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SARS-CoV-2 spike protein (SARS-2-S) induced cell-cell fusion in uninfected cells may occur in long COVID-19 syndrome, as circulating SARS-2-S or extracellular vesicles containing SARS-2-S (S-EVs) were found to be prevalent in post-acute sequelae of COVID-19 (PASC) for up to 12 months after diagnosis. Although isolated recombinant SARS-2-S protein has been shown to increase the SASP in senescent ACE2-expressing cells, the direct linkage of SARS-2-S syncytia with senescence in the absence of virus infection and the degree to which SARS-2-S syncytia affect pathology in the setting of cardiac dysfunction are unknown. Here, we found that the senescent outcome of SARS-2-S induced syncytia exacerbated heart failure progression. We first demonstrated that syncytium formation in cells expressing SARS-2-S delivered by DNA plasmid or LNP-mRNA exhibits a senescence-like phenotype. Extracellular vesicles containing SARS-2-S (S-EVs) also confer a potent ability to form senescent syncytia without de novo synthesis of SARS-2-S. However, it is important to note that currently approved COVID-19 mRNA vaccines do not induce syncytium formation or cellular senescence. Mechanistically, SARS-2-S syncytia provoke the formation of functional MAVS aggregates, which regulate the senescence fate of SARS-2-S syncytia by TNFα. We further demonstrate that senescent SARS-2-S syncytia exhibit shrinked morphology, leading to the activation of WNK1 and impaired cardiac metabolism. In pre-existing heart failure mice, the WNK1 inhibitor WNK463, anti-syncytial drug niclosamide, and senolytic dasatinib protect the heart from exacerbated heart failure triggered by SARS-2-S. Our findings thus suggest a potential mechanism for COVID-19-mediated cardiac pathology and recommend the application of WNK1 inhibitor for therapy especially in individuals with post-acute sequelae of COVID-19.
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COVID-19 , Senescência Celular , Células Gigantes , Insuficiência Cardíaca , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/virologia , Animais , Células Gigantes/virologia , Células Gigantes/metabolismo , Células Gigantes/patologia , COVID-19/metabolismo , COVID-19/complicações , COVID-19/virologia , COVID-19/patologia , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Camundongos , Vesículas Extracelulares/metabolismoRESUMO
PURPOSE: The overexpression of mitotic kinase monopolar spindle 1 (Mps1) has been identified in many tumor types, and targeting Mps1 for tumor therapy has shown great promise in multiple preclinical cancer models. However, the role played by Mps1 in tamoxifen (TAM) resistance in breast cancer has never been reported. METHODS: The sensitivity of breast cancer cells to tamoxifen was analysed in colony formation assays and wound healing assays. Enhanced transactivational activity of estrogen receptor α (ERα) led by Mps1 overexpression was determined by luciferase assays. The interaction between Mps1 and ERα was verified by co-immunoprecipitation and proximity ligation assay. Phosphorylation of ERα by Mps1 was detected by in vitro kinase assay and such phosphorylation process in vivo was proven by co-immunoprecipitation. The potential phosphorylation site(s) of ERα were analyzed by mass spectrometry. RESULTS: Mps1 determines the sensitivity of breast cancer cells to tamoxifen treatment. Mps1 overexpression rendered breast cancer cells more resistant to tamoxifen, while an Mps1 inhibitor or siMps1 oligos enabled cancer cells to overcome tamoxifen resistance. Mechanistically, Mps1 interacted with estrogen receptor α and stimulated its transactivational activity in a kinase activity-dependent manner. Mps1 was critical for ERα phosphorylation at Thr224 amino acid site. Importantly, Mps1 failed to enhance the transactivational activity of the ERα-T224A mutant. CONCLUSION: Mps1 contributes to tamoxifen resistance in breast cancer and is a potential therapeutic that can overcome tamoxifen resistance in breast cancer.
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Noticeable morbidity and mortality can be caused by influenza A virus in humans. Conventional live attenuated influenza vaccine (LAIV) is one of the main strategies to control the spread of influenza, but its protective efficacy is often limited by its suboptimal immunogenicity and safety. Therefore, a new type of LAIV that can overcome the shortage of existing vaccines is urgently needed. Here, we report a novel method to construct the recombinant influenza A virus (IAV) regulated by small molecules. By inserting 4-hydroxytamoxifen (4-HT)-dependent intein into the polymerase acidic (PA) protein of IAV, a series of 4-HT-dependent recombinant viruses were generated and screened. Among them, the S218 recombinant virus strain showed excellent 4-HT dependent replication characteristics both in vitro and in vivo. Further immunological evaluation indicated that the 4-HT-dependent viruses were highly attenuated in the host and could elicit robust humoral, mucosal, and cellular immunity against the challenge of homologous viruses. The attenuated strategies presented here could also be broadly applied to the development of vaccines against other pathogens.
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Vírus da Influenza A , Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Humanos , Inteínas , Vírus da Influenza A/genética , Vacinas AtenuadasRESUMO
Introduction: Inflammation play important roles in the initiation and progression of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), septic shock, clotting dysfunction, or even death associated with SARS-CoV-2 infection. However, the pathogenic mechanisms underlying SARS-CoV-2-induced hyperinflammation are still largely unknown. Methods: The animal model of septic shock and ALI was established after LPS intraperitoneal injection or intratracheal instillation. Bone marrow-derived macrophages (BMDMs) from WT and BPOZ-2 KO mouse strains were harvested from the femurs and tibias of mice. Immunohistology staining, ELISA assay, coimmunoprecipitation, and immunoblot analysis were used to detect the histopathological changes of lung tissues and the expression of inflammatory factors and protein interaction. Results and conclusions: We show a distinct mechanism by which the SARS-CoV-2 N (SARS-2-N) protein targets Bood POZ-containing gene type 2 (BPOZ-2), a scaffold protein for the E3 ubiquitin ligase Cullin 3 that we identified as a negative regulator of inflammatory responses, to promote NLRP3 inflammasome activation. We first demonstrated that BPOZ-2 knockout (BPOZ-2 KO) mice were more susceptible to lipopolysaccharide (LPS)-induced septic shock and ALI and showed increased serum IL-1ß levels. In addition, BMDMs isolated from BPOZ-2 KO mice showed increased IL-1ß production in response to NLRP3 stimuli. Mechanistically, BPOZ-2 interacted with NLRP3 and mediated its degradation by recruiting Cullin 3. In particular, the expression of BPOZ-2 was significantly reduced in lung tissues from mice infected with SARS-CoV-2 and in cells overexpressing SARS-2-N. Importantly, proinflammatory responses triggered by the SARS-2-N were significantly blocked by BPOZ-2 reintroduction. Thus, we concluded that BPOZ-2 is a negative regulator of the NLPR3 inflammasome that likely contributes to SARS-CoV-2-induced hyperinflammation.
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Lesão Pulmonar Aguda , COVID-19 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Nucleares , Choque Séptico , Animais , Camundongos , Lesão Pulmonar Aguda/metabolismo , Proteínas Culina , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , SARS-CoV-2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Golgi protein 73 (GP73), also called Golgi membrane protein 1 (GOLM1), is a resident Golgi type II transmembrane protein and is considered as a serum marker for the detection of a variety of cancers. A recent work revealed the role of the secreted GP73 in stimulating liver glucose production and systemic glucose homeostasis. Since exaggerated hepatic glucose production plays a key role in the pathogenesis of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), GP73 may thus represent a potential therapeutic target for treating diabetic patients with pathologically elevated levels. Here, in this study, we found that the circulating GP73 levels were significantly elevated in T2DM and positively correlated with hemoglobin A1c. Notably, the aberrantly upregulated GP73 levels were indispensable for the enhanced protein kinase A signaling pathway associated with diabetes. In diet-induced obese mouse model, GP73 siRNA primarily targeting liver tissue was potently effective in alleviating abnormal glucose metabolism. Ablation of GP73 from whole animals also exerted a profound glucose-lowering effect. Importantly, neutralizing circulating GP73 improved glucose metabolism in streptozotocin (STZ) and high-fat diet/STZ-induced diabetic mice. We thus concluded that GP73 was a feasible therapeutic target for the treatment of diabetes.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Camundongos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Experimental/patologia , Fígado/metabolismo , Glucose/metabolismo , HomeostaseRESUMO
As a critical immune checkpoint molecule, PD-L1 is expressed at significantly higher levels in multiple neoplastic tissues compared to normal ones. PD-L1/PD-1 axis is a critical target for tumor immunotherapy, blocking the PD-L1/PD-1 axis is recognized and has achieved unprecedented success in clinical applications. However, the clinical efficacy of therapies targeting the PD-1/PD-L1 pathway remains limited, emphasizing the need for the mechanistic elucidation of PD-1/PD-L1 expression. In this study, we found that RNF125 interacted with PD-L1 and regulated PD-L1 protein expression. Mechanistically, RNF125 promoted K48-linked polyubiquitination of PD-L1 and mediated its degradation. Notably, MC-38 and H22 cell lines with RNF125 knockout, transplanted in C57BL/6 mice, exhibited a higher PD-L1 level and faster tumor growth than their parental cell lines. In contrast, overexpression of RNF125 in MC-38 and H22 cells had the opposite effect, resulting in lower PD-L1 levels and delayed tumor growth compared with parental cell lines. In addition, immunohistochemical analysis of MC-38 tumors with RNF125 overexpression showed significantly increased infiltration of CD4+, CD8+ T cells and macrophages. Consistent with these findings, analyses using The Cancer Genome Atlas (TCGA) public database revealed a positive correlation of RNF125 expression with CD4+, CD8+ T cell and macrophage tumor infiltration. Moreover, RNF125 expression was significantly downregulated in several human cancer tissues, and was negatively correlated with the clinical stage of these tumors, and patients with higher RNF125 expression had better clinical outcomes. Our findings identify a novel mechanism for regulating PD-L1 expression and may provide a new strategy to increase the efficacy of immunotherapy.
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Severe cases of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with elevated blood glucose levels and metabolic complications. However, the molecular mechanisms for how SARS-CoV-2 infection alters glycometabolic control are incompletely understood. Here, we connect the circulating protein GP73 with enhanced hepatic gluconeogenesis during SARS-CoV-2 infection. We first demonstrate that GP73 secretion is induced in multiple tissues upon fasting and that GP73 stimulates hepatic gluconeogenesis through the cAMP/PKA signaling pathway. We further show that GP73 secretion is increased in cultured cells infected with SARS-CoV-2, after overexpression of SARS-CoV-2 nucleocapsid and spike proteins and in lungs and livers of mice infected with a mouse-adapted SARS-CoV-2 strain. GP73 blockade with an antibody inhibits excessive glucogenesis stimulated by SARS-CoV-2 in vitro and lowers elevated fasting blood glucose levels in infected mice. In patients with COVID-19, plasma GP73 levels are elevated and positively correlate with blood glucose levels. Our data suggest that GP73 is a glucogenic hormone that likely contributes to SARS-CoV-2-induced abnormalities in systemic glucose metabolism.
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COVID-19/complicações , COVID-19/virologia , Glucose/metabolismo , Hiperglicemia/etiologia , Hiperglicemia/metabolismo , Proteínas de Membrana/metabolismo , SARS-CoV-2 , Animais , Biomarcadores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Jejum , Expressão Gênica , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Interações Hospedeiro-Patógeno , Humanos , Hiperglicemia/sangue , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos/genéticaRESUMO
The prevalence of non-obese nonalcoholic fatty liver disease (NAFLD) is increasing worldwide with unclear etiology and pathogenesis. Here, we show GP73, a Golgi protein upregulated in livers from patients with a variety of liver diseases, exhibits Rab GTPase-activating protein (GAP) activity regulating ApoB export. Upon regular-diet feeding, liver-GP73-high mice display non-obese NAFLD phenotype, characterized by reduced body weight, intrahepatic lipid accumulation, and gradual insulin resistance development, none of which can be recapitulated in liver-GAP inactive GP73-high mice. Common and specific gene expression signatures associated with GP73-induced non-obese NAFLD and high-fat diet (HFD)-induced obese NAFLD are revealed. Notably, metformin inactivates the GAP activity of GP73 and alleviates GP73-induced non-obese NAFLD. GP73 is pathologically elevated in NAFLD individuals without obesity, and GP73 blockade improves whole-body metabolism in non-obese NAFLD mouse model. These findings reveal a pathophysiological role of GP73 in triggering non-obese NAFLD and may offer an opportunity for clinical intervention.
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Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/complicações , Fosfoproteínas/metabolismo , Animais , Apolipoproteína B-100/metabolismo , Peso Corporal , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Resistência à Insulina , Fígado/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfoproteínas/genética , TranscriptomaRESUMO
Introduction. Shigella flexneri is an intracellular bacterial pathogen that utilizes a type III secretion apparatus to inject effector proteins into host cells.Hypothesis/Gap Statement. The T3SS effector IpaH4.5 is important for the virulence of Shigella.Aim. This study aimed to elucidate the molecular mechanism and host target of the IpaH4.5 as well as its roles in S. flexneri infection.Methodology. The GAP assay was used to identify substrate Rab GTPases of IpaH4.5. A coimmunoprecipitation assay was applied to identify the interaction of Rab GTPases with IpaH4.5. A confocal microscopy analysis was used to assess the effects of IpaH4.5 on mannose 6-phosphate receptor (MPR) trafficking. To identify the effects of IpaH4.5 GAP activity on the activity of lysosomal cathepsin B, the Magic Red-RR assay was used. Finally, the intracellular persistence assay was used to identify IpaH4.5 GAP activity in S. flexneri intracellular growth.Results. We found that the effector IpaH4.5 disrupts MPR trafficking and lysosomal function, thereby counteracting host lysosomal degradation. IpaH4.5 harbours TBC-like dual-finger motifs and exhibits potent RabGAP activities towards Rab31. IpaH4.5 disrupts the transport of the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the Golgi to the endosome by targeting Rab31, thereby attenuating lysosomal function. As a result, the intracellular persistence of S. flexneri requires IpaH4.5 TBC-like GAP activity to mediate bacterial escape from host lysosome-mediated elimination.Conclusion. We identified an unknown function of IpaH4.5 and its potential role in S. flexneri infection.
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Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Lisossomos/metabolismo , Shigella flexneri/patogenicidade , Proteínas rab de Ligação ao GTP/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Catepsina B/metabolismo , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Shigella flexneri/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Responsible for the ongoing coronavirus disease 19 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding of the viral spike protein (SARS-2-S) to the cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Here we show that the high-density lipoprotein (HDL) scavenger receptor B type 1 (SR-B1) facilitates ACE2-dependent entry of SARS-CoV-2. We find that the S1 subunit of SARS-2-S binds to cholesterol and possibly to HDL components to enhance viral uptake in vitro. SR-B1 expression facilitates SARS-CoV-2 entry into ACE2-expressing cells by augmenting virus attachment. Blockade of the cholesterol-binding site on SARS-2-S1 with a monoclonal antibody, or treatment of cultured cells with pharmacological SR-B1 antagonists, inhibits HDL-enhanced SARS-CoV-2 infection. We further show that SR-B1 is coexpressed with ACE2 in human pulmonary tissue and in several extrapulmonary tissues. Our findings reveal that SR-B1 acts as a host factor that promotes SARS-CoV-2 entry and may help explain viral tropism, identify a possible molecular connection between COVID-19 and lipoprotein metabolism, and highlight SR-B1 as a potential therapeutic target to interfere with SARS-CoV-2 infection.
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COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno , Lipoproteínas HDL/metabolismo , SARS-CoV-2/fisiologia , Receptores Depuradores Classe B/metabolismo , Internalização do Vírus , Linhagem Celular , Colesterol/metabolismo , Suscetibilidade a Doenças , Humanos , Ligação Proteica , Receptores Virais , Glicoproteína da Espícula de Coronavírus/metabolismo , Tropismo Viral , Ligação ViralRESUMO
Activation of the NLRP3 inflammasome requires the expression of NLRP3, which is strictly regulated by its capacity to directly recognize microbial-derived substances. Even though the involvement of caspase-1 activation in macrophages via NLRP3 and NLRC4 has been discovered, the accurate mechanisms by which Shigella infection triggers NLRP3 activation remain inadequately understood. Here, we demonstrate that IpaH4.5, a Shigella T3SS effector, triggers inflammasome activation by regulating NLRP3 expression through the E3 ubiquitin ligase activity of IpaH4.5. First, we found that IpaH4.5 interacted with NLRP3. As a result, IpaH4.5 modulated NLRP3 protein stability and inflammasome activation. Bacteria lacking IpaH4.5 had dramatically reduced ability to induce pyroptosis. Our results identify a previously unrecognized target of IpaH4.5 in the regulation of inflammasome signaling and clarify the molecular basis for the cytosolic response to the T3SS effector.
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Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Shigella , Interleucina-1beta , Macrófagos , PiroptoseRESUMO
The Gram-negative bacterial pathogen Yersinia delivers six effector proteins into the host cells to block the host innate immune response. One of the effectors, YopT, is a potent cysteine protease that causes the disruption of the actin cytoskeleton to inhibit phagocytosis of the pathogen; however, its molecular mechanism and relevance to pathogenesis need further investigation. In this report, we show that RIG-I is a novel target of the YopT protein. Remarkably, YopT interacts with RIG-I and inhibits rat liver homogenate-mediated nuclear factor-κB and interferon regulatory factor-3 activation. Further studies revealed a YopT-dependent increase in the K48-polymerized ubiquitination of RIG-I. These findings suggest that YopT negatively regulates RIG-I-mediated cellular antibacterial response by targeting RIG-I.
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Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Fator Regulador 3 de Interferon/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Yersinia/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Cisteína Endopeptidases/genética , Células HEK293 , Humanos , Camundongos , NF-kappa B/genética , Fagocitose , Células RAW 264.7 , Fator de Transcrição RelA , Yersinia/genéticaRESUMO
Recent reports have shown the critical role of the mitochondrial antiviral signaling (MAVS) protein in virus-induced apoptosis, but the involvement of MAVS in tumorigenesis is still poorly understood. Herein, we report that MAVS is a key regulator of p53 activation and is critical for protecting against tumorigenesis. We find that MAVS promotes p53-dependent cell death in response to DNA damage. MAVS interacts with p53 and mediates p53 mitochondrial recruitment under genotoxic stress. Mechanistically, MAVS inhibits p53 ubiquitination by blocking the formation of the p53-murine double-minute 2 (MDM2) complex, leading to the stabilization of p53. Notably, compared with their wild-type littermates, MAVS knockout mice display decreased resistance to azoxymethane (AOM) or AOM/dextran sulfate sodium salt (DSS)-induced colon cancer. MAVS expression is significantly downregulated in human colon cancer tissues. These results unveil roles for MAVS in DNA damage response and tumor suppression.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteínas Mitocondriais/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Dano ao DNA , Progressão da Doença , Células HCT116 , Células HEK293 , Humanos , Inflamação/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Fenótipo , Estabilidade Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , UbiquitinaçãoRESUMO
Viral infection triggers the formation of mitochondrial antiviral signaling protein (MAVS) aggregates, which potently promote immune signaling. Autophagy plays an important role in controlling MAVS-mediated antiviral signaling; however, the exact molecular mechanism underlying the targeted autophagic degradation of MAVS remains unclear. Here, we investigated the mechanism by which RNF34 regulates immunity and mitophagy by targeting MAVS. RNF34 binds to MAVS in the mitochondrial compartment after viral infection and negatively regulates RIG-I-like receptor (RLR)-mediated antiviral immunity. Moreover, RNF34 catalyzes the K27-/K29-linked ubiquitination of MAVS at Lys 297, 311, 348, and 362 Arg, which serves as a recognition signal for NDP52-dependent autophagic degradation. Specifically, RNF34 initiates the K63- to K27-linked ubiquitination transition on MAVS primarily at Lys 311, which facilitates the autophagic degradation of MAVS upon RIG-I stimulation. Notably, RNF34 is required for the clearance of damaged mitochondria upon viral infection. Thus, we elucidated the mechanism by which RNF34-mediated autophagic degradation of MAVS regulates the innate immune response, mitochondrial homeostasis, and infection.
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Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Viroses/imunologia , Proteína DEAD-box 58/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata , Lisina/metabolismo , Mitofagia , Proteólise , Receptores Imunológicos , Transdução de Sinais , Células THP-1 , Ubiquitinação , Viroses/metabolismoRESUMO
The unfolded protein response (UPR) signal in tumor cells activates UPR signaling in neighboring macrophages, which leads to tumor-promoting inflammation by up-regulating UPR target genes and proinflammatory cytokines. However, the molecular basis of this endoplasmic reticulum (ER) stress transmission remains largely unclear. Here, we identified the secreted form of Golgi protein 73 (GP73), a Golgi-associated protein functional critical for hepatocellular carcinoma (HCC) growth and metastasis, is indispensable for ER stress transmission. Notably, ER stressors increased the cellular secretion of GP73. Through GRP78, the secreted GP73 stimulated ER stress activation in neighboring macrophages, which then released cytokines and chemokines involved in the tumor-associated macrophage (TAM) phenotype. Analysis of HCC patients revealed a positive correlation of GP73 with glucose-regulated protein 78 (GRP78) expression and TAM density. High GP73 and CD206 expression was associated with poor prognosis. Blockade of GP73 decreased the density of TAMs, inhibited tumor growth, and prolonged survival in two mouse HCC models. Conclusion: Our findings provide insight into the molecular mechanisms of extracellular GP73 in the amplification and transmission of ER stress signals.
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Carcinoma Hepatocelular/patologia , Estresse do Retículo Endoplasmático/genética , Neoplasias Hepáticas/genética , Fosfoproteínas/genética , Microambiente Tumoral/genética , Análise de Variância , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Xenoenxertos , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos , Transdução de Sinais/genética , Estatísticas não Paramétricas , Análise de Sobrevida , Regulação para Cima/genéticaRESUMO
Increased GP73 expression in hepatocytes from patients with acute hepatitis, through disease progression to cirrhosis and chronic liver disease suggests that progressive tissue remodeling and fibrogenesis are driving forces for GP73 upregulation. Nevertheless, details about regulation of GP73 expression and its biological functions remain elusive and await further characterization. In this study, we demonstrate that GP73 is a direct target of TGF-ß1 transcriptional regulation. Its induced expression inhibits TGF-ß-Smad mediated growth suppression. On the other hand, elevated GP73 results in upregulation of ERK/Akt signaling induced by TGF-ß1. Mechanistically, upregulation of lipid raft and caveolae-1 induced by GP73 overexpression mediates its regulatory effect on TGF-ß1 signaling. Notably, lipid raft expression is elevated in HCC tumors and tissues with higher GP73 expression yield more intensive Flotillin staining. Our results establish the linkage between GP73 and TGF-ß signaling, indicating that GP73 may promote HCC tumorigenesis by selectively regulating TGF-ß signaling through lipid raft modulation.
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Proteínas de Membrana/metabolismo , Transdução de Sinais , Proteínas Smad/antagonistas & inibidores , Fator de Crescimento Transformador beta1/fisiologia , Animais , Carcinoma Hepatocelular/metabolismo , Caveolina 1/metabolismo , Linhagem Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Transcrição GênicaRESUMO
Stringent control of the type I interferon signaling pathways is critical to effective host immune responses, however, the molecular mechanisms that negatively regulate these pathways are still poorly understood. Here, we show that apoptosis speck-like protein (ASC), an adaptor protein of inflammasome complex, can inhibit IFN-ß signaling response by interacting with mitochondrial antiviral signaling protein (MAVS). Importantly, ASC-specific siRNA knockdown enhanced virus-induced type I interferon production, with consequent reduction of virus replication. Taken together, these results suggest that ASC, as a negative regulator of the MAVS-mediated innate immunity, may play an important role in host protection upon virus infection.
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Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Adaptadoras de Sinalização CARD/fisiologia , Imunidade Inata/fisiologia , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Inflamassomos/fisiologia , Interferon beta/genética , Interferon beta/metabolismo , Células MCF-7 , Camundongos , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Viroses/imunologia , Viroses/prevenção & controle , Replicação Viral/fisiologiaRESUMO
Elevated Golgi phosphoprotein 2 (GP73, also known as GOLPH2 or GOLM1) expression in serum and liver, which can be induced by viral infection and cytokine treatments, is intimately connected with liver disease, including acute hepatitis, cirrhosis and hepatocellular carcinoma (HCC). However, its pathogenic roles in hepatic diseases have never been clarified in detail. Here, we showed that the upregulated GP73 is indispensable for SREBPs activation and lipogenesis. Notably, GP73 overexpression enhanced SCAP-SREBPs binding and its Golgi trafficking even under cholesterol sufficiency. Consistent with these functional findings, GP73 blockage could alleviate tunicamycin-induced liver steatosis by reducing SREBPs activation. A significant positive correlation of GP73 with genes in lipid metabolism pathway was also identified in liver cancer based on data from The Cancer Genome Atlas (TCGA) dataset. Our findings revealed previously unrecognized role of GP73 in lipid metabolism.
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Fígado Gorduroso/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Animais , Fígado Gorduroso/patologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Ativação Transcricional , Regulação para CimaRESUMO
Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily controlling energy homeostasis; however, its precise role in regulating antiviral innate immunity remains to be clarified. Here, we showed that ERRα deficiency conferred resistance to viral infection both in vivo and in vitro. Mechanistically, ERRα inhibited the production of type-I interferon (IFN-I) and the expression of multiple interferon-stimulated genes (ISGs). Furthermore, we found that viral infection induced TBK1-dependent ERRα stabilization, which in turn associated with TBK1 and IRF3 to impede the formation of TBK1-IRF3, IRF3 phosphorylation, IRF3 dimerization, and the DNA binding affinity of IRF3. The effect of ERRα on IFN-I production was independent of its transcriptional activity and PCG-1α. Notably, ERRα chemical inhibitor XCT790 has broad antiviral potency. This work not only identifies ERRα as a critical negative regulator of antiviral signaling, but also provides a potential target for future antiviral therapy.