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Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, but the molecular mechanisms underlying IBD are incompletely understood. In this study, we explored the role and regulating mechanism of otubain 2 (OTUB2), a deubiquitinating enzyme, in IBD. Methods: To study the function of OTUB2 in IBD, we generated Otub2–/– mice and treated them with dextran sulfate sodium (DSS) to induce experimental colitis. Bone marrow transplantation was performed to identify the cell populations that were affected by OTUB2 in colitis. The molecular mechanism of OTUB2 in signal transduction was studied by various biochemical methods. Results: OTUB2 was highly expressed in colon‐infiltrating macrophages in both humans with IBD and mice with DSS‐induced experimental colitis. Colitis was significantly aggravated in Otub2–/– mice and bone marrow chimeric mice receiving Otub2–/– bone marrow. OTUB2‐deficiency impaired the production of cytokines and chemokines in macrophages in response to the NOD2 agonist muramyl dipeptide (MDP). Upon MDP stimulation, OTUB2 promoted NOD2 signaling by stabilizing RIPK2. Mechanistically, OTUB2 inhibited the proteasomal degradation of RIPK2 by removing K48‐linked polyubiquitination on RIPK2, which was mediated by the active C51 residue in OTUB2. In mice, OTUB2 ablation abolished the protective effects of MDP administration in colitis. Conclusion: This study identified OTUB2 as a novel regulator of intestinal inflammation. [ABSTRACT FROM AUTHOR]

Abstract Background Colon cancer is one of the most prevalent tumors in the digestive tract, and its stemness feature significantly contribute to chemoresistance, promote the epithelial-mesenchymal transition (EMT) process, and ultimately lead to tumor metastasis. Therefore, it is imperative for researchers to elucidate the molecular mechanisms underlying the enhancement of stemness feature, chemoresistance, and EMT in colon cancer. Methods Sphere-formation and western blotting assays were conducted to assess the stemness feature. Edu, flow cytometry, and cell viability assays were employed to evaluate the chemoresistance. Immunofluorescence and western blotting assays were utilized to detect EMT. Immunoprecipitation, ubiquitination, agarose gel electrophoresis, chromatin immunoprecipitation followed by quantitative PCR (chip-qPCR), and dual luciferase reporter gene assays were employed for mechanistic investigations. Results We demonstrated a markedly higher expression level of OTUB2 in colon cancer tissues compared to adjacent tissues. Furthermore, elevated OTUB2 expression was closely associated with poor prognosis and distant tumor metastasis. Functional experiments revealed that knockdown of OTUB2 attenuated stemness feature of colon cancer, enhanced its sensitivity to oxaliplatin, inhibited its EMT process, ultimately reduced the ability of tumor metastasis. Conversely, overexpression of OTUB2 exerted opposite effects. Mechanistically, we identified OTUB2 as a deubiquitinase for SP1 protein which bound specifically to SP1 protein, thereby inhibiting K48 ubiquitination of SP1 protein. The SP1 protein functioned as a transcription factor for the GINS1, exerting its regulatory effect by binding to the 1822–1830 region of the GINS1 promoter and enhancing its transcriptional activity. Ultimately, alterations in GINS1 expression directly regulated stemness feature, chemosensitivity, and EMT progression in colon cancer. Conclusion Collectively, the OTUB2/SP1/GINS1 axis played a pivotal role in driving stemness feature, chemoresistance, and EMT in colon cancer. These results shed new light on understanding chemoresistance and metastasis mechanisms involved in colon cancer.

Background: Colon cancer is one of the most prevalent tumors in the digestive tract, and its stemness feature significantly contribute to chemoresistance, promote the epithelial-mesenchymal transition (EMT) process, and ultimately lead to tumor metastasis. Therefore, it is imperative for researchers to elucidate the molecular mechanisms underlying the enhancement of stemness feature, chemoresistance, and EMT in colon cancer. Methods: Sphere-formation and western blotting assays were conducted to assess the stemness feature. Edu, flow cytometry, and cell viability assays were employed to evaluate the chemoresistance. Immunofluorescence and western blotting assays were utilized to detect EMT. Immunoprecipitation, ubiquitination, agarose gel electrophoresis, chromatin immunoprecipitation followed by quantitative PCR (chip-qPCR), and dual luciferase reporter gene assays were employed for mechanistic investigations. Results: We demonstrated a markedly higher expression level of OTUB2 in colon cancer tissues compared to adjacent tissues. Furthermore, elevated OTUB2 expression was closely associated with poor prognosis and distant tumor metastasis. Functional experiments revealed that knockdown of OTUB2 attenuated stemness feature of colon cancer, enhanced its sensitivity to oxaliplatin, inhibited its EMT process, ultimately reduced the ability of tumor metastasis. Conversely, overexpression of OTUB2 exerted opposite effects. Mechanistically, we identified OTUB2 as a deubiquitinase for SP1 protein which bound specifically to SP1 protein, thereby inhibiting K48 ubiquitination of SP1 protein. The SP1 protein functioned as a transcription factor for the GINS1, exerting its regulatory effect by binding to the 1822–1830 region of the GINS1 promoter and enhancing its transcriptional activity. Ultimately, alterations in GINS1 expression directly regulated stemness feature, chemosensitivity, and EMT progression in colon cancer. Conclusion: Collectively, the OTUB2/SP1/GINS1 axis played a pivotal role in driving stemness feature, chemoresistance, and EMT in colon cancer. These results shed new light on understanding chemoresistance and metastasis mechanisms involved in colon cancer. [ABSTRACT FROM AUTHOR]

The regulatory potential of long noncoding RNA (lncRNA) FBXL19‐AS1 has been highlighted in various cancers, but its effect on triple‐negative breast cancer (TNBC) remains unclear. Here, we aimed to elucidate the role of FBXL19‐AS1 in TNBC and its underlying mechanism. RT‐qPCR was employed to detect the expressions of FBXL19‐AS1 and miR‐378a‐3p in tissues and cells. Immunohistochemical staining and western blot were utilized to detect the expression levels of proteins. Cell activities were detected using flow cytometry, CCK‐8, and transwell assay. Dual‐luciferase reporter and RNA immunoprecipitation (RIP) assays were deployed to investigate interactions of different molecules. Protein–protein interaction (PPI) network, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways were used to analyze the downstream pathway. In vivo xenograft model was conducted to detect the effect of FBXL19‐AS1 on tumor growth. FBXL19‐AS1 was overexpressed in TNBC tissues and cell lines compared with counterparts. FBXL19‐AS1 knockdown suppressed TNBC cell activities, whereas its overexpression exhibited the opposite effect. Mechanistically, FBXL19‐AS1 was found to interact with miR‐378a‐3p. Further analysis revealed that miR‐378a‐3p exerted tumor‐suppressive effects in TNBC cells. Additionally, miR‐378a‐3p targeted and downregulated the expression of ubiquitin aldehyde binding 2 (OTUB2), a deubiquitinase associated with TNBC progression. In vivo experiments substantiated the inhibitory effects of FBXL19‐AS1 knockdown on TNBC tumorigenesis, and a miR‐378a‐3p inhibitor partially rescued these effects. The downstream pathway of the miR‐378a‐3p/OTUB2 axis was explored, revealing connections with proteins involved in modifying other proteins, removing ubiquitin molecules, and influencing signaling pathways, including the Hippo signaling pathway. Western blot analysis confirmed changes in YAP and TAZ expression levels, indicating a potential regulatory network. In summary, FBXL19‐AS1 promotes exacerbation in TNBC by suppressing miR‐378a‐3p, leading to increased OTUB2 expression. The downstream mechanism may be related to the Hippo signaling pathway. These findings propose potential therapeutic targets for TNBC treatment. Significance statement: Studies have highlighted the regulatory potential of lncRNA‐FBXL19‐AS1 in various cancers; however, its effect on TNBC remains elusive. The present study elucidated the functional role of FBXL19‐AS1 in TNBC and its underlying molecular mechanism. The in‐vitro and in‐vivo models demonstrated that FBXL19‐AS1 promotes exacerbation in TNBC by suppressing miR‐378a‐3p, leading to increased OTUB2 expression, consequently activating the Hippo signaling pathway. Our study provides novel insights into the function of FBXL19‐AS1 in regulating TNBC, thus representing a promising target for TNBC clinical treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, but the molecular mechanisms underlying IBD are incompletely understood. In this study, we explored the role and regulating mechanism of otubain 2 (OTUB2), a deubiquitinating enzyme, in IBD. Methods To study the function of OTUB2 in IBD, we generated Otub2–/– mice and treated them with dextran sulfate sodium (DSS) to induce experimental colitis. Bone marrow transplantation was performed to identify the cell populations that were affected by OTUB2 in colitis. The molecular mechanism of OTUB2 in signal transduction was studied by various biochemical methods. Results OTUB2 was highly expressed in colon‐infiltrating macrophages in both humans with IBD and mice with DSS‐induced experimental colitis. Colitis was significantly aggravated in Otub2–/– mice and bone marrow chimeric mice receiving Otub2–/– bone marrow. OTUB2‐deficiency impaired the production of cytokines and chemokines in macrophages in response to the NOD2 agonist muramyl dipeptide (MDP). Upon MDP stimulation, OTUB2 promoted NOD2 signaling by stabilizing RIPK2. Mechanistically, OTUB2 inhibited the proteasomal degradation of RIPK2 by removing K48‐linked polyubiquitination on RIPK2, which was mediated by the active C51 residue in OTUB2. In mice, OTUB2 ablation abolished the protective effects of MDP administration in colitis. Conclusion This study identified OTUB2 as a novel regulator of intestinal inflammation.

In cancer research, oncogenesis can be affected by modulating the deubiquitination pathway. Ubiquitination regulates proteins post-translationally in variety of physiological processes. The Otubain Subfamily includes OTUB1 (ovarian tumor-associated proteinase B1) and OTUB2(ovarian tumor-associated proteinase B2). They are deubiquitinating enzymes, which are research hotspots in tumor immunotherapy, with their implications extending across the spectrum of tumor development. Understanding their important role in tumorigenesis, includ-ing hepatocellular carcinoma (HCC) is crucial. HCC has alarming global incidence rates and mortality statistics, ranking among the top five prevalent cancers in Malaysia11 https://gco.iarc.fr/en. Numerous studies have consistently indicated significant expression of OTUB1 and OTUB2 in HCC cells. In addition, OTUB1 has important biological functions in cancer, suggesting its important role in tumorigenesis. However, the mechanism underlying the action of OTUB1 and OTUB2 in liver cancer remains inadequately explored. Therefore, Otubain Subfamily, as potential molecular target, holds promise for advancing HCC treatments. However, further clinical studies are required to verify its efficacy and application prospects.

Background: Ovarian cancer (OVCA) is prevalent in female reproductive organs. Despite recent advances, clinical outcomes remain poor, warranting fresh treatment avenues. Honokiol has an inhibitory effect on proliferation, invasion, and survival of cancer cells in vitro and in vivo. Therefore, this study intended to explore specific molecular mechanism by which honokiol affected OVCA progression. Methods: Bioinformatics analyzed the drug honokiol that bound to OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2). Cellular thermal shift assay (CETSA) verified the binding relationship between honokiol and OTUB2. Cell counting kit 8 (CCK‐8) tested the IC50 value and cell viability of OVCA cells after honokiol treatment. Corresponding assay kits determined malonic dialdehyde (MDA) and Fe2+ levels in OVCA cells. Flow cytometry measured reactive oxygen species levels. Western blot detected OTUB2, SLC7A11, and transcriptional co‐activators Yes‐associated protein (YAP) expression, and quantitative polymerase chain reaction (qPCR) detected OTUB2 expression. Immunohistochemistry (IHC) detected the expression level of Ki67 protein in tumor tissues. Results: Honokiol was capable of inducing ferroptosis in OVCA cells. CETSA confirmed that honokiol could bind to OTUB2. Further cell functional and molecular experiments revealed that honokiol induced ferroptosis in OVCA cells via repression of YAP signaling pathway through binding to OTUB2. In addition, in vivo experiments have confirmed that honokiol could inhibit the growth of OVCA. Conclusion: Honokiol induced ferroptosis in OVCA cells via repression of YAP signaling pathway through binding to OTUB2, implicating that OTUB2 may be an effective target for OVCA treatment, and our study results may provide new directions for development of more effective OVCA treatment strategies. [ABSTRACT FROM AUTHOR]

Cervical cancer (CC) is a deadly gynecological tumor worldwide. Otubain 2 (OTUB2) has been recently identified as an oncogene in human malignancies. However, its expression and function remain unclear. This work aims to explore the role of OTUB2 in CC progression. Herein, The Cancer Genome Atlas data revealed that OTUB2 expression was significantly upregulated in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and gradually increased with CESC progression; moreover, OTUB2 expression predicted poor outcomes of CESC patients. Then, RT‐qPCR and Western blotting were applied to determine mRNA and protein expression in CC and normal cells. Our results confirmed that OTUB2 was highly expressed in CC cell lines. As indicated by CCK‐8, Transwell, and flow cytometry results, OTUB2 silencing attenuated proliferative and metastatic capacities of CC cells but promoted CC cell apoptosis. Then, RBM15, an N6‐methyladenosine (m6A) methyltransferase "writer," was also demonstrated to be upregulated in CESC and CC cells. Mechanistically, m6A RNA immunoprecipitation (Me‐RIP) results showed that RBM15 inhibition reduced the m6A methylation level of OTUB2 in CC cells, leading to the decline of OTUB2 expression. In addition, OTUB2 inhibition deactivated the AKT/mTOR signaling in CC cells. Furthermore, SC‐79 (AKT/mTOR activator) partially abated the inhibitory effects of OTUB2 knockdown on the AKT/mTOR signaling pathway and the malignant phenotypes of CC cells. In summary, this work showed that RBM15‐mediated m6A modification led to OTUB2 upregulation, thereby promoting malignant behaviors of CC cells via the AKT/mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Objective The effects of Otubain-2 (OTUB2) on the proliferation, invasion, and migration of esophageal squamous cell carcinoma (ESCC) were investigated by interfering with OTUB2 expression. Methods Bioinformatics analysis was used to analyze OTUB2 expression in esophageal carcinoma and interactions between OTUB2 and YAP1/TAZ. Paraffin-embedded ESCC tissues (n = 183) were selected for immunohistochemical staining to detect OTUB2, YAP1, TAZ, CTGF and their relationship with clinicopathological parameters, then the survival prognosis of ESCC patients was analyzed. Immunofluorescence, western blotting, and qRT-PCR were used to evaluate OTUB2 in ESCC cell lines. Cell lines with the highest expression of OTUB2 were transfected with lentivirus to knockdown OTUB2 levels. Changes in KYSE150 cell proliferation, migration, and invasion were measured using CCK-8, wound healing, and clone formation assays. The Transwell test and flow cytometry identified OTUB2 targets and explored roles and mechanisms involved in ESCC. Effects of OTUB2 on YAP1/TAZ signaling were also observed. Results Bioinformatics analysis revealed OTUB2 was highly expressed in esophageal cancer and was associated with YAP1/TAZ. Immunohistochemistry showed that OTUB2 expression was increased in ESCC samples compared to parcancerous tissue. YAP1 and TAZ were higher expression in ESCC tissues, mainly localized in the nucleus. Compared with controls, the proliferation, migration, and invasion ability of KYSE150 cells after OTUB2 knockdown were significantly reduced (P

Endometrial carcinoma (EC) morbidity and mortality have been increasing in recent years. Otubain 2 (OTUB2) was shown to be upregulated in EC patients, so the aim of this study was to explore the role of OTUB2 in EC. Cell Counting Kit‐8 (CCK‐8), colony formation, enzyme‐linked immunosorbent assay, the wound healing assay, and Transwell invasion assays were used to investigate the specific role of OTUB2 in EC tumorigenesis. Real‐time polymerase chain reaction and western blot analysis were used to detect the expression of OTUB2 in EC tissues and cells. OTUB2 is upregulated in EC patients and cell lines and is associated with a poor prognosis. The overexpression of OTUB2 promoted glycolysis and induced the proliferation, migration, and invasion of endometrial cancer cells. The silencing of OTUB2 had the opposite effect. In addition, the silencing of OTUB2 significantly suppressed the expression levels of PKM2. Importantly, inhibition of the PKM2/PI3K/AKT signaling pathway significantly reversed the promoting effect of OTUB2 overexpression on EC. OTUB2 regulated the proliferation and invasion of EC cells by regulating the PKM2/PI3K/AKT signaling pathway. OTUB2 may serve as a potential prognostic and therapeutic target in EC. [ABSTRACT FROM AUTHOR]

In cancer research, oncogenesis can be affected by modulating the deubiquitination pathway. Ubiquitination regulates proteins post-translationally in variety of physiological processes. The Otubain Subfamily includes OTUB1 (ovarian tumor-associated proteinase B1) and OTUB2(ovarian tumor-associated proteinase B2). They are deubiquitinating enzymes, which are research hotspots in tumor immunotherapy, with their implications extending across the spectrum of tumor development. Understanding their important role in tumorigenesis, includ-ing hepatocellular carcinoma (HCC) is crucial. HCC has alarming global incidence rates and mortality statistics, ranking among the top five prevalent cancers in Malaysia 1 1 https://gco.iarc.fr/en. Numerous studies have consistently indicated significant expression of OTUB1 and OTUB2 in HCC cells. In addition, OTUB1 has important biological functions in cancer, suggesting its important role in tumorigenesis. However, the mechanism underlying the action of OTUB1 and OTUB2 in liver cancer remains inadequately explored. Therefore, Otubain Subfamily, as potential molecular target, holds promise for advancing HCC treatments. However, further clinical studies are required to verify its efficacy and application prospects. • Otubains subfamily's complicated roles in hepatocellular carcinoma (HCC) progression. • OTUB1 and OTUB2 are linked to poor prognosis in HCC patients. • Potential Otubain Subfamily-related therapeutic targets were identified in HCC. • Deubiquitinating Enzymes Inhibit Tumor Growth and Immune Escape in HCC. • Novel Insights into the Role of OTUB1/2 in TGF-β and NF-κB Signaling Pathways. [ABSTRACT FROM AUTHOR]

Objective: The effects of Otubain-2 (OTUB2) on the proliferation, invasion, and migration of esophageal squamous cell carcinoma (ESCC) were investigated by interfering with OTUB2 expression. Methods: Bioinformatics analysis was used to analyze OTUB2 expression in esophageal carcinoma and interactions between OTUB2 and YAP1/TAZ. Paraffin-embedded ESCC tissues (n = 183) were selected for immunohistochemical staining to detect OTUB2, YAP1, TAZ, CTGF and their relationship with clinicopathological parameters, then the survival prognosis of ESCC patients was analyzed. Immunofluorescence, western blotting, and qRT-PCR were used to evaluate OTUB2 in ESCC cell lines. Cell lines with the highest expression of OTUB2 were transfected with lentivirus to knockdown OTUB2 levels. Changes in KYSE150 cell proliferation, migration, and invasion were measured using CCK-8, wound healing, and clone formation assays. The Transwell test and flow cytometry identified OTUB2 targets and explored roles and mechanisms involved in ESCC. Effects of OTUB2 on YAP1/TAZ signaling were also observed. Results: Bioinformatics analysis revealed OTUB2 was highly expressed in esophageal cancer and was associated with YAP1/TAZ. Immunohistochemistry showed that OTUB2 expression was increased in ESCC samples compared to parcancerous tissue. YAP1 and TAZ were higher expression in ESCC tissues, mainly localized in the nucleus. Compared with controls, the proliferation, migration, and invasion ability of KYSE150 cells after OTUB2 knockdown were significantly reduced (P < 0.05). The protein expression levels of YAP1, TAZ and CTGF decreased after knocking down the expression of OTUB2 (P < 0.05). OTUB2 knockdown in ESCC cell lines suppressed YAP1/TAZ signaling. Conclusions: OTUB2 regulated the protein expression of YAP1/TAZ to promote cell proliferation, migration, invasion, and tumor development. Therefore, OTUB2 may represent a biomarker for ESCC and a potential target for ESCC treatment. [ABSTRACT FROM AUTHOR]

Red-spotted grouper nervous necrosis virus (RGNNV) is a major viral pathogen of grouper and is able to antagonize interferon responses through multiple strategies, particularly evading host immune responses by inhibiting interferon responses. Ovarian tumor (OTU) family proteins are an important class of DUBs and the underlying mechanisms used to inhibit interferon pathway activation are unknown. In the present study, primers were designed based on the transcriptome data, and the ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and OTUB2 genes of Epinephelus coioides (EcOTUB1 and EcOTUB2) were cloned and characterized. The homology alignment showed that both EcOTUB1 and EcOTUB2 were most closely related to E. lanceolatus with 98 % identity. Both EcOTUB1 and EcOTUB2 were distributed to varying degrees in grouper tissues, and the transcript levels were significantly up-regulated following RGNNV stimulation. Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro , and inhibited the promoter activities of interferon stimulated response element (ISRE), nuclear transcription factors kappaB (NF-κB) and IFN3, and the expression levels of interferon related genes and proinflammatory factors. Co-immunoprecipitation experiments showed that both EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, indicating that EcOTUB1 and EcOTUB2 may play important roles in interferon signaling pathway. The results will provide a theoretical reference for the development of novel disease prevention and control techniques. • EcOTUB1 and EcOTUB2 were distributed in all the tested tissues. • Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro and decreased host interferon immune responses. • EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, respectively. [ABSTRACT FROM AUTHOR]

Ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein Otubain2 (OTUB2) was a functional cysteine protease in the OTU family with deubiquitinase activity. In recent years, with the wide application of molecular biology techniques, molecular mechanism regulation at multiple levels of cell signaling pathways has been gradually known, such as ubiquitin-mediated protein degradation and phosphorylation-mediated protein activation. OTUB2 is involved in the deubiquitination of many key proteins in different cell signaling pathways, and the effect of OTUB2 on human health or disease is not clear. OTUB2 is likely to cause cancer and other malignant diseases while maintaining normal human development and physiological function. Therefore, it is of great value to comprehensively understand the regulatory mechanism of OTUB2 and regard it as a target for the treatment of diseases. This review makes a general description and appropriate analysis of OTUB2’s regulation in different cell signaling pathways, and connects OTUB2 with cancer from the research hotspot perspective of DNA damage repair and immunity, laying the theoretical foundation for future research.

Abstract The deubiquitinase otubain 2 (OTUB2) has been reported to play significant roles in the tumorigenesis of several cancers, but the role of OTUB2 in liver cancer is not investigated yet. In the present study, OTUB2 was found significantly upregulated in liver cancer tumor tissues and cell lines, and elevated OTUB2 indicated as a negative index for the overall survival of liver cancer patients. At the cellular level, knockdown of OTUB2 markedly inhibited liver cancer cell growth. Our further investigations revealed that knockdown of OTUB2 significantly suppressed NF‐κB‐driving luciferase activity, and markedly inhibited the phosphorylation of NF‐κB p65 in liver cancer cells, which indicated that OTUB2 mediated liver cancer cell growth by regulating NF‐κB signaling. Additionally, we found that liver cancer cell lines harboring higher OTUB2 expression were more sensitive to NF‐κB inhibitors, and overexpression of OTUB2 could significantly reduce the antitumor effects of NF‐κB inhibitors in liver cancer cells. This study indicated that OTUB2 could be a promising target for the treatment of liver cancer in the future.

Background: Breast cancer is a prevalent disease that has a dismal prognosis for patients and a bad outlook for treatments. Ubiquitination is a reversible biological process that regulates protein production and degradation, as well as plays a vital role in protein transport, localization, and biological activity., Methods: We obtained the breast cancer patient sample data and used a machine learning technique to create a novel index called Deubiquitinating enzyme related index (DUBRI) by gathering genes associated to deubiquitinating enzymes. Based on DUBRI, we systematically analyze patients' prognosis, clinical characteristics, tumor immune microenvironment, chemotherapy response and immunotherapy response. Finally, the function of OTUB2 was explored in breast cancer cells., Results: DUBRI, which consists of five deubiquitinating enzyme genes (OTUB2, USP41, MINDY2, YOD1, and PSMD7), is a reliable predictor of survival in breast cancer patients. We found that the high DUBRI group presented higher levels of immune cell infiltration. We performed molecular docking prediction of core target proteins in deubiquitinating enzymes. In vitro experiments verified that knockdown of OTUB2 could inhibit the proliferation and migration of breast cancer., Conclusions: The DUBRI discovered in this research may effectively evaluate the outlook of breast cancer patients and identify groups of patients who would gain advantages from immunotherapy, offering vital knowledge for the future targeted treatment of breast cancer patients.

Otubain 2 (OTUB2), a deubiquitinating enzyme, overexpression is considered to predict poor outcome in various cancers. However, the function and potential regulatory mechanisms of OTUB2 in gastric cancer (GC) progression remains unclear. To determine how OTUB2 participate in GC progression, the gain and loss of-function experiments were conducted in vivo and in vitro. We found that OTUB2 was upregulated in GC samples (n=140) and cells. Moreover, the overall, first progression and post progression survival rates of GC patients with high OTUB2 expression showed a poorer prognosis than that in those patients with low OTUB2 expression. Down-regulation of OTUB2 suppressed sphere formation and reduced expression of stem cell markers in GC cells. Furthermore, OTUB2-silenced GC cells also showed a decreased proliferation, invasion, migration, and in vivo tumorigenic ability. However, OTUB2 overexpression showed the opposite effects. Notably, we demonstrated that OTUB2 increased lysine-specific histone demethylase 1A (KDM1A) expression through deubiquitination. KDM1A, a demethylase known to promote demethylation of downstream genes, was identified to promote the maintenance of cancer stem cell characteristics. Moreover, the alterations caused by OTUB2 overexpression were partly inversed by KDM1A knockdown and in turn KDM1A overexpression reversed the changes induced by OTUB2 shRNA. Taken together, we demonstrate that OTUB2 may serve as a vital driver in GC tumorigenesis by enhancing KDM1A-mediated stem cell-like properties. [ABSTRACT FROM AUTHOR]

Otubain 2 (OTUB2), a deubiquitinating enzyme, overexpression is considered to predict poor outcome in various cancers. However, the function and potential regulatory mechanisms of OTUB2 in gastric cancer (GC) progression remains unclear. To determine how OTUB2 participate in GC progression, the gain and loss of-function experiments were conducted in vivo and in vitro. We found that OTUB2 was upregulated in GC samples (n=140) and cells. Moreover, the overall, first progression and post progression survival rates of GC patients with high OTUB2 expression showed a poorer prognosis than that in those patients with low OTUB2 expression. Down-regulation of OTUB2 suppressed sphere formation and reduced expression of stem cell markers in GC cells. Furthermore, OTUB2-silenced GC cells also showed a decreased proliferation, invasion, migration, and in vivo tumorigenic ability. However, OTUB2 overexpression showed the opposite effects. Notably, we demonstrated that OTUB2 increased lysine-specific histone demethylase 1A (KDM1A) expression through deubiquitination. KDM1A, a demethylase known to promote demethylation of downstream genes, was identified to promote the maintenance of cancer stem cell characteristics. Moreover, the alterations caused by OTUB2 overexpression were partly inversed by KDM1A knockdown and in turn KDM1A overexpression reversed the changes induced by OTUB2 shRNA. Taken together, we demonstrate that OTUB2 may serve as a vital driver in GC tumorigenesis by enhancing KDM1A-mediated stem cell-like properties.

Yanfei Ma, Yu Sun Department of Fourth Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China Background: Aberrant expression of microRNAs (miRNAs) is closely involved in cancer development. Downregulation of miR-29a-3p and its tumor suppressive roles in cancer have been revealed by multiple reporters. However, study of its expression pattern and function in papillary thyroid carcinoma (PTC) is rare. Materials and methods: The expression of miR-29a-3p in PTC tissues and cells was detected by qPCR. CCK-8, plate clone formation, transwell invasion, Western blot, immunohistochemistry, and luciferase reporter assays were carried out to identify the target of miR-29a-3p and explore its roles and mechanisms in PTC. Results: Deregulated miR-29a-3p in PTC tissues and cell lines were revealed by qPCR. Restoring miR-29a-3p expression significantly inhibited growth, proliferation, and invasion of PTC cells demonstrated by CCK-8, plate clone formation, and transwell assays. Luciferase reporter assays showed miR-29a-3p can directly target OTUB2 in PTC cells. Ectopic expression of OTUB2 can antagonize the effects of miR-29a-3p on cell growth, proliferation, and invasion of PTC. Mechanistically, OTUB2 overexpression can activate NF-κB signaling mostly by stabilizing TRAF6. Upregulated OTUB2 expression was observed in PTC tissues via immunohistochemistry analysis. Moreover, OTUB2 showed a positive correlation to metastatic status and showed a negative correlation to the overall survival rate in PTC patients. Conclusion: Deregulated miR-29a-3p can promote cell growth, proliferation, and invasion in PTC. OTUB2 is a direct downstream target of miR-29a-3p in PTC, and it mediates the effects of deregulated miR-29a-3p by activating TRAF6-associated NF-κB signaling in PTC. Keywords: miR-29a-3p, PTC, OTUB2, NF-κB signaling

Background: Esophageal squamous cell carcinoma (ESCC) is a gastrointestinal malignancy with high incidence. This study aimed to reveal the complete circRNA-miRNA-mRNA regulatory network in ESCC and validate its function mechanism., Method: Expression of OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 2 (OTUB2) in ESCC was analyzed by bioinformatics to find the binding sites between circRNA6448-14 and miR-455-3p, as well as miR-455-3p and OTUB2. The binding relationships were verified by RNA Immunoprecipitation (RIP) and dual-luciferase assay. The expressions of circRNA6448-14, miR-455-3p, and OTUB2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). MTT assay measured cell viability, and the spheroid formation assay assessed the ability of stem cell sphere formation. Western blot (WB) determined the expression of marker proteins of stem cell surface and rate-limiting enzyme of glycolysis. The Seahorse XFe96 extracellular flux analyzer measured the rate of extracellular acidification rate and cellular oxygen consumption. Corresponding assay kits assessed cellular glucose consumption, lactate production, and adenosine triphosphate (ATP) generation., Results: In ESCC, circRNA6448-14 and OTUB2 were highly expressed in contrast to miR-455-3p. Knocking down circRNA6448-14 could prevent the glycolysis and stemness of ESCC cells. Additionally, circRNA6448-14 enhanced the expression of OTUB2 by sponging miR-455-3p. Overexpression of OTUB2 or silencing miR-455-3p reversed the inhibitory effect of knockdown of circRNA6448-14 on ESCC glycolysis and stemness., Conclusion: This research demonstrated that the circRNA6448-14/miR-455-3p/OTUB2 axis induced the glycolysis and stemness of ESCC cells. Our study revealed a novel function of circRNA6448-14, which may serve as a potential therapeutic target for ESCC.

The deubiquitinase otubain 2 (OTUB2) has been reported to play significant roles in the tumorigenesis of several cancers, but the role of OTUB2 in liver cancer is not investigated yet. In the present study, OTUB2 was found significantly upregulated in liver cancer tumor tissues and cell lines, and elevated OTUB2 indicated as a negative index for the overall survival of liver cancer patients. At the cellular level, knockdown of OTUB2 markedly inhibited liver cancer cell growth. Our further investigations revealed that knockdown of OTUB2 significantly suppressed NF‐κB‐driving luciferase activity, and markedly inhibited the phosphorylation of NF‐κB p65 in liver cancer cells, which indicated that OTUB2 mediated liver cancer cell growth by regulating NF‐κB signaling. Additionally, we found that liver cancer cell lines harboring higher OTUB2 expression were more sensitive to NF‐κB inhibitors, and overexpression of OTUB2 could significantly reduce the antitumor effects of NF‐κB inhibitors in liver cancer cells. This study indicated that OTUB2 could be a promising target for the treatment of liver cancer in the future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: OTULIN, OTUB1 and OTUB2 are deubiquitinases, the enzymes responsible for reversing ubiquitination process that occupies key roles in numerous cellular processes. The ubiquitination protein-protein interaction (PPI) network has been extensively explored in order to unravel the complexity of ubiquitin pathway. However, many significant challenges remain to develop a network-based understanding of the ubiquitination complexity including incompleteness of human interactome. Therefore, we aim to construct a pair of yeast two-hybrid (Y2H) vectors using pDEST32/pDEST22 vector system as a preparation for screening OTULIN-, OTUB1- and OTUB2-interacting proteins from human cDNA library, with ultimate aim of expanding the PPI network in human ubiquitome. Methods: OTULIN, OTUB1 and OTUB2 were cloned into entry vector using pCR™8/GW/TOPO® TA Cloning® system and shuttled into pDEST™32 bait vector by LR recombination reaction. To generate Y2H prey library clones, cDNA library was synthesized from HEK293 cells and cloned into donor vector pDONR™222 before transferred into destination vector pDEST™22. Results: DNA sequencing analysis confirmed the correct sequence of OTULIN, OTUB1 and OTUB2 inserts in pDEST32. Meanwhile, generation of cDNA library in pDEST22 produced 5.2 x 106 clones. Randomly picked pDEST22-cDNA clones showed that the recombination rate was 83% and gel electrophoresis indicated that the inserts length ranged from 0.45 to 3.4 kb. Conclusion: OTULIN, OTUB1, OTUB2 and cDNA library were successfully cloned into Y2H bait and prey vectors. The clones have been transfected into competent yeast Saccharomyces cerevisiae strain MaV203 and Y2H experiment to screen novel OTULIN-, OTUB1- and OTUB2-interacting protein from human cDNA library is underway. [ABSTRACT FROM AUTHOR]

Objectives: Ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein Otubain2 (OTUB2) is an important cysteine protease with deubiquitinase activity in the OTU family. However, the role of OTUB2 in cervical cancer (CC) has not been investigated., Methods: OTUB2 expression was analyzed employing the CC data from The Cancer Genome Atlas (TCGA) database. Western blot and qRT-PCR analysis were performed to identify OTUB2 expression in CC. The oncogenic function of OTUB2 was identified through a series of in vitro and in vivo experiments. Tandem Mass Tag™ Quantitative Proteomics examination was used to identify potential targets of OTUB2., Results: OTUB2 was overexpressed in CC and was related to poor prognosis of patients. In our in-house cohort, we also showed that OTUB2 was overexpressed in tumor tissues of CC compared to para-tumor. Knockdown of OTUB2 suppressed CC cell growth whereas OTUB2 upregulation fostered the proliferation of cancer cells. Forkhead box M1 (FOXM1) was found to be a target of OTUB2. FOXM1 can be positively regulated by OTUB2 in CC cells. In human CC tissues, protein level of FOXM1 was positively correlated with OTUB2. FOXM1 was found to play a critical role in OTUB2-mediated CC cell growth. Mechanistically, OTUB2 could bind FOXM1 and deubiquitinate FOXM1 to stabilize it., Conclusion: OTUB2 promotes CC progression through deubiquitinating and stabilizing FOXM1., Competing Interests: None., (AJTR Copyright © 2024.)

The urgent need for novel and effective drugs against the SARS-CoV-2 coronavirus pandemic has stimulated research worldwide. The Papain-like protease (PLpro), which is essential for viral replication, shares a similar active site structural architecture to other cysteine proteases. Here, we have used representatives of the Ovarian Tumor Domain deubiquitinase family OTUB1 and OTUB2 along with the PLpro of SARS-CoV-2 to validate and rationalize the binding of inhibitors from previous SARS-CoV candidate compounds. By forming a new chemical bond with the cysteine residue of the catalytic triad, covalent inhibitors irreversibly suppress the protein’s activity. Modeling covalent inhibitor binding requires detailed knowledge about the compounds’ reactivities and binding. Molecular Dynamics refinement simulations of top poses reveal detailed ligand-protein interactions and show their stability over time. The recently discovered selective OTUB2 covalent inhibitors were used to establish and validate the computational protocol. Structural parameters and ligand dynamics are in excellent agreement with the ligand-bound OTUB2 crystal structures. For SARS-CoV-2 PLpro, recent covalent peptidomimetic inhibitors were simulated and reveal that the ligand-protein interaction is very dynamic. The covalent and non-covalent docking plus subsequent MD refinement of known SARS-CoV inhibitors into DUBs and the SARS-CoV-2 PLpro point out a possible approach to target the PLpro cysteine protease from SARS-CoV-2. The results show that such an approach gives insight into ligand-protein interactions, their dynamic character, and indicates a path for selective ligand design.

In the effort to identify deubiquitinating enzymes required for the growth of colorectal cancer (CRC) cells, we found that OTUB2 knockdown markedly inhibited the viability of these cancer cells in culture and in xenografted mice. It was also found that the level of OTUB2 was elevated in primary CRCs, and its high expression was a poor prognostic indicator for the patients. Interestingly, immunoprecipitation and LC-MS/MS analyses suggested that β-Catenin was an OTUB2-interacting protein, and there was a positive correlation between OTUB2 and β-Catenin expression in both CRC tissues and cell lines. We then performed reciprocal co-immunoprecipitations and demonstrated that OTUB2 and β-Catenin bound to each other. Enforced expression of OTUB2 decreased ubiquitination of β-Catenin and increased the half-life and intracellular level of β-Catenin, whereas the catalytic inactive OTUB2 did not. OTUB2 also enhanced β-Catenin-mediated transactivation as measured by TCF-luciferase and expression of endogenous CCND1 and MYC in CRC cells. These results indicated that OTUB2 was a potential target for therapeutic intervention for CRC., Competing Interests: None., (AJCR Copyright © 2023.)

Cervical cancer (CC) is a deadly gynecological tumor worldwide. Otubain 2 (OTUB2) has been recently identified as an oncogene in human malignancies. However, its expression and function remain unclear. This work aims to explore the role of OTUB2 in CC progression. Herein, The Cancer Genome Atlas data revealed that OTUB2 expression was significantly upregulated in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and gradually increased with CESC progression; moreover, OTUB2 expression predicted poor outcomes of CESC patients. Then, RT-qPCR and Western blotting were applied to determine mRNA and protein expression in CC and normal cells. Our results confirmed that OTUB2 was highly expressed in CC cell lines. As indicated by CCK-8, Transwell, and flow cytometry results, OTUB2 silencing attenuated proliferative and metastatic capacities of CC cells but promoted CC cell apoptosis. Then, RBM15, an N6-methyladenosine (m 6 A) methyltransferase "writer," was also demonstrated to be upregulated in CESC and CC cells. Mechanistically, m 6 A RNA immunoprecipitation (Me-RIP) results showed that RBM15 inhibition reduced the m 6 A methylation level of OTUB2 in CC cells, leading to the decline of OTUB2 expression. In addition, OTUB2 inhibition deactivated the AKT/mTOR signaling in CC cells. Furthermore, SC-79 (AKT/mTOR activator) partially abated the inhibitory effects of OTUB2 knockdown on the AKT/mTOR signaling pathway and the malignant phenotypes of CC cells. In summary, this work showed that RBM15-mediated m 6 A modification led to OTUB2 upregulation, thereby promoting malignant behaviors of CC cells via the AKT/mTOR signaling pathway., (© 2023 Wiley Periodicals LLC.)

Abstract The transcription factor Gli2 plays crucial roles in the transduction of Hedgehog (Hh) signals, yet the mechanisms that control Gli2 degradation remain unclear. Here we have identified the eubiquitinating enzyme otubain2 (OTUB2) as a regulator of Gli2 protein degradation. We found that OTUB2 was coimmunoprecipitated with Gli2. Knockdown of OTUB2 decreased Gli2 protein level while the proteasome inhibitor MG-132 treatment restored Gli2 expression. Additionally, OTUB2 overexpression stabilized Gli2 protein in U2OS cells and extended the half-life of Gli2. We also found that knockdown of OTUB2 reduced deubiquitination of Gli2 in vivo. In vitro deubiquitination assay showed that ubiquitinated Gli2 was decreased by wild-type OTUB2 but not OTUB2 mutations. We also found that OTUB2 knockdown suppressed the ALP activity and the expression of the common markers BMP2 and RUNX2 during osteogenesis of MSCs in response to Shh and Smo agonists, which indicated OTUB2 may have effect on osteogenic differentiation by regulating Hh signaling. Highlights • OTUB2 stabilizes Gli2 protein. • OTUB2 deubiquitinates Gli2. • OTUB2 promotes osteogenesis of MSCs. [ABSTRACT FROM AUTHOR]

Aberrant regulation of ubiquitination is an essential fundamental process in tumors, especially intrahepatic cholangiocarcinoma (iCCA). We reported that OTUB2, an OTU deubiquitinase, is upregulated in iCCA and stabilizes the CTNNB1-ZEB1 axis, resulting in epithelial–mesenchymal transition (EMT) and iCCA metastasis. Mechanistically, OTUB2 promotes CTNNB1 expression by interacting with the E3 ligase TRAF6. OTUB2 inhibits the lysosomal degradation of CTNNB1 by interacting with TRAF6 and thus regulates the progression of iCCA through ZEB1. Clinically, high OTUB2 expression is related to increased ZEB1 expression and activity and reduced overall survival in iCCA patients. Therefore, advanced iCCA patients may benefit from drugs targeting OTUB2 and its pathway. [Display omitted] • High expression of OTUB2 is closely associated with poor survival in intrahepatic cholangiocarcinoma (iCCA) patients. • Downregulation of OTUB2 suppresses iCCA cell proliferation, migration and invasion by inducing ZEB1 expression. • OTUB2 regulates the level of CTNNB1 through the interaction with TRAF6, and regulates the iCCA progression via ZEB1 consequently. [ABSTRACT FROM AUTHOR]

Oral and esophageal squamous cell carcinomas (SCCs) are associated with high mortality, yet the molecular mechanisms underlying these malignancies are largely unclear. We show that DNA hypermethylation of otubain 2 (OTUB2), a previously recognized oncogene, drives tongue and esophageal SCC initiation and drug resistance. Mechanistically, OTUB2 promotes the deubiquitination and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and subsequently regulates the transcription of calmodulin-like protein 3 (CALML3). Activation of CALML3-mediated mitochondrial calcium signaling promotes oxidative phosphorylation (OXPHOS) and the synthesis of phosphatidylserine (PS). In mouse models, orally administered soybean-derived PS inhibits SCC initiation in cells with low OTUB2 expression and increases their sensitivity to chemotherapy. Our study indicates that the OTUB2/STAT1/CALML3/PS axis plays tumor-suppressive roles and shows the potential of PS administration as a strategy for the treatment and prevention of tongue and esophageal SCCs. [Display omitted] • OTUB2 suppresses tongue and esophageal SCC development and progression via CALML3 • OTUB2 deubiquitinates STAT1 to promote STAT1 phosphorylation and dimerization • The OTUB2/CALML3 axis activates Ca2+ signaling to promote PS synthesis • Oral administration of PS suppresses SCC initiation and enhances chemosensitivity of SCC Chang et al. show that OTUB2, a previously recognized oncoprotein, functions as a tumor suppressor in tongue and esophageal squamous cell carcinomas (SCCs). OTUB2 deubiquitinates STAT1 and promotes STAT1 phosphorylation and dimerization to activate CALML3/Ca2+/phosphatidylserine signaling. Oral administration of soybean-derived phosphatidylserine inhibits SCC initiation and progression with low OTUB2 expression. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Pro-inflammatory cytokines induce death of beta cells and hamper engraftment of transplanted islet mass. Our aim was to reveal novel genes involved in this process, as a platform for innovative therapeutic approaches. Methods: Small interfering RNA (siRNA) high-throughput screening (HTS) of primary human islets was employed to identify novel genes involved in cytokine-induced beta cell apoptosis. Dispersed human islets from nine human donors, treated with a combination of TNF-α, IL-1β and IFN-γ were transfected with ∼730 different siRNAs. Caspase-3/7 activity was measured, results were analysed and potential anti- and pro-apoptotic genes were identified. Results: Dispersed human pancreatic islets appeared to be suitable targets for performance of siRNA HTS. Using this methodology we found a number of potential pro- and anti-apoptotic target hits that have not been previously associated with pancreatic beta cell death. One such hit was the de-ubiquitinating enzyme otubain 2 (OTUB2). OTUB2 knockdown increased caspase-3/7 activity in MIN6 cells and primary human islets and inhibited insulin secretion and increased nuclear factor-κB (NF-κB) activity both under basal conditions and following cytokine treatment. Conclusions: Use of dispersed human islets provides a new platform for functional HTS in a highly physiological system. Employing this technique enabled the identification of OTUB2 as a novel promoter of viability and insulin secretion in human beta cells. OTUB2 acts through the inhibition of NF-κB signalling, which is deleterious to beta cell survival. siRNA screens of human islets may therefore identify new targets, such as OTUB2, for therapeutic intervention in type 1 diabetes and islet transplantation. [ABSTRACT FROM AUTHOR]

Ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein Otubain2 (OTUB2) was a functional cysteine protease in the OTU family with deubiquitinase activity. In recent years, with the wide application of molecular biology techniques, molecular mechanism regulation at multiple levels of cell signaling pathways has been gradually known, such as ubiquitin-mediated protein degradation and phosphorylation-mediated protein activation. OTUB2 is involved in the deubiquitination of many key proteins in different cell signaling pathways, and the effect of OTUB2 on human health or disease is not clear. OTUB2 is likely to cause cancer and other malignant diseases while maintaining normal human development and physiological function. Therefore, it is of great value to comprehensively understand the regulatory mechanism of OTUB2 and regard it as a target for the treatment of diseases. This review makes a general description and appropriate analysis of OTUB2's regulation in different cell signaling pathways, and connects OTUB2 with cancer from the research hotspot perspective of DNA damage repair and immunity, laying the theoretical foundation for future research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Zhang, Li, Hong, Meng and Ouyang.)

The urgent need for novel and effective drugs against the SARS-CoV-2 coronavirus pandemic has stimulated research worldwide. The Papain-like protease (PLpro), which is essential for viral replication, shares a similar active site structural architecture to other cysteine proteases. Here, we have used representatives of the Ovarian Tumor Domain deubiquitinase family OTUB1 and OTUB2 along with the PLpro of SARS-CoV-2 to validate and rationalize the binding of inhibitors from previous SARS-CoV candidate compounds. By forming a new chemical bond with the cysteine residue of the catalytic triad, covalent inhibitors irreversibly suppress the protein's activity. Modeling covalent inhibitor binding requires detailed knowledge about the compounds' reactivities and binding. Molecular Dynamics refinement simulations of top poses reveal detailed ligand-protein interactions and show their stability over time. The recently discovered selective OTUB2 covalent inhibitors were used to establish and validate the computational protocol. Structural parameters and ligand dynamics are in excellent agreement with the ligand-bound OTUB2 crystal structures. For SARS-CoV-2 PLpro, recent covalent peptidomimetic inhibitors were simulated and reveal that the ligand-protein interaction is very dynamic. The covalent and non-covalent docking plus subsequent MD refinement of known SARS-CoV inhibitors into DUBs and the SARS-CoV-2 PLpro point out a possible approach to target the PLpro cysteine protease from SARS-CoV-2. The results show that such an approach gives insight into ligand-protein interactions, their dynamic character, and indicates a path for selective ligand design. [ABSTRACT FROM AUTHOR]

• Otubains (OTUB1 and OTUB2) play crucial roles in cancers and other human diseases. • Otubains act as tumor promoters in different types of human cancers. However, in some special cases, OTUB1 functions as a tumor suppressor. • Otubains are regulated at transcriptional and post-translational levels. OTU domain-containing ubiquitin aldehyde-binding proteins Otubain1 (OTUB1) and Otubain2 (OTUB2) were initially identified as OTU deubiquitinases (DUBs). Recently, Otubains have emerged as essential regulators of diverse physiological processes, such as immune signaling and DNA damage response. Dysregulation of those processes is likely to increase the risk in multiple aspects of aging-related diseases, including cancers, neurodegenerative disorders, chronic kidney diseases, bone dysplasia and pulmonary fibrosis. Consistently, Otubains are aberrantly expressed in cancers and have been identified to be both tumor suppressors and tumor promoters in different types of cancers. Therefore, the regulatory mechanism of the activity and expression of Otubains is very important for better understanding of Otubains-associated biological networks and human diseases. This review provides a comprehensive description of functions and regulatory axis of Otubains, highlighting experimental evidences indicating Otubains as potential therapeutic targets against aging-related disorders. [ABSTRACT FROM AUTHOR]

Summary The transcriptional regulators YAP and TAZ play important roles in development, physiology, and tumorigenesis and are negatively controlled by the Hippo pathway. It is yet unknown why the YAP/ TAZ proteins are frequently activated in human malignancies in which the Hippo pathway is still active. Here, by a gain-of-function cancer metastasis screen, we discovered OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ. We found OTUB2 to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ. We also identified a yet-unknown SUMO-interacting motif (SIM) in YAP and TAZ required for their association with SUMOylated OTUB2. Importantly, EGF and oncogenic KRAS induce OTUB2 poly-SUMOylation and thereby activate YAP/TAZ. Our results establish OTUB2 as an essential modulator of YAP/TAZ and also reveal a novel mechanism via which YAP/TAZ activity is induced by oncogenic KRAS. Graphical Abstract Highlights • OTUB2 enhances metastasis through Hippo-independent activation of YAP/TAZ signaling • Poly-SUMOylation is required for OTUB2 to interact with and deubiquitinate YAP/TAZ • YAP/TAZ contain a SUMO-interacting motif (SIM) required for binding SUMOylated OTUB2 • EGF treatment or KRAS mutation stabilizes YAP/TAZ by stimulating OTUB2 SUMOylation Zhang et al. identified OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ independent of the Hippo signaling. OTUB2 was induced by EGF/KRAS to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ through a yet-unknown SUMO-interacting motif (SIM). [ABSTRACT FROM AUTHOR]

Increasing evidence has confirmed that deubiquitinating enzymes play an important role in lung cancer progression. In the current study, we investigated the expression profile of deubiquitinating enzymes in non-small cell lung cancer (NSCLC) tissues and identified OTUB2 as an upregulated deubiquitinating enzyme. The role of OTUB2 in NSCLC is unknown. Methods: Quantitative, real-time PCR and Western blot were used to detect OTUB2 and U2AF2 expression in NSCLC tissues. The correlations between OTUB2 and U2AF2 expression and clinicopathologic features were then analyzed. We used In vitro Cell Counting Kit-8 (CCK-8) , colony formation , and trans-well invasion assays to investigate the function of OTUB2 and U2AF2 in tumorigenesis. The regulation of glycolysis by OTUB2 and U2AF2 was assessed by determining the extracellular acid ratio, glucose consumption, and lactate production. The mechanism of OTUB2 was explored through co-immunoprecipitation and mass spectrometry analyses. A xenograft model was also used to study the tumorigenesis role of OTUB2 In vivo . Results: OTUB2 expression was significantly upregulated in primary NSCLC tissues and greatly associated with metastasis, advanced tumor stages, poor survival, and recurrence. In NSCLC cell lines, OTUB2 promoted cell growth, colony formation, migration, and invasive activities. Mechanistic investigations showed that OTUB2 stimulated the Warburg effect and induced the activation of the serine/threonine kinase/mechanistic target of rapamycin kinase (AKT/mTOR) pathway in different NSCLC cells. More importantly, OTUB2 promoted NSCLC progression, which was largely dependent on the direct binding to and deubiquitination of U2AF2, at least in NSCLC cells. U2AF2 expression was also significantly upregulated in primary NSCLC tissues and dramatically associated with metastasis, advanced tumor stages, poor survival, and recurrence. Importantly, a positive correlation between the protein expression of OTUB2 and U2AF2 in NSCLC tissues was found. In vivo experiments indicated that OTUB2 promoted xenograft tumor growth of NSCLC cell. In addition, our results suggest that high expression of OTUB2, U2AF2 and PGK1 is significantly associated with worse prognosis in NSCLC patients. Conclusion: Taken together, the present study provides the first evidence that OTUB2 acts as a pivotal driver in NSCLC tumorigenesis by stabilizing U2AF2 and activating the AKT/mTOR pathway and the Warburg effect. It may serve as a new potential prognostic indicator and therapeutic target in NSCLC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.