RESUMO
Despite recent advances in systemic therapy for hepatocellular carcinoma (HCC), the prognosis of hepatitis B virus (HBV)-induced HCC patients remains poor. By screening a sgRNA library targeting human deubiquitinases, we find that ubiquitin-specific peptidase 26 (USP26) deficiency impairs HBV-positive HCC cell proliferation. Genetically engineered murine models with Usp26 knockout confirm that Usp26 drives HCC tumorigenesis. Mechanistically, we find that the HBV-encoded protein HBx binds to the promoter and induces the production of USP26, which is an X-linked gene exclusively expressed in the testis. HBx consequently promotes the association of USP26 with SIRT1 to synergistically stabilize SIRT1 by deubiquitination, which promotes cell proliferation and impedes cell apoptosis to accelerate HCC tumorigenesis. In patients with HBV-positive HCC, USP26 is robustly induced, and its levels correlate with SIRT1 levels and poor prognosis. Collectively, our study highlights a causative link between HBV infection, deubiquitinase induction and development of HCC, identifying a druggable target, USP26.
Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Epigênese Genética , Vírus da Hepatite B , Neoplasias Hepáticas , Sirtuína 1 , Transativadores , Proteínas Virais Reguladoras e Acessórias , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Animais , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Vírus da Hepatite B/genética , Camundongos , Sirtuína 1/metabolismo , Sirtuína 1/genética , Transativadores/metabolismo , Transativadores/genética , Masculino , Proliferação de Células/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Carcinogênese/genética , Hepatite B/virologia , Hepatite B/complicações , Hepatite B/genética , Hepatite B/metabolismo , Linhagem Celular Tumoral , Camundongos Knockout , Regulação Neoplásica da Expressão Gênica , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Apoptose/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Circular RNAs (circRNAs) are a type of regulatory RNA that feature covalently closed single-stranded loops. Evidence suggested that circRNAs play important roles in the progression and development of various cancers. However, the impact of circRNA on autophagy-mediated progression of colorectal cancer (CRC) remains unclear. The objective of this project was to investigate the influence of circSEC24B on autophagy and its underlying mechanisms in CRC. To validate the presence and circular structure of circSEC24B in CRC cells and tissues, PCR and Sanger sequencing techniques were employed. Drug resistance and invasive phenotype of CRC cells were evaluated using CCK8, transwell, and Edu assays. Gain- and loss-of-function experiments were conducted to assess the effects of circSEC24B and its protein partner on the growth, invasion, and metastasis of CRC cells in vitro and in vivo. Interactions between circSEC24B, OTUB1, and SRPX2 were analyzed through immunofluorescence, RNA-pulldown, and RIP assays. Mass spectrometry analysis was used to identify potential binding proteins of circRNA in CRC cells. Vectors were constructed to investigate the specific structural domain of the deubiquitinating enzyme OTUB1 that binds to circSEC24B. Results showed that circSEC24B expression was increased in CRC tissues and cell lines, and it enhanced CRC cell proliferation and autophagy levels. Mechanistically, circSEC24B promoted CRC cell proliferation by regulating the protein stability of SRPX2. Specifically, circSEC24B acted as a scaffold, facilitating the binding of OTUB1 to SRPX2 and thereby enhancing its protein stability. Additionally, evidence suggested that OTUB1 regulated SRPX2 expression through an acetylation-dependent mechanism. In conclusion, this study demonstrated that circSEC24B activated autophagy and induced chemoresistance in CRC by promoting the deubiquitination of SRPX2, mediated by the deubiquitinating enzyme OTUB1.
Assuntos
Autofagia , Neoplasias Colorretais , Cisteína Endopeptidases , Enzimas Desubiquitinantes , Resistencia a Medicamentos Antineoplásicos , Proteínas de Membrana , RNA Circular , Ubiquitinação , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Linhagem Celular Tumoral , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Nus , Animais , Camundongos , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Camundongos Endogâmicos BALB C , Masculino , FemininoRESUMO
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.
Assuntos
Neoplasias da Mama , Enzimas Desubiquitinantes , Imunoterapia , Humanos , Neoplasias da Mama/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Feminino , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Prognóstico , Imunoterapia/métodos , Microambiente Tumoral/imunologia , Proliferação de Células , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Simulação de Acoplamento Molecular , Ubiquitinação , Aprendizado de Máquina , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genéticaRESUMO
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.
Assuntos
Neoplasias do Colo , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas , Humanos , Transição Epitelial-Mesenquimal/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Masculino , Animais , Feminino , Oxaliplatina/farmacologia , Ubiquitinação , Pessoa de Meia-Idade , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/genética , Camundongos Nus , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , CamundongosRESUMO
Deubiquitinases (DUBs) are essential for the maintenance of protein homeostasis and assembly of proteins into functional complexes. Despite growing interest in DUBs biological functions, the roles of DUBs in regulating intestinal stem cells (ISCs) and gut homeostasis remain largely unknown. Here, we perform an in vivo RNAi screen through induced knock-down of DUBs expression in adult midgut ISCs and enteroblasts (EBs) to identify DUB regulators of intestinal homeostasis in Drosophila. We screen 43 DUBs and identify 8 DUBs that are required for ISCs homeostasis. Knocking-down of usp1, CG7857, usp5, rpn8, usp10 and csn5 decreases the number of ISCs/EBs, while knocking-down of CG4968 and usp8 increases the number of ISCs/EBs. Moreover, knock-down of usp1, CG4968, CG7857, or rpn8 in ISCs/EBs disrupts the intestinal barrier integrity and shortens the lifespan, indicating the requirement of these DUBs for the maintenance of gut homeostasis. Furthermore, we provide evidences that USP1 mediates ISC lineage differentiation via modulating the Notch signaling activity. Our study identifies, for the first time, the deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila, and provide new insights into the functional links between the DUBs and intestinal homeostasis.
Assuntos
Enzimas Desubiquitinantes , Proteínas de Drosophila , Homeostase , Intestinos , Interferência de RNA , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células-Tronco/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genéticaRESUMO
As the largest organ in the human body, skeletal muscle is essential for breathing support, movement initiation, and maintenance homeostasis. It has been shown that programmed cell death (PCD), which includes autophagy, apoptosis, and necrosis, is essential for the development of skeletal muscle. A novel form of PCD called ferroptosis is still poorly understood in relation to skeletal muscle. In this study, we observed that the activation of ferroptosis significantly impeded the differentiation of C2C12 myoblasts into myotubes and concurrently suppressed the expression of OTUB1, a crucial deubiquitinating enzyme. OTUB1-silenced C2C12 mouse myoblasts were used to investigate the function of OTUB1 in ferroptosis. The results show that OTUB1 knockdown in vitro significantly increased C2C12 ferroptosis and inhibited myogenesis. Interestingly, the induction of ferroptosis resulting from OTUB1 knockdown was concomitant with the activation of autophagy. Furthermore, OTUB1 interacted with the P62 protein and stabilized its expression by deubiquitinating it, thereby inhibiting autophagy-dependent ferroptosis and promoting myogenesis. All of these findings demonstrate the critical role that OTUB1 plays in controlling ferroptosis, and we suggest that focusing on the OTUB1-P62 axis may be a useful tactic in the treatment and prevention of disorders involving the skeletal muscle.
Assuntos
Autofagia , Diferenciação Celular , Cisteína Endopeptidases , Ferroptose , Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Mioblastos , Animais , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Ferroptose/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Mioblastos/metabolismo , Mioblastos/citologia , Linhagem Celular , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Ubiquitinação , Humanos , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genéticaRESUMO
The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.
Assuntos
Enzimas Desubiquitinantes , Subunidade alfa do Fator 1 Induzível por Hipóxia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Mutagênese , Sistemas CRISPR-Cas , Células HEK293 , Transdução de Sinais , UbiquitinaçãoRESUMO
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.
Assuntos
Bass , Doenças dos Peixes , Proteínas de Peixes , Imunidade Inata , Nodaviridae , Infecções por Vírus de RNA , Replicação Viral , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Imunidade Inata/genética , Nodaviridae/fisiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Bass/imunologia , Filogenia , Regulação da Expressão Gênica/imunologia , Sequência de Aminoácidos , Alinhamento de Sequência/veterinária , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/imunologia , Perfilação da Expressão Gênica/veterináriaRESUMO
Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification and functional characterization in teleosts are still rarely known. In this work, we cloned the otub1 of miiuy croaker (Miichthys miiuy), analyzed its sequence, structure, and evolution at genetic and protein levels, and determined its function in the antiviral immune response. The complete open reading frame (ORF) of miiuy croaker otub1 is 843 bp in length, encoding 280 amino acids. Miiuy croaker Otub1 has an OTU domain at the carboxyl terminus, which is a common functional domain that exists in OTU deubiquitinases. Molecular characteristics and evolution analysis results indicated that miiuy croaker Otub1, especially its functional domain, is highly conserved during evolution. The luciferase reporter assays showed that miiuy croaker Otub1 could significantly inhibit the poly(I:C) and Irf3-induced IFN1 and IFN-stimulated response element (ISRE) activation. Further experiments showed that miiuy croaker Otub1 decreases Irf3 protein abundance by promoting its proteasomal degradation. These data suggest that the evolutionarily conserved Otub1 acts as a suppressor in controlling antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker.
Assuntos
Proteínas de Peixes , Fator Regulador 3 de Interferon , Perciformes , Complexo de Endopeptidases do Proteassoma , Proteólise , Animais , Perciformes/imunologia , Perciformes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Peixes/imunologia , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Imunidade Inata , Evolução Molecular , Poli I-C/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Clonagem Molecular , FilogeniaRESUMO
Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by EBV, is a lytic cycle protein with deubiquitinating activity has been found to participate in disrupting repair of DNA damage. We first confirmed that BPLF1 gene in gastric cancer (GC) significantly increased the DNA double strand breaks (DSBs). Ubiquitination mass spectrometry identified histones as BPLF1 interactors and potential substrates, and co-immunoprecipitation and in vitro experiments verified that BPLF1 regulates H2Bub by targeting Rad6. Over-expressing Rad6 restored H2Bub but partially reduced γ-H2AX, suggesting that other downstream DNA repair processes were affected. mRNA expression of BRCA2 were significantly down-regulated by next-generation sequencing after over-expression of BPLF1, and over-expression of p65 facilitated the repair of DSBs. We demonstrated BPLF1 may lead to the accumulation of DSBs by two pathways, reducing H2B ubiquitination (H2Bub) and blocking homologous recombination which may provide new ideas for the treatment of gastric cancer.
Assuntos
Quebras de DNA de Cadeia Dupla , Infecções por Vírus Epstein-Barr , Instabilidade Genômica , Herpesvirus Humano 4 , Histonas , Neoplasias Gástricas , Ubiquitinação , Humanos , Herpesvirus Humano 4/genética , Neoplasias Gástricas/virologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/etiologia , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Histonas/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Regulação Neoplásica da Expressão Gênica , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Carcinogênese/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
BACKGROUND: There is growing evidence indicating that deubiquitinating enzymes may contribute to tumor progression and can serve as promising therapeutic targets. METHODS: The overexpression of deubiquitinase OTUD6B in lung adenocarcinoma (LUAD) and its adjacent tissues was analyzed by immunohistochemistry and TCGA/GO database. Survival analysis further supported OTUD6B as a potential target for LUAD treatment. We assessed the effect of OTUD6B on LUAD cell growth using cell viability assays and conducted TUNEL staining, migration, and invasion experiments to investigate the impact of OTUD6B on the apoptosis and metastasis of LUAD cells. Additionally, we established a transplanted tumor model in nude mice to validate our findings in vivo. Finally, using IP mass spectrometry and co-IP experiments, we screened and confirmed the influence of RIPK1 as a substrate of OTUD6B in LUAD. RESULTS: OTUD6B is highly overexpressed in human LUAD and predicts poor prognosis in LUAD patients. OTUD6B knockdown inhibited the proliferation of LUAD cells and enhanced apoptosis and inhibited metastasis in LUAD cells suppressed. A549 xenografts revealed that OTUD6B deletion can slow down tumour growth. Additionally, OTUD6B can bind to RIPK1, reduce its ubiquitination level and increase its protein stability. CONCLUSIONS: Our results suggest that OTUD6B is a promising clinical target for LUAD treatment and that targeting OTUD6B may constitute an effective anti-LUAD strategy.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Camundongos Nus , Proteína Serina-Treonina Quinases de Interação com Receptores , Humanos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Progressão da Doença , Proliferação de Células , Apoptose , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Células A549 , Ubiquitinação , Estabilidade Proteica , Endopeptidases/metabolismo , Endopeptidases/genéticaRESUMO
Recent studies have shown the crucial role of podocyte injury in the development of diabetic kidney disease (DKD). Deubiquitinating modification of proteins is widely involved in the occurrence and development of diseases. Here, we explore the role and regulating mechanism of a deubiquitinating enzyme, OTUD5, in podocyte injury and DKD. RNA-seq analysis indicates a significantly decreased expression of OTUD5 in HG/PA-stimulated podocytes. Podocyte-specific Otud5 knockout exacerbates podocyte injury and DKD in both type 1 and type 2 diabetic mice. Furthermore, AVV9-mediated OTUD5 overexpression in podocytes shows a therapeutic effect against DKD. Mass spectrometry and co-immunoprecipitation experiments reveal an inflammation-regulating protein, TAK1, as the substrate of OTUD5 in podocytes. Mechanistically, OTUD5 deubiquitinates K63-linked TAK1 at the K158 site through its active site C224, which subsequently prevents the phosphorylation of TAK1 and reduces downstream inflammatory responses in podocytes. Our findings show an OTUD5-TAK1 axis in podocyte inflammation and injury and highlight the potential of OTUD5 as a promising therapeutic target for DKD.
Assuntos
Nefropatias Diabéticas , Inflamação , MAP Quinase Quinase Quinases , Camundongos Knockout , Podócitos , Ubiquitinação , Animais , Humanos , Masculino , Camundongos , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/genética , Células HEK293 , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/genética , Camundongos Endogâmicos C57BL , Fosforilação , Podócitos/metabolismo , Podócitos/patologia , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genéticaRESUMO
Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by "wet lab" experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.
Assuntos
Enzimas Desubiquitinantes , Proteoma , Ubiquitinação , Humanos , Proteoma/metabolismo , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Aprendizado Profundo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/química , Especificidade por Substrato , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Aprendizado de Máquina , Ligação Proteica , Sequência de Aminoácidos , Tioléster HidrolasesRESUMO
Plant cells need to respond to environmental stimuli and developmental signals accurately and promptly. Ubiquitylation is a reversible posttranslational modification that enables the adaptation of cellular proteostasis to internal or external factors. The different topologies of ubiquitin linkages serve as the structural basis for the ubiquitin code, which can be interpreted by ubiquitin-binding proteins or readers in specific processes. The ubiquitylation status of target proteins is regulated by ubiquitylating enzymes or writers, as well as deubiquitylating enzymes (DUBs) or erasers. DUBs can remove ubiquitin molecules from target proteins. Arabidopsis (A. thaliana) DUBs belong to 7 protein families and exhibit a wide range of functions and play an important role in regulating selective protein degradation processes, including proteasomal, endocytic, and autophagic protein degradation. DUBs also shape the epigenetic landscape and modulate DNA damage repair processes. In this review, we summarize the current knowledge on DUBs in plants, their cellular functions, and the molecular mechanisms involved in the regulation of plant DUBs.
Assuntos
Arabidopsis , Ubiquitina , Ubiquitinação , Ubiquitina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Deubiquitinating enzymes (DUBs) cleave ubiquitin on substrate molecules to maintain protein stability. DUBs reportedly participate in the tumorigenesis and tumour progression of hepatocellular carcinoma (HCC). OTU deubiquitinase 5 (OTUD5), a DUB family member, has been recognized as a critical regulator in bladder cancer, breast cancer and HCC. However, the expression and biological function of OTUD5 in HCC are still controversial. RESULTS: We determined that the expression of OTUD5 was significantly upregulated in HCC tissues. High levels of OTUD5 were also detected in most HCC cell lines. TCGA data analysis demonstrated that high OTUD5 expression indicated poorer overall survival in HCC patients. OTUD5 silencing prominently suppressed HCC cell proliferation, while its overexpression markedly enhanced the proliferation of HCC cells. Mass spectrometry analysis revealed solute carrier family 38 member 1 (SLC38A1) as a candidate downstream target protein of OTUD5. Coimmunoprecipitation analysis confirmed the interaction between OTUD5 and SLC38A1. OTUD5 knockdown reduced and OTUD5 overexpression increased SLC38A1 protein levels in HCC cells. However, OTUD5 alteration had no effect on SLC38A1 mRNA expression. OTUD5 maintained SLC38A1 stability by preventing its ubiquitin-mediated proteasomal degradation. SLC38A1 silencing prominently attenuated the OTUD5-induced increase in HCC cell proliferation. Finally, OTUD5 knockdown markedly suppressed the growth of HCC cells in vivo. CONCLUSIONS: OTUD5 is an oncogene in HCC. OTUD5 contributes to HCC cell proliferation by deubiquitinating and stabilizing SLC38A1. These results may provide a theoretical basis for the development of new anti-HCC drugs.
Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: CDC6 is an oncogenic protein whose expression level fluctuates during the cell cycle. Although several E3 ubiquitin ligases responsible for the ubiquitin-mediated proteolysis of CDC6 have been identified, the deubiquitination pathway for CDC6 has not been investigated. METHODS: The proteome-wide deubiquitinase (DUB) screening was used to identify the potential regulator of CDC6. Immunofluorescence, protein half-life and deubiquitination assays were performed to determine the protein stability of CDC6. Gain- and loss-of-function experiments were implemented to analyse the impacts of OUTD6A-CDC6 axis on tumour growth and chemosensitivity in vitro. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced conditional Otud6a knockout (CKO) mouse model and tumour xenograft model were performed to analyse the role of OTUD6A-CDC6 axis in vivo. Tissue specimens were used to determine the association between OTUD6A and CDC6. RESULTS: OTUD6A interacts with, depolyubiquitinates and stabilizes CDC6 by removing K6-, K33-, and K48-linked polyubiquitination. Moreover, OTUD6A promotes cell proliferation and decreases sensitivity to chemotherapy by upregulating CDC6. CKO mice are less prone to BCa tumorigenesis induced by BBN, and knockdown of OTUD6A inhibits tumour progression in vivo. Furthermore, OTUD6A protein level has a positive correlation with CDC6 protein level, and high protein levels of OTUD6A and CDC6 are associated with poor prognosis in patients with bladder cancer. CONCLUSIONS: We reveal an important yet missing piece of novel DUB governing CDC6 stability. In addition, our findings propose a model for the OTUD6A-CDC6 axis that provides novel insights into cell cycle and chemosensitivity regulation, which may become a potential biomarker and promising drug target for cancer treatment.
Assuntos
Proteínas de Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares , Ubiquitinação , Animais , Humanos , Camundongos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Camundongos Knockout , Ensaios Antitumorais Modelo de Xenoenxerto , Regulação Neoplásica da Expressão Gênica , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Modelos Animais de DoençasRESUMO
Ubiquitin-specific proteases (UBPs), the largest subfamily of deubiquitinating enzymes, regulate ubiquitin homeostasis and play diverse roles in eukaryotes. Ubp4 is essential for the growth, development, and pathogenicity of various fungal pathogens. However, its functions in the growth, stress responses, and virulence of entomopathogenic fungi remain unclear. In this study, we elucidated the role of the homolog of Ubp4, MrUbp4, in the entomopathogenic fungus Metarhizium robertsii. Deletion of MrUbp4 led to a notable increase in ubiquitination levels, demonstrating the involvement of MrUbp4 in protein deubiquitination. Furthermore, the ΔMrUbp4 mutant displayed a significant reduction in conidial yield, underscoring the pivotal role of MrUbp4 in conidiation. Additionally, the mutant exhibited heightened resistance to conidial heat treatment, emphasizing the role of MrUbp4 in thermotolerance. Notably, insect bioassays unveiled a substantial impairment in the virulence of the ΔMrUbp4 mutant. This was accompanied by a notable decrease in cuticle penetration ability and appressorium formation upon further analysis. In summary, our findings highlight the essential role of MrUbp4 in regulating the conidial yield, thermotolerance, and contributions to the virulence of M. robertsii.
Assuntos
Metarhizium , Esporos Fúngicos , Termotolerância , Metarhizium/patogenicidade , Metarhizium/genética , Metarhizium/fisiologia , Virulência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismoRESUMO
Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes.
Assuntos
Enzimas Desubiquitinantes , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/genética , Humanos , Proteases Virais/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitinação , Animais , Replicação Viral , Antivirais/farmacologia , Inibidores de Proteases/farmacologia , Vírus/efeitos dos fármacos , Vírus/enzimologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Ubiquitina/metabolismo , Imunidade InataRESUMO
The Epstein-Barr virus (EBV) is implicated in several cancers, including EBV-associated gastric cancer (EBVaGC). This study focuses on EBV-encoded BALF1 (BamH1 A fragment leftward reading frame 1), a key apoptosis regulator in EBV-related cancers, whose specific impact on EBVaGC was previously unknown. Our findings indicate that BALF1 overexpression in gastric cancer cells significantly enhances their proliferation, migration, and resistance to chemotherapy-induced apoptosis, confirming BALF1's oncogenic potential. A novel discovery is that BALF1 undergoes degradation via the ubiquitin-proteasome pathway. Through analysis of 69 deubiquitinating enzymes (DUBs), ovarian tumor protease (OTU) domain-containing protein 1 (OTUD1) emerged as a vital regulator for maintaining BALF1 protein stability. Furthermore, BALF1 was found to play a role in regulating the stability of the B-cell lymphoma-2 (Bcl-2) protein, increasing its levels through deubiquitination. This mechanism reveals BALF1's multifaceted oncogenic role in gastric cancer, as it contributes both directly and indirectly to cancer progression, particularly by stabilizing Bcl-2, known for its anti-apoptotic characteristics. These insights significantly deepen our understanding of EBV's involvement in the pathogenesis of gastric cancer. The elucidation of OTUD1's role in BALF1 regulation and its influence on Bcl-2 stabilization provide new avenues for therapeutic intervention in EBVaGC, bridging the gap between viral oncogenesis and cellular protein regulation and offering a more holistic view of gastric cancer development under the influence of EBV.
Assuntos
Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Neoplasias Gástricas , Ubiquitinação , Humanos , Neoplasias Gástricas/patologia , Neoplasias Gástricas/virologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Linhagem Celular Tumoral , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proliferação de Células , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/genética , Estabilidade Proteica , Movimento Celular , Animais , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
Dysregulation of deubiquitination contributes to various diseases, including cancer, and aberrant expression of deubiquitinating enzymes is involved in carcinoma progression. As a member of the ovarian tumor (OTU) deubiquitinases, OTUD4 is considered a tumor suppressor in many kinds of malignancies. The biological characteristics and mechanisms of OTUD4 in clear cell renal cell carcinoma (ccRCC) remain unclear. The downregulation of OTUD4 in ccRCC was confirmed based on the TCGA database and a validation cohort of 30-paired ccRCC and para-carcinoma samples. Moreover, OTUD4 expression was detected by immunohistochemistry in 50 cases of ccRCC tissues, and patients with lower levels of OTUD4 showed larger tumor size (p = 0.015). TCGA data revealed that patients with high expression of OTUD4 had a longer overall survival rate. In vitro and in vivo studies revealed that downregulation of OTUD4 was essential for tumor cell growth and metastasis in ccRCC, and OTUD4 overexpression inhibited these malignant phenotypes. We further found that OTUD4 sensitized ccRCC cells to Erastin-induced ferroptosis, and ferrostain-1 inhibited OTUD4-induced ferroptotic cell death. Mechanistic studies indicated that OTUD4 functioned as an anti-proliferative and anti-metastasic factor through the regulation of RNA-binding protein 47 (RBM47)-mediated activating transcription factor 3 (ATF3). OTUD4 directly interacted with RBM47 and promoted its stability via deubiquitination events. RBM47 was critical in ccRCC progression by regulating ATF3 mRNA stability, thereby promoting ATF3-mediated ferroptosis. RBM47 interference abolished the suppressive role of OTUD4 overexpression in ccRCC. Our findings provide mechanistic insight into OTUD4 of ccRCC progression and indicate a novel critical pathway OTUD4/RBM47/ATF3 may serve as a potential therapeutic pathway for ccRCC.