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OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.
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Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Endopeptidases/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Poliubiquitina/metabolismo , Adulto , Animais , Endopeptidases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Fator 2 de Diferenciação de Crescimento/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Mutantes , Mutação , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Telangiectasia Hemorrágica Hereditária , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The phosphoinositide 3-kinase (PI3K)-Akt axis is one of the most frequently activated pathways and is demonstrated as a therapeutic target in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC). Targeting the PI3K-Akt pathway has been a challenging undertaking through the decades. Here we unveiled an essential role of E3 ligase SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated phosphoinositide-dependent protein kinase 1 (PDK1) neddylation in PI3K-Akt signaling and tumorigenesis. Upon growth factor stimulation, Smurf1 immediately triggers PDK1 neddylation and the poly-neural precursor cell expressed developmentally downregulated protein 8 (poly-Nedd8) chains recruit methyltransferase SET domain bifurcated histone lysine methyltransferase 1 (SETDB1). The cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS) consisting of PDK1, Smurf1 and SETDB1 directs Akt membrane attachment and T308 phosphorylation. Smurf1 deficiency dramatically reduces CRC tumorigenesis in a genetic mouse model. Furthermore, we developed a highly selective Smurf1 degrader, Smurf1-antagonizing repressor of tumor 1, which exhibits efficient PDK1-Akt blockade and potent tumor suppression alone or combined with PDK1 inhibitor in KRAS-mutated CRC. The findings presented here unveil previously unrecognized roles of PDK1 neddylation and offer a potential strategy for targeting the PI3K-Akt pathway and KRAS mutant cancer therapy.
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BACKGROUND & AIMS: Liver fibrosis is an intrinsic wound-healing response to chronic injury and the major cause of liver-related morbidity and mortality worldwide. However, no effective diagnostic or therapeutic strategies are available, owing to its poorly characterized molecular etiology. We aimed to elucidate the mechanisms underlying liver fibrogenesis. METHODS: We performed a quantitative proteomic analysis of clinical fibrotic liver samples to identify dysregulated proteins. Further analyses were performed on the sera of 164 patients with liver fibrosis. Two fibrosis mouse models and several biochemical experiments were used to elucidate liver fibrogenesis. RESULTS: We identified cathepsin S (CTSS) up-regulation as a central node for extracellular matrix remodeling in the human fibrotic liver by proteomic screening. Increased serum CTSS levels efficiently predicted liver fibrosis, even at an early stage. Secreted CTSS cleaved collagen 18A1 at its C-terminus, releasing endostatin peptide, which directly bound to and activated hepatic stellate cells via integrin α5ß1 signaling, whereas genetic ablation of Ctss remarkably suppressed liver fibrogenesis via endostatin reduction in vivo. Further studies identified macrophages as the main source of hepatic CTSS, and splenectomy effectively attenuated macrophage infiltration and CTSS expression in the fibrotic liver. Pharmacologic inhibition of CTSS ameliorated liver fibrosis progression in the mouse models. CONCLUSIONS: CTSS functions as a novel profibrotic factor by remodeling extracellular matrix proteins and may represent a promising target for the diagnosis and treatment of liver fibrosis.
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Endostatinas , Proteômica , Camundongos , Animais , Humanos , Endostatinas/metabolismo , Endostatinas/farmacologia , Fígado/metabolismo , Cirrose Hepática/metabolismo , Fibrose , Modelos Animais de Doenças , Células Estreladas do Fígado/metabolismo , Matriz Extracelular , Macrófagos/metabolismoRESUMO
As an important post-translational modification, ubiquitination mediates â¼80% of protein degradation in eukaryotes. The degree of protein ubiquitination is tightly determined by the delicate balance between specific ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase-mediated deubiquitination. In 2017, we developed UbiBrowser 1.0, which is an integrated database for predicted human proteome-wide E3-substrate interactions. Here, to meet the urgent requirement of proteome-wide E3/deubiquitinase-substrate interactions (ESIs/DSIs) in multiple organisms, we updated UbiBrowser to version 2.0 (http://ubibrowser.ncpsb.org.cn). Using an improved protocol, we collected 4068/967 known ESIs/DSIs by manual curation, and we predicted about 2.2 million highly confident ESIs/DSIs in 39 organisms, with >210-fold increase in total data volume. In addition, we made several new features in the updated version: (i) it allows exploring proteins' upstream E3 ligases and deubiquitinases simultaneously; (ii) it has significantly increased species coverage; (iii) it presents a uniform confidence scoring system to rank predicted ESIs/DSIs. To facilitate the usage of UbiBrowser 2.0, we also redesigned the web interface for exploring these known and predicted ESIs/DSIs, and added functions of 'Browse', 'Download' and 'Application Programming Interface'. We believe that UbiBrowser 2.0, as a discovery tool, will contribute to the study of protein ubiquitination and the development of drug targets for complex diseases.
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Bases de Dados Genéticas , Enzimas Desubiquitinantes/genética , Software , Ubiquitina-Proteína Ligases/genética , Enzimas Desubiquitinantes/classificação , Células Eucarióticas/metabolismo , Proteoma/genética , Especificidade por Substrato/genética , Ubiquitina-Proteína Ligases/classificaçãoRESUMO
BACKGROUND: Renal ischemia-reperfusion injury (IRI) is one reason for renal transplantation failure. Recent studies have shown that mitochondrial dynamics is closely related to IRI, and that inhibition or reversal of mitochondrial division protects organs against IRI. Optic atrophy protein 1 (OPA1), an important factor in mitochondrial fusion, has been shown to be upregulated by sodium-glucose cotransporter 2 inhibitor (SGLT2i). Also, the antiinflammatory effects of SGLT2i have been demonstrated in renal cells. Thus, we hypothesized that empagliflozin could prevent IRI through inhibiting mitochondrial division and reducing inflammation. METHODS: Using hematoxylin-eosin staining, enzyme linked immunosorbent assay (ELISA), flow cytometry, immunofluorescent staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, real-time PCR, RNA-sequencing, and western blot, we analyzed renal tubular tissue from in vivo and in vitro experiments. RESULTS: Through animal experiments and sequencing analysis, we first confirmed the protection against IRI and the regulation of mitochondrial dynamics-related factors and inflammatory factors by empagliflozin pretreatment. Then, through hypoxia/reoxygenation (H/R) cellular experiments, we confirmed that empagliflozin could inhibit mitochondrial shortening and division and upregulate OPA1 in human renal tubular epithelial cell line (HK-2) cells. Subsequently, we knocked down OPA1, and mitochondrial division and shortening were observed, which could be alleviated by empagliflozin treatment. Combined with the previous results, we concluded that OPA1 downregulation leads to mitochondrial division and shortening, and empagliflozin can alleviate the condition by upregulating OPA1. We further explored the pathway through which empagliflozin functions. Related studies have shown the activation of AMPK pathway by empagliflozin and the close correlation between the AMPK pathway and OPA1. In our study, we blocked the AMPK pathway, and OPA1 upregulation by empagliflozin was not observed, thus demonstrating the dependence of empagliflozin on the AMPK pathway. CONCLUSION: The results indicated that empagliflozin could prevent or alleviate renal IRI through antiinflammatory effects and the AMPK-OPA1 pathway. Ischemia-reperfusion injury is an inevitable challenge in organ transplantation. It is necessary to develop a new therapeutic strategy for IRI prevention in addition to refining the transplantation process. In this study, we confirmed the preventive and protective effects of empagliflozin in renal ischemia-reperfusion injury. Based on these findings, empagliflozin is promising to be a preventive agent for renal ischemia-reperfusion injury and can be applied for preemptive administration in kidney transplantation.
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Dinâmica Mitocondrial , Traumatismo por Reperfusão , Animais , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Rim , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Apoptose , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/farmacologiaRESUMO
Smad ubiquitination regulatory factor 1 (Smurf1) and Smurf2 are HECT-type E3 ubiquitin ligases, and both Smurfs were initially identified to regulate Smad protein stability in the TGF-ß/BMP signaling pathway. In recent years, Smurfs have exhibited E3 ligase-dependent and -independent activities in various kinds of cells. Smurfs act as either potent tumor promoters or tumor suppressors in different tumors by regulating biological processes, including metastasis, apoptosis, cell cycle, senescence and genomic stability. The regulation of Smurfs activity and expression has therefore emerged as a hot spot in tumor biology research. Further, the Smurf1- or Smurf2-deficient mice provide more in vivo clues for the functional study of Smurfs in tumorigenesis and development. In this review, we summarize these milestone findings and, in turn, reveal new avenues for the prevention and treatment of cancer by regulating Smurfs.
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Neoplasias/patologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Genes Supressores de Tumor , Humanos , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
INTRODUCTION: Bone loss is a major health concern for astronauts during long-term spaceflight and for patients during prolonged bed rest or paralysis. It is essential to develop therapeutic strategies to combat the bone loss occurring in people afflicted with disuse atrophy on earth as well as in astronauts in space, especially during prolonged missions. Although several drugs have been demonstrated for treating postmenopausal osteoporosis or bone-related diseases, their effects on microgravity-induced bone loss are still unclear. MATERIALS AND METHODS: Here, we employed the hindlimb-unloading (HLU) tail suspension model and compared the preventive efficiencies of five agents including alendronate (ALN), raloxifene (Rox), teriparatide (TPTD), anti-murine RANKL monoclonal antibody (anti-RANKL) and proteasome inhibitor bortezomib (Bzb) on mechanical unloading-induced bone loss. Bone mineral density (BMD) was measured by quantitative computed tomography. The osteoblastic and osteoclastic activity were measured by serum ELISA, histology analysis, and histomorphometric analysis. RESULTS: Compared to the control, ALN and anti-RANKL antibody could restore bone mass close to sham levels by inhibiting bone resorption. Bzb could increase the whole bone mass and strength by inhibiting bone resorption and promoting bone formation simultaneously. Meanwhile, Rox did not affect bone loss caused by HLU. TPTD stimulated cortical bone formation but the total bone mass was not increased significantly. CONCLUSIONS: We demonstrated for the first time that anti-RANKL antibody and Bzb had a positive effect on preventing mechanical unloading-induced bone loss. This finding puts forward the potential use of anti-RANKL and Bzb on bone loss therapies or prophylaxis of astronauts in spaceflight.
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Reabsorção Óssea , Teriparatida , Animais , Anticorpos Monoclonais , Densidade Óssea , Reabsorção Óssea/tratamento farmacológico , Bortezomib , Humanos , CamundongosRESUMO
OBJECTIVE: Myelin oligodendrocyte glycoprotein-associated disorders (MOGADs) are a rare new neurological autoimmune disease with unclear pathogenesis. Since a linkage of the disease to the human leucocyte antigen (HLA) has not been shown, we here investigated whether MOGAD is associated with the HLA locus. METHODS: HLA genotypes of 95 patients with MOGADs, assessed between 2016 and 2018 from three academic centres, were compared with 481 healthy Chinese Han individuals. Patients with MOGADs included 51 paediatric-onset and 44 adult-onset cases. All patients were seropositive for IgG targeting the myelin oligodendrocyte glycoprotein (MOG). RESULTS: Paediatric-onset MOGAD was associated with the DQB1*05:02-DRB1*16:02 alleles (OR=2.43; OR=3.28) or haplotype (OR=2.84) of HLA class II genes. The prevalence of these genotypes in patients with paediatric-onset MOGAD was significantly higher than healthy controls (padj=0.0154; padj=0.0221; padj=0.0331). By contrast, adult-onset MOGAD was not associated with any HLA genotype. Clinically, patients with the DQB1*05:02-DRB1*16:02 haplotype exhibited significantly higher expanded disability status scale scores at onset (p=0.004) and were more likely to undergo a disease relapse (p=0.030). HLA-peptide binding prediction algorithms and computational docking analysis provided supporting evidence for the close relationship between the MOG peptide subunit and DQB1*05:02 allele. In vitro results indicated that site-specific mutations of the predicted target sequence reduced the antigen-antibody binding, especially in the paediatric-onset group with DQB1*05:02 allele. CONCLUSIONS: This study demonstrates a possible association between specific HLA class II alleles and paediatric-onset MOGAD, providing evidence for the conjecture that different aetiology and pathogenesis likely underlie paediatric-onset and adult-onset cases of MOGAD.
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Doenças Autoimunes/genética , Genótipo , Antígenos HLA/genética , Glicoproteína Mielina-Oligodendrócito/imunologia , Adolescente , Adulto , Idoso , Alelos , Doenças Autoimunes/imunologia , Criança , Pré-Escolar , China , Estudos de Coortes , Feminino , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The bone morphogenetic protein (BMP)-Smad signaling pathway plays a crucial role in the control of bone homeostasis by regulating osteoblast activity. It is known that the ubiquitin ligase Smad ubiquitination regulatory factor (Smurf)1 is a master negative regulator of BMP signaling, but how its stability and activity are regulated remains poorly understood. Our study showed that valosin-containing protein/p97, the mutations of which lead to rare forms of Paget's disease of bone (PDB)-like syndrome-such as inclusion body myopathy (IBM) associated with Paget's disease of bone and frontotemporal dementia (IBM-PFD)-together with its adaptor nuclear protein localization (NPL)4, specifically interact with Smurf1 and deliver the ubiquitinated Smurf1 for degradation. Depletion of either p97 or NPL4 resulted in the elevation of Smurf1 protein level and decreased BMP signaling accordingly. Mechanically, a typical proline, glutamic acid, serine, and threonine motif specifically existing in Smurf1 is necessary for its recognition and degradation by p97, and this process is dependent on p97 ATPase activity. More importantly, compared with p97 WT, PDB-associated mutation of p97 (mainly A232E) harboring the higher ATPase activity of p97 further promoted Smurf1 degradation, thus increasing BMP signaling activity. Our findings first establish a link between p97 and Smurf1, providing an in-depth understanding of how Smurf1 is regulated, as well as the mechanism of p97-related bone diseases.-Li, H., Cui, Y., Wei, J., Liu, C., Chen, Y., Cui, C.-P., Li, L., Zhang, X., Zhang, L. VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation.
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Adenosina Trifosfatases/metabolismo , Proteína Morfogenética Óssea 1/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/metabolismo , Adenosina Trifosfatases/genética , Proteína Morfogenética Óssea 1/genética , Humanos , Mutação , Proteínas Nucleares/genética , Osteíte Deformante/genética , Osteíte Deformante/metabolismo , Osteíte Deformante/patologia , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Proteína com Valosina/genéticaRESUMO
Stem cells can remain quiescent, self-renewal, and differentiate into many types of cells and even cancer stem cells. The coordination of these complex processes maintains the homeostasis of the organism. Ubiquitination is an important posttranslational modification process that regulates protein stability and activity. The ubiquitination levels of stem cell-associated proteins are closely related with stem cell characteristics. Cullin-RING Ligases (CRLs) are the largest family of E3 ubiquitin ligases, accounting for approximately 20% of proteins degraded by proteasome. In this review, we discuss the role of CRLs in stem cell homeostasis, self-renewal, and differentiation and expound their ubiquitination substrates. In addition, we also discuss the effect of CRLs on the formation of cancer stem cells that may provide promising therapy strategies for cancer.
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Proteínas Culina/metabolismo , Células-Tronco/metabolismo , Animais , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Células-Tronco/citologia , UbiquitinaçãoRESUMO
The tumor suppressor phosphatase and tensin homolog (PTEN) plays a central role in regulating phosphatidylinositol 3-kinase (PI3K) signaling, and its gene is very frequently mutated in various human cancers. Numerous studies have revealed that PTEN levels are tightly regulated by both transcriptional and posttranslational modifications, with especially ubiquitylation significantly regulating PTEN protein levels. Although several ubiquitin ligases have been reported to mediate PTEN ubiquitylation in vitro, the ubiquitin ligase that promotes PTEN degradation in vivo has not been reported. Here we took advantage of specific knockout mouse models to demonstrate that WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) promotes PTEN degradation under physiological conditions, whereas another ubiquitin ligase, carboxyl terminus of Hsp70-interacting protein (CHIP), had no such effect. WWP2 knockout mice exhibited reduced body size, elevated PTEN protein levels, and reduced phosphorylation levels of the serine/threonine kinase and PTEN target AKT. In contrast, we observed no elevation of PTEN protein levels in CHIP knockout tissues and mouse embryonic fibroblasts. Furthermore, PTEN protein levels in CHIP/WWP2 double knockout mice were very similar to those in WWP2 single knockout mice and significantly higher than in WT and CHIP knockout mice. Our results demonstrate that WWP2, rather than CHIP, is an ubiquitin ligase that promotes PTEN degradation in vivo Considering PTEN's significant role in tumor development, we propose that WWP2 may be a potential target for fine-tuning PTEN levels in anticancer therapies.
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PTEN Fosfo-Hidrolase/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genéticaRESUMO
Testosterone is essential for spermatogenesis and the maintenance of secondary sexual characteristics in males. An important transcription factor, LIM-homeobox gene 9 (Lhx9) is indispensable for testis development and testosterone production; however, post-translational modifications of Lhx9 are largely unknown. Here, for the first time to our knowledge, we demonstrate that the level of Lhx9 protein increases in human chorionic gonadotropin-exposed Leydig cells and can be polyubiquitylated. We found that Smad ubiquitylation regulatory factor 1 (Smurf1), an E3 ubiquitin ligase, targets Lhx9 for ubiquitin-mediated proteasome degradation, thereby negatively modulating its function. Increasing Smurf1 decreases the level of Lhx9 and inhibits the Lhx9 transactivation capacity of steroidogenic factor 1 [nuclear receptor subfamily 5, group A, member 1 (NR5A1)]. In contrast, the depletion of Smurf1 leads to increased expression of Lhx9 protein and enhances testosterone biosynthesis-related gene transcripts [NR5A1, steroidogenic acute regulatory protein, CYP17A1, hydroxy-δ-5-steroid dehydrogenase, hydroxy-δ-5-steroid dehydrogenase isomerase 6, and hydroxysteroid (17-ß) dehydrogenase 3] and testosterone production in Leydig cells. Furthermore, we found that Smurf1 knockout mice exhibit higher levels of Lhx9 protein and steroidogenesis, which leads to increased serum testosterone concentration. These findings reveal that Smurf1 promotes Lhx9 ubiquitylation and is involved in testosterone production in Leydig cells directly. Our results provide new insights into the molecular events that play a role in the homeostasis of testosterone levels and may provide a new target for testosterone regulation.-Hu, F., Zhu, Q., Sun, B., Cui, C., Li, C., Zhang, L. Smad ubiquitylation regulatory factor 1 promotes LIM-homeobox gene 9 degradation and represses testosterone production in Leydig cells.
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Proteínas com Homeodomínio LIM/genética , Células Intersticiais do Testículo/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Animais , Genes Homeobox/genética , Humanos , Masculino , Camundongos Knockout , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Testosterona/metabolismo , Testosterona/farmacologia , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: We investigated the effect and mechanism of hypoxic microenvironment and hypoxia-inducible factors (HIFs) on hepatocellular carcinoma (HCC) cancer stemness. DESIGN: HCC cancer stemness was analysed by self-renewal ability, chemoresistance, expression of stemness-related genes and cancer stem cell (CSC) marker-positive cell population. Specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) mRNA level was examined with quantitative PCR in human paired HCCs. Immunoprecipitation was used to examine the binding of proteins and chromatin immunoprecipitation assay to detect the binding of HIFs with hypoxia response element sequence. In vivo characterisation was performed in immunocompromised mice and stem cell frequency was analysed. RESULTS: We showed that hypoxia enhanced the stemness of HCC cells and hepatocarcinogenesis through enhancing HIF-1α deSUMOylation by SENP1 and increasing stabilisation and transcriptional activity of HIF-1α. Furthermore, we demonstrated that SENP1 is a direct target of HIF-1/2α and a previously unrecognised positive feedback loop exists between SENP1 and HIF-1α. CONCLUSIONS: Taken together, our findings suggest the significance of this positive feedback loop between HIF-1α and SENP1 in contributing to the increased cancer stemness in HCC and hepatocarcinogenesis under hypoxia. Drugs that specifically target SENP1 may offer a potential novel therapeutic approach for HCC.
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Carcinoma Hepatocelular/metabolismo , Cisteína Endopeptidases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína SUMO-1/metabolismo , Animais , Western Blotting , Carcinoma Hepatocelular/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Imunoprecipitação , Neoplasias Hepáticas/patologia , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/patologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microambiente TumoralRESUMO
The deregulation of HGF/c-Met signaling is implicated in epithelial-mesenchymal transition (EMT) and progress of hepatocellular carcinoma (HCC). However, the epigenetic mechanisms that HGF/c-Met regulates EMT and metastasis of HCC cells are less explored. In this study, we demonstrated that HCC cells express a high level of SUMO/sentrin-specific protease 1 (Senp1) which is induced by HGF/c-Met signals. Lentivirus-mediated small hairpin RNA (shRNA) transduction results in Senp1 silence in HCC cells. Senp1 silence reduces the HGF-induced proliferation and migration of HCC cells. Senp1 inhibition also induces HCC cell apoptosis and growth arrest. Furthermore, Senp1 knockdown inhibits epithelial-to-mesenchymal transition, with increase of E-cadherin and ZO-1 expression, decrease of fibronectin and N-cadherin expression. The EMT-related transcription factor Zeb1 was SUMO-modified and decreased in Senp1-silenced HCC cells. These results delineate that senp1 might play an important role in the regulation of HGF-induced invasion and migration of HCC cells.
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Carcinoma Hepatocelular/patologia , Endopeptidases/fisiologia , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/fisiologia , Apoptose , Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Pontos de Checagem do Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Cisteína Endopeptidases , Endopeptidases/genética , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-met/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Sumoilação , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
AIM: miRNA-486 (miR-486) was first identified from the human fetal liver cDNA library and considered to be associated with hepatocellular carcinoma (HCC) development. Its roles in regulation of HCC metastasis and chemosensitivity have not been explored yet. METHODS: miR-486 expression in HCC tissues, cell lines and serum was evaluated by real-time polymerase chain reaction. miR-486 overexpression or downregulation in the cell lines SMMC-7721/LM3 was conducted by lentivirus transfection. Cell proliferation, migration and apoptosis were quantitated using commercial assays. Matrix metalloproteinase activity was quantitated by gelatin zymography. The target genes of miR-486 were screened by 3'-untranslated region luciferase report assays and their function was validated by small RNA interference. RESULTS: We show here that miR-486 is frequently down-expressed in HCC tissues and cell lines. Lentivirus-mediated restoration of miR-486 in HCC cell lines resulted in significant reduction in the ability of cell growth, colony formation and migration. However, miR-486 inhibition enhances proliferation and invasion of HCC cells. Two genes, CITRON and CLDN10 which regulate cell proliferation and invasion, respectively, were identified as the direct targets of miR-486 in HCC cells. CITRON and CLDN10 knockdown by siRNA results in similar phenotypes of miR-486 restoration in HCC cell lines. In addition, miR-486 enhances the chemosensitivity of HCC cells to sorafenib. CONCLUSION: Our data indicated that miR-486 may function as a novel tumor suppressor in HCC.
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The hepatic growth factor hepatopoietin Cn (HPPCn) prevents liver injury induced by carbon tetrachloride in rats. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK). S1P and S1P receptors (S1PRs) are involved in liver fibrogenesis and oxidative injury. This work sought to understand the mechanism by which SphK/S1P/S1PRs are involved in the protective effects of HPPCn on ethanol-induced liver injury and fibrosis. Transgenic mice with liver-specific overexpression of HPPCn (HPPCn(liver) (+/+)) were generated. Two ethanol feeding protocols were used to assess the protective effect of HPPCn on acute and chronic liver injury in mice. Specific inhibitors of S1PR1, S1PR2 and S1PR3 and siRNA were used to examine the roles of S1PRs in hepatic stellate cell (HSC) activation and hepatocyte apoptosis. Increased HPPCn expression in transgenic mice attenuated fibrosis induced by ethanol and carbon tetrachloride (CCl4). Treatment with recombinant human HPPCn prevented human hepatocyte apoptosis and HSC activation. JTE-013 or S1PR2-siRNA attenuated the effect of HPPCn on HSC activation induced by tumour necrosis factor-α (TNF-α). Consistent with the effect of N,N-dimethylsphingosine (DMS), suramin or S1PR3-siRNA treatment blocked HPPCn-induced Erk1/2 phosphorylation in human hepatocytes. This study demonstrated that HPPCn attenuated oxidative injury and fibrosis induced by ethanol feeding and that the SphK1/S1P/S1PRs signalling pathway contributes to the protective effect of HPPCn on hepatocyte apoptosis and HSC activation.
Assuntos
Etanol , Fator de Crescimento de Hepatócito/metabolismo , Cirrose Hepática Alcoólica/prevenção & controle , Fígado/enzimologia , Proteínas Nucleares/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Células Estreladas do Fígado/enzimologia , Células Estreladas do Fígado/patologia , Fator de Crescimento de Hepatócito/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Alcoólica/enzimologia , Cirrose Hepática Alcoólica/etiologia , Cirrose Hepática Alcoólica/genética , Cirrose Hepática Alcoólica/patologia , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/genética , Estresse Oxidativo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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
Linear ubiquitination catalyzed by HOIL-1-interacting protein (HOIP), the key component of the linear ubiquitination assembly complex, plays fundamental roles in tissue homeostasis by executing domain-specific regulatory functions. However, a proteome-wide analysis of the domain-specific interactome of HOIP across tissues is lacking. Here, we present a comprehensive mass spectrometry-based interactome profiling of four HOIP domains in nine mouse tissues. The interaction dataset provides a high-quality HOIP interactome resource with an average of approximately 90 interactors for each bait per tissue. HOIP tissue interactome presents a systematic understanding of linear ubiquitination functions in each tissue and also shows associations of tissue functions to genetic diseases. HOIP domain interactome characterizes a set of previously undefined linear ubiquitinated substrates and elucidates the cross-talk among HOIP domains in physiological and pathological processes. Moreover, we show that linear ubiquitination of Integrin-linked protein kinase (ILK) decreases focal adhesion formation and promotes the detachment of Shigella flexneri-infected cells. Meanwhile, Hoip deficiency decreases the linear ubiquitination of Smad ubiquitination regulatory factor 1 (SMURF1) and enhances its E3 activity, finally causing a reduced bone mass phenotype in mice. Overall, our work expands the knowledge of HOIP-interacting proteins and provides a platform for further discovery of linear ubiquitination functions in tissue homeostasis.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Camundongos , Homeostase , NF-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The ubiquitin-proteasome system (UPS) controls protein turnover, and its dysfunction contributes to human diseases including cancer. Deubiquitinating enzymes (DUBs) remove ubiquitin from proteins to maintain their stability. Inhibition of DUBs could induce the degradation of selected oncoproteins and has therefore become a potential therapeutic strategy for cancer. The deubiquitylase OTUD3 was reported to promote lung tumorigenesis by stabilizing oncoprotein GRP78, implying that inhibition of OTUD3 may be a therapeutic strategy for lung cancer. Here, we report a small-molecule inhibitor of OTUD3 (named OTUDin3) by computer-aided virtual screening and biological experimental verification. OTUDin3 exhibited pronounced antiproliferative and proapoptotic effects by inhibiting deubiquitinating activity of OTUD3 in non-small-cell lung cancer (NSCLC) cell lines. Moreover, OTUDin3 efficaciously inhibited growth of lung cancer xenografts in mice. In summary, our results support OTUDin3 as a potent inhibitor of OTUD3, the inhibition of which may be a promising therapeutic strategy for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Camundongos , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , CarcinogêneseRESUMO
Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Dysregulation of this process leads to multiple diseases, including osteoporosis. However, the underlying molecular mechanisms are not fully understood. Here, we show that the global and conditional osteoblast knockout of a deubiquitinase Otub1 result in low bone mass and poor bone strength due to defects in osteogenic differentiation and mineralization. Mechanistically, the stability of FGFR2, a crucial regulator of osteogenesis, is maintained by OTUB1. OTUB1 attenuates the E3 ligase SMURF1-mediated FGFR2 ubiquitination by inhibiting SMURF1's E2 binding. In the absence of OTUB1, FGFR2 is ubiquitinated excessively by SMURF1, followed by lysosomal degradation. Consistently, adeno-associated virus serotype 9 (AAV9)-delivered FGFR2 in knee joints rescued the bone mass loss in osteoblast-specific Otub1-deleted mice. Moreover, Otub1 mRNA level was significantly downregulated in bones from osteoporotic mice, and restoring OTUB1 levels through an AAV9-delivered system in ovariectomy-induced osteoporotic mice attenuated osteopenia. Taken together, our results suggest that OTUB1 positively regulates osteogenic differentiation and mineralization in bone homeostasis by controlling FGFR2 stability, which provides an optical therapeutic strategy to alleviate osteoporosis.