Ovarian Tumor Domain Research Articles

Ovarian tumor domain proteases in pathogen infection.

With the aim of overcoming host immune responses, and to permit persistence, numerous bacterial and viral pathogens have evolved effective strategies to control the activity of ovarian tumor domain proteases (OTUs), a group of deubiquitinylases crucial for regulating ubiquitin-modified proteins. Due to the important role of eukaryotic OTUs in cellular physiology, it is not surprising that pathogens have evolutionarily developed effector proteins which mimic host OTUs. Here, we focus on recent findings that illustrate how pathogen-encoded OTUs modulate eukaryotic host proteins and how they are implicated in cellular dysregulation. Further, we discuss the biological effects of OTUs in the context of structural features and pharmacological targeting. We point out the potentiality of selective OTU inhibitors, which shield ubiquitin-binding sites, as pharmacologic targets to treat harmful infections.

LncRNA OTUD6B-AS1 inhibits many cellular processes in colorectal cancer by sponging miR-21-5p and regulating PNRC2.

Accumulating evidence has revealed that long noncoding RNAs (lncRNAs) play essential roles in regulating cellular process of various cancers. There have been many studies on the biological functions of lncRNAs in colorectal cancer (CRC). In this research, we explored the role and mechanism of lncRNA ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) in CRC. Here, we detected OTUD6B-AS1 expression in CRC tissues and cells by RT-qPCR. Functional experiments were performed to test alterations in different cellular processes. Moreover, to verify the binding ability among the indicated RNA molecules, we carried out RIP, RNA pull-down and luciferase reporter assays. According to our data, OTUD6B-AS1 expression was low in CRC tissues and cells. Functionally, overexpression of OTUD6B-AS1 inhibited cell proliferation, migration, invasion and EMT, and promoted cell apoptosis. Bioinformatic analysis and mechanistical experiments confirmed that OTUD6B-AS1 could act as a competitive endogenous RNA (ceRNA) to upregulate Proline-Rich Nuclear Receptor Coactivator 2 (PNRC2) expression by sequestering miR-21-5p. Further rescue experiments validated the inhibitory function of the OTUD6B-AS1/miR-21-5p/PNRC2 axis in cellular process of CRC. Overall, OTUD6B-AS1 inhibits cellular development in CRC by sponging miR-21-5p and upregulating PNRC2, providing a novel insight into the exploration on CRC treatment.

Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.

Long non-coding RNA OTUD6B-AS1 overexpression inhibits the proliferation, invasion and migration of colorectal cancer cells via downregulation of microRNA-3171.

Colorectal cancer (CRC) is a common digestive system malignancy and a major cause of cancer-associated mortality worldwide. Aberrant expression of long non-coding RNAs has been reported in several types of cancer. The aim of the present study was to investigate the role of ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) in CRC and its underlying mechanisms. OTUD6B-AS1 expression in CRC cell lines was examined using reverse transcription-quantitative PCR. Furthermore, The Cancer Genome Atlas database was utilized to examine the expression levels of OTUD6B-AS1 in CRC tissues. Following OTUD6B-AS1 overexpression, Cell Counting Kit-8 and colony formation assays were used to detect the proliferation ability of HCT116 cells. The expression levels of proliferation-related protein Ki67 were determined using immunofluorescence staining. Subsequently, Transwell and wound healing assays were used to evaluate the invasion and migration of HCT116 cells, respectively. The expression levels of migration-related proteins (MMP2 and MMP9) were measured using western blotting. Additionally, a luciferase reporter assay was used to verify the potential interaction between OTUD6B-AS1 and microRNA-3171 (miR-3171). Subsequently, rescue assays were performed to clarify the regulatory effects of OTUD6B-AS1 and miR-3171 on CRC development. The results demonstrated that OTUD6B-AS1 expression was low in CRC cells and tissues. Overexpression of OTUD6B-AS1 inhibited the proliferation, invasion and migration of HCT116 cells. Furthermore, miR-3171 was demonstrated to be a direct target of OTUD6B-AS1 using a luciferase reporter assay. The rescue assays revealed that miR-3171 mimics markedly reversed the inhibitory effects of OTUD6B-AS1 overexpression on proliferation, invasion and migration of CRC cells. Overall, these findings demonstrated that OTUD6B-AS1 overexpression inhibited the proliferation, invasion and migration of HCT116 cells via downregulation of miR-3171, suggesting that OTUD6B-AS1 may serve as a novel biomarker for CRC treatment.

The N-terminal cysteine protease domain of rice stripe tenuivirus Pc1 possesses deubiquitinating enzyme activity.

Virus encoded deubiquitinating enzyme (DUB) plays important roles in viral replication and the regulation of host innate immunity. Bioinformatics-based analysis revealed the presence of an ovarian tumor (OTU) protease domain in the N terminus of rice stripe tenuivirus (RSV) Pc1. Many viral OTU domains have been reported to possess DUB activity, which suggests that RSV OTU probably also have DUB activity. To confirm this prediction, we first expressed and purified RSV OTU domain (the N-terminal 200 amino acids of Pc1) and its three mutants (D42A, C45A and H148A) from Escherichia coli and analyzed its DUB activity in vitro. The purified RSV OTU hydrolyzed both K48-linked and K63-linked polyubiquitin chains, indicating RSV OTU domain has DUB enzyme activity in vitro. The mutations of the predicted catalytic sites (Asp42, Cys45 and His148) resulted in the loss of DUB activity, demonstrating these three residues were required for enzyme activity. Then, RSV OTU and its mutants were expressed in insect cells and assayed their DUB activities in vivo by co-transfection with HA-tagged ubiquitin. RSV OTU dramatically reduced ubiquitin-conjugated cellular proteins compared to control and the mutants, showing that RSV OTU also displays DUB activity in vivo. Characterization of RSV OTU DUB enzyme activity and its key catalytic residues will facilitate the development of novel antiviral reagents against RSV.

Flipping the substrate preference of Hazara virus ovarian tumour domain protease through structure-based mutagenesis.

Nairoviruses are arthropod-borne viruses with a nearly global geographical distribution. Several are known causative agents of human disease, including Crimean-Congo hemorrhagic fever virus (CCHFV), which has a case fatality rate that can exceed 30%. Nairoviruses encode an ovarian tumour domain protease (OTU) that can suppress the innate immune response by reversing post-translational modifications by ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15). As a result, the OTU has been identified as a potential target for the development of CCHFV therapeutics. Despite sharing the same general fold, nairoviral OTUs show structural and enzymatic diversity. The CCHFV OTU, for example, possesses activity towards both Ub and ISG15, while the Hazara virus (HAZV) OTU interacts exclusively with Ub. Virology studies focused on the OTU have mostly been restricted to CCHFV, which requires BSL-4 containment facilities. Although HAZV has been proposed as a BSL-2 alternative, differences in the engagement of substrates by CCHFV and HAZV OTUs may present complicating factors when trying to model one using the other. To understand the molecular underpinnings of the differences in activity, a 2.78 Å resolution crystal structure of HAZV OTU bound to Ub was solved. Using structure-guided site-directed mutagenesis, HAZV OTUs were engineered with altered or eliminated deubiquitinase activity, including one with an exclusive activity for ISG15. Additionally, analysis of the structure yielded insights into the difference in inhibition observed between CCHFV and HAZV OTUs with a Ub-based inhibitor. These new insights present opportunities to utilize HAZV as a model system to better understand the role of the OTU in the context of infection.

Association of Clinical Phenotypes in Haploinsufficiency A20 (HA20) With Disrupted Domains of A20.

Background: Haploinsufficiency A20 (HA20) is a newly described monogenic disease characterized by a wide spectrum of manifestations and caused by heterozygous mutations in TNFAIP3 which encodes A20 protein. TNFAIP3 mutation leads to disruption of the A20 ovarian tumor (OTU) domain and/or the zinc finger (ZnF) domain. This study aims at exploring the association between the various manifestations of HA20 and different domains disruption of A20.Methods: We reviewed the HA20 cases in previous literature and summarized the clinical features, TNFAIP3 mutation loci and the disrupted domains caused by different sites and patterns of mutations. Patients were classified into three groups according to the A20 domains disruption.Results: A total of 89 patients from 39 families with a genetic diagnosis of HA20 were included. Overall, the age at onset of HA20 was early (median:5.92, IQR:1-10). Patients in the ZnF group showed the earliest onset (median:2.5, IQR:0.6-5), followed by patients in the OTU+ZnF group (median:6, IQR:1-10) and patients in the OTU group (median:10, IQR:8-14). The main manifestations of HA20 patients were recurrent oral ulcers (70%), recurrent fever (42%), gastrointestinal ulcers (40%), skin lesion (38%), genital ulcers (36%), and musculoskeletal disorders (34%). The percentage of patients with musculoskeletal disorders was significantly different among the three groups (p = 0.005). Patients in the OTU+ZnF group and ZnF group were more likely to develop musculoskeletal disorders than patients in the OTU group (p = 0.002 and p = 0.035, respectively). Besides, forty-three percent of HA20 patients were initially diagnosed as Behcet's disease (BD). Compared to the ZnF group, the OTU+ZnF group and OTU group had a higher percentage of patients initially diagnosed as BD (p = 0.006 and p < 0.001, respectively).Conclusion: HA20 is characterized by early-onset and the most common symptoms of HA20 are recurrent oral ulcers, fever and gastrointestinal ulcers. The onset of HA20 in patients with the ZnF domain disruption is earlier than patients with the OTU domain disruption. Compared to the OTU domain, the ZnF domain may be more closely related to musculoskeletal disorders.

ISG15 overexpression compensates the defect of Crimean-Congo hemorrhagic fever virus polymerase bearing a protease-inactive ovarian tumor domain.

Crimean-Congo Hemorrhagic Fever virus (CCHFV; family Nairoviridae) is an extremely pathogenic member of the Bunyavirales order. Previous studies have shown that the N-terminal domain of the CCHFV polymerase (L) contains an ovarian tumor-type protease (OTU) domain with the capability to remove both ubiquitin and ISG15 molecules from proteins. The approximately 200 amino acids-long OTU domain, if ectopically expressed, can interfere with both the induction of antiviral type I interferons (IFN) as well as the IFN-stimulated signaling. A OTU protease mutant (C40A), by contrast, was inactive in that respect. However, the effect of the OTU protease activity in the context of the full-length L protein (approximately 4000 amino acids) is only poorly characterized, and recombinant CCHFV with the C40A mutation could not be rescued. Here, we employed transcriptionally active virus-like particles (tc-VLPs) to investigate the interaction between the L-embedded OTU protease and the IFN system. Our data show a cis requirement of the OTU protease for optimal CCHFV polymerase activity in human HuH-7 cells. The L-embedded OTU did not influence IFN signaling, the sensitivity to IFN, or IFN induction. Moreover, the attenuation of OTU C40A-mutated L could not be relieved by inactivating the IFN response, but after overexpression of conjugation-competent ISG15 the polymerase activity recovered to wild-type levels. Consequently, ISG15 was used to produce OTU-deficient tc-VLPs, a potential vaccine candidate. Our data thus indicate that in the context of full-length L the OTU domain is important for the regulation of CCHFV polymerase by ISG15.

LncRNA OTUD6B-AS1 Exacerbates As2O3-Induced Oxidative Damage in Bladder Cancer via miR-6734-5p-Mediated Functional Inhibition of IDH2.

Arsenic trioxide (As2O3) is a promising effective chemotherapeutic agent for cancer treatment; however, how and through what molecular mechanisms the oxidative damage of As2O3 is controlled remains poorly understood. Recently, the involvement of dysregulated long noncoding RNA ovarian tumor domain containing 6B antisense RNA1 (lncRNA OTUD6B-AS1) in tumorigenesis is established. Here, for the first time, we characterize the regulation of As2O3 in the oxidative damage against bladder cancer via lncRNA OTUD6B-AS1. As2O3 could activate lncRNA OTUD6B-AS1 transcription in bladder cancer cells, and these findings were validated in a xenograft tumor model. Functional assays showed that lncRNA OTUD6B-AS1 dramatically exacerbated As2O3-mediated oxidative damage by inducing oxidative stress. Mechanistically, As2O3 increased levels of metal-regulatory transcription factor 1 (MTF1), which regulates lncRNA OTUD6B-AS1, in response to oxidative stress. Further, lncRNA OTUD6B-AS1 inhibited mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) expression by stabilizing miR-6734-5p, which contributed to cytotoxicity by enhancing oxidative stress. Together, our findings offer new insights into the mechanism of As2O3-induced oxidative damage and identify important factors in the pathway, As2O3/lncRNA OTUD6B-AS1/miR-6734-5p/IDH2, expanding the knowledge of activity of As2O3 as cancer treatment.

The long noncoding RNA OTUD6B-AS1 enhances cell proliferation and the invasion of hepatocellular carcinoma cells through modulating GSKIP/Wnt/β-catenin signalling via the sequestration of miR-664b-3p

Ovarian tumour domain containing 6B antisense RNA1 (OTUD6B-AS1), a newly identified long noncoding RNA (lncRNA), has been reported as a key cancer-related lncRNA. However, the detailed relevance of OTUD6B-AS1 in hepatocellular carcinoma (HCC) remains undetermined. This study was designed to determine the functional significance and regulatory mechanism of OTUD6B-AS1 in HCC. We found that the expression of OTUD6B-AS1 was up-regulated in HCC tissues, and patients with high levels of OTUD6B-AS1 expression had shorter survival rates than those with low OTUD6B-AS1 expression. Elevated expression of the lncRNA was also found in multiple HCC cell lines and the silencing of OTUD6B-AS1 significantly decreased proliferation, colony formation and invasion. Correspondingly, OTUD6B-AS1 overexpression had the opposite effect on HCC cell invasion, colony formation and proliferation. Notably, OTUD6B-AS1 was identified as a molecular sponge of microRNA-664b-3p (miR-664b-3p). The down-regulation of miR-664b-3p was detected in HCC tissues and cell lines, and the up-regulation of miR-664b-3p repressed proliferation and invasion in HCC cells by targeting the glycogen synthase kinase-3β interaction protein (GSKIP). Moreover, OTUD6B-AS1 knockdown or miR-664b-3p up-regulation exerted a suppressive effect on Wnt/β-catenin signalling via the down-regulation of GSKIP. In addition, GSKIP overexpression markedly reversed OTUD6B-AS1 knockdown- or miR-664b-3p overexpression-induced antitumour effects in HCC. Further data confirmed that OTUD6B-AS1 knockdown exerted a tumour-inhibition role in HCC in vivo. Overall, these findings indicate that the lncRNA OTUD6B-AS1 accelerates the proliferation and invasion of HCC cells by enhancing GSKIP/Wnt/β-catenin signalling via the sequestration of miR-664b-3p. Our study reveals a novel molecular mechanism, mediated by lncRNA OTUD6B-AS1, which may play a key role in regulating the progression of HCC.

Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein.

BackgroundThe Crimean-Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity.Methodology/Principle findingsRecombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2’-deoxy-2’-fluoro-CTP (FdC) and 2’-deoxy-2’-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, the ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions.Conclusions/SignificanceThe requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors.

Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267 In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

OTUD1 Negatively Regulates Type I IFN Induction by Disrupting Noncanonical Ubiquitination of IRF3

IFN regulatory factor 3 (IRF3) is critical for the transcription of type I IFNs in defensing virus and promoting inflammatory responses. Although several kinds of posttranslational modifications have been identified to modulate the activity of IRF3, whether atypical ubiquitination participates in the function regulation, especially the DNA binding capacity of IRF3, is unknown. In this study, we found that the ovarian tumor domain containing deubiquitinase OTUD1 deubiquitinated IRF3 and attenuated its function. An atypical ubiquitination, K6-linked ubiquitination, was essential for the DNA binding capacity of IRF3 and subsequent induction of target genes. Mechanistically, OTUD1 cleaves the viral infection-induced K6-linked ubiquitination of IRF3, resulting in the disassociation of IRF3 from the promoter region of target genes, without affecting the protein stability, dimerization, and nuclear translocation of IRF3 after a viral infection. Otud1 -/- cells as well as Otud1 -/- mice produced more type I IFNs and proinflammatory cytokines after viral infection. Otud1 -/- mice were more resistant to lethal HSV-1 and VSV infection. Consistent with the former investigations that IRF3 promoted inflammatory responses in LPS-induced sepsis, Otud1 -/- mice were more susceptible to LPS stimulation. Taken together, our findings revealed that the DNA binding capacity of IRF3 in the innate immune signaling pathway was modulated by atypical K6-linked ubiquitination and deubiquitination process, which was regulated by the deubiquitinase OTUD1.

OTUB1-mediated deubiquitination of FOXM1 up-regulates ECT-2 to promote tumor progression in renal cell carcinoma

BackgroundOTUB1 (ovarian tumor domain protease domain-containing ubiquitin aldehyde-binding proteins)-mediated deubiquitination of FOXM1 (Forkhead box M1) participates in carcinogenesis of various tumors. We aim to investigate the effect and mechanism of OTUB1/FOXM1 on RCC (renal cell carcinoma) progression. Expression levels of OTUB1 in RCC tissues and cell lines were examined by qRT-PCR (quantitative real-time polymerase chain reaction) and immunohistochemistry. Cell proliferation was measured with CCK8 (Cell Counting Kit-8) and colony formation assays. Wound healing and transwell assays were used to determine cell migration and invasion, respectively. The effect of OTUB1 on FOXM1 ubiquitination was examined by Immunoprecipitation. Western blot was used to uncover the underlying mechanism. In vivo subcutaneous xenotransplanted tumor model combined with immunohistochemistry and western blot were used to examine the tumorigenic function of OTUB1.ResultsOTUB1 was up-regulated in RCC tissues and cell lines, and was associated with poor prognosis of RCC patients. Knockdown of OTUB1 inhibited cell viability and proliferation, as well as migration and invasion of RCC cells. Mechanistically, knockdown of OTUB1 down-regulated FOXM1 expression by promoting its ubiquitination. Down-regulation of FOXM1 inhibited ECT2 (epithelial cell transforming 2)-mediated Rho signaling. Moreover, the inhibition of RCC progression caused by OTUB1 knockdown was reversed by FOXM1 over-expression. In vivo subcutaneous xenotransplanted tumor model also revealed that knockdown of OTUB1 could suppress in vivo RCC growth via down-regulation of FOXM1-mediated ECT2 expression.ConclusionsOTUB1-mediated deubiquitination of FOXM1 up-regulates ECT-2 to promote tumor progression in RCC, providing a new potential therapeutic target for RCC treatment.

OTUB2 Promotes Homologous Recombination Repair Through Stimulating Rad51 Expression in Endometrial Cancer

Genetic instability, raised from dysregulation of DNA repair, is involved in tumor development. OTUB2 (ovarian tumor domain protease domain-containing ubiquitin aldehyde-binding protein 2), which is responsible for DNA double-strand break (DSB), is implicated in carcinogenesis of various tumors. The effect of OTUB2 on endometrial cancer progression was then investigated. First, OTUB2 was found to be upregulated in endometrial cancer tissues and cell lines, and was closely associated with overall survival of endometrial cancer patients. Cell Counting Kit-8 and flow cytometry assay results revealed that overexpression of OTUB2 enhanced cell viability of endometrial cancer cells, while knockdown of OTUB2 inhibited cell viability. Moreover, as demonstrated by promoting cell viability and suppression of cell apoptosis, cisplatin-induced cell damage was reversed by OTUB2. Mechanistically, OTUB2 could activate Yes-associated protein/transcriptional co-activator with PDZ-binding motif (TAZ) to promote homologous recombination repair via depletion of γH2AX (phosphorylation of histone H2AX) and accumulation of Rad51. In vivo xenograft model also showed that silence of OTUB2 suppressed the growth of endometrial cancer and increased tumor sensitivity to antitumor drugs. In conclusion, OTUB2 promoted homologous recombination repair in endometrial cancer via YAP/TAZ-mediated Rad51 expression, providing a potential therapeutic target for endometrial cancer.

Determining the molecular drivers of species-specific interferon-stimulated gene product 15 interactions with nairovirus ovarian tumor domain proteases.

Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species’ ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species’ ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection.

Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase.

OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.

Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity.

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.

Insights into the Porcine Reproductive and Respiratory Syndrome Virus Viral Ovarian Tumor Domain Protease Specificity for Ubiquitin and Interferon Stimulated Gene Product 15.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread economically devastating disease caused by PRRS virus (PRRSV). First recognized in the late 1980s, PRRSV is known to undergo somatic mutations and high frequency viral recombination, which leads to many diverse viral strains. This includes differences within viral virulence factors, such as the viral ovarian tumor domain (vOTU) protease, also referred to as the papain-like protease 2. These proteases down-regulate innate immunity by deubiquitinating proteins targeted by the cell for further processing and potentially also acting against interferon-stimulated genes (ISGs). Recently, vOTUs from vaccine derivative Ingelvac PRRS modified live virus (MLV) and the highly pathogenic PRRSV strain JXwn06 were biochemically characterized, revealing a marked difference in activity toward K63 linked polyubiquitin chains and a limited preference for interferon-stimulated gene product 15 (ISG15) substrates. To extend our research, the vOTUs from NADC31 (low virulence) and SDSU73 (moderately virulent) were biochemically characterized using a myriad of ubiquitin and ISG15 related assays. The K63 polyubiquitin cleavage activity profiles of these vOTUs were found to track with the established pathogenesis of MLV, NADC31, SDSU73, and JXwn06 strains. Fascinatingly, NADC31 demonstrated significantly enhanced activity toward ISG15 substrates compared to its counterparts. Utilizing this information and strain-strain differences within the vOTU encoding region, sites were identified that can modulate K63 polyubiquitin and ISG15 cleavage activities. This information represents the basis for new tools to probe the role of vOTUs in the context of PRRSV pathogenesis.