaviridae) associated with high case fatality disease outbreaks in regions of Africa, Europe, and Asia. The CCHFV genome consists of three negative-strand RNA segments, S, M, and L. The unusually large virus L polymerase protein and the need for biosafety level 4 (BSL-4) containment conditions for work with infectious virus have hampered the study of CCHFV replication. The L protein has an ovarian tumor (OTU) protease domain located in the N terminus, which has led to speculation that the protein may be autoproteolytically cleaved to generate the active virus L polymerase and additional functions. We report the successful development of efficient CCHFV helper virus-independent S, M, and L segment minigenome systems for analysis of virus RNA and protein features involved in replication. The virus RNA segment S, M, and L untranslated regions were found to be similar in support of replication of the respective minigenomes. In addition, the OTU domain located in the N terminus of the expressed virus L protein was shown to be a functional protease. However, no evidence of L protein autoproteolytic processing was found, and the OTU protease activity was dispensable for virus RNA replication. Finally, physiologically relevant doses of ribavirin inhibited CCHFV minigenome replication. These results demonstrated the utility of the minigenome system for use in BSL-2 laboratory settings to analyze CCHFV biology and in antiviral drug discovery programs for this important public health and bioterrorism threat.

Evidence for Bidentate Substrate Binding as the Basis for the K48 Linkage Specificity of Otubain 1

Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination.

Zinc fingers protect the kidney from ischemia/reperfusion injury

A pivotal transcription factor influencing both gene expression and apoptosis is the nuclear factor-kappa B (NF-κB). NF-κB is a central regulator of inflammation by inducing adhesion molecules and other pro-inflammatory components. In ischemia/reperfusion injury, NF-κB upregulates the genes encoding these molecules. Attenuating the process could be important in decreasing myocardial infarct size or diminishing the likelihood of acute renal failure when the heart or kidneys are subject to ischemia. In this issue, Lutz et al. [1] show that an endogenous protector can suppress the pro-inflammatory activation of NF-κB. This protector is the NF-κB-induced zinc finger regulator, termed A20. The authors first studied human endothelial cells that were exposed to 1% O2 for 4 h followed by reoxygenation. They observed that E-selectin, vascular cell adhesion molecule, and interleukin (IL-8) expression were transiently increased, accompanied by sustained A20 expression. The authors next used an adenovirus to introduce A20 and found that the endothelial cells exhibited blunted NF-κB activation of the above-mentioned genes in response to hypoxia/re-oxygenation. They next subjected rats to renal ischemia/reperfusion by 45-min renal pedicle clamping. Pretreatment of the animals with adenovirus carrying A20 reduced renal inflammation and injury. The authors concluded that A20 maintains homeostasis in kidneys exposed to ischemia/reperfusion, a form of endogenous damage control. What is A20 [2]? A20 is a cytoplasmic protein that contains an N-terminal ovarian tumor (OTU) domain and seven novel zinc finger structures with a characteristic CysXaa2–4-Cys-Xaa11-Cys-Xaa2–Cys motif. What are zinc fingers? A zinc finger is a protein domain that can bind to DNA. A zinc finger consists of two anti-parallel β sheets and an α-helix. The zinc ion is crucial for the stability of this domain type; in the absence of the metal ion the domain unfolds, since it is too small to have a hydrophobic core. Zinc fingers are important in regulation because, when interacted with DNA and zinc ion, they provide a unique structural motif for DNA-binding proteins. The structure of each individual finger is highly conserved and consists of about 30 amino acid residues, constructed as a ββα-fold and held together by a zinc ion. The α-helix occurs at the C-terminal part of the finger, while the β-sheet occurs at the N-terminal part. Many transcription factors, regulatory proteins, and other proteins that interact with DNA contain zinc fingers, as does A20. These proteins typically interact with the major groove along the double helix of DNA in which case the zinc fingers are arranged around the DNA strand in such a way that the α-helix of each finger contacts the DNA, forming an almost continuous stretch of α-helices around the DNA molecule. How does A20 regulate NF-κB and influence apoptosis [2, 3]? To deal with this complicated question, a prototype model of NF-κB and common activators should be presented first (Fig. 1). Perhaps, the best model of NF-κB activation, although by no means the only model, is via tumor necrosis factor (TNF) and members of the IL-1/toll receptor family. TNF binds to its receptor (TNFR1) and activates the recruitment of TNF-receptor-associated factor (TRAF2) and the receptor-interacting protein (RIP). This combination binds to the TNFR via the adapter protein known as TNFRSF1A-associated via death domain (TRADD). TRADD in turn recruits Fas-associated protein with death domain (FADD), which is an adaptor molecule J Mol Med (2008) 86:1297–1300 DOI 10.1007/s00109-008-0411-6

Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains

A key effector of the canonical Wnt pathway is beta-catenin, which binds to TCF/LEF factors to promote the transcription of Wnt target genes. In the absence of Wnt stimulation, beta-catenin is phosphorylated constitutively, and modified with K48-linked ubiquitin for subsequent proteasomal degradation. Here, we identify Trabid as a new positive regulator of Wnt signaling in mammalian and Drosophila cells. Trabid show a remarkable preference for binding to K63-linked ubiquitin chains with its three tandem NZF fingers (Npl4 zinc finger), and it cleaves these chains with its OTU (ovarian tumor) domain. These activities of Trabid are required for efficient TCF-mediated transcription in cells with high Wnt pathway activity, including colorectal cancer cell lines. We further show that Trabid can bind to and deubiquitylate the APC tumor suppressor protein, a negative regulator of Wnt-mediated transcription. Epistasis experiments indicate that Trabid acts below the stabilization of beta-catenin, and that it may affect the association or activity of the TCF-beta-catenin transcription complex. Our results indicate a role of K63-linked ubiquitin chains during Wnt-induced transcription.