RESUMEN
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.
Asunto(s)
ARN Polimerasa Dependiente de ARN de Coronavirus , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico , Interacciones Microbiota-Huesped , SARS-CoV-2 , Proteínas no Estructurales Virales , Replicación Viral , Humanos , Adenosina Trifosfatasas/metabolismo , Antivirales/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , COVID-19/virología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Helicasas/metabolismo , Concentración 50 Inhibidora , ARN Helicasas/metabolismo , SARS-CoV-2/clasificación , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , SARS-CoV-2/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Femenino , Animales , RatonesRESUMEN
Hepatocellular carcinoma (HCC) exhibits a high mortality rate due to its high invasion and metastatic nature, and the acidic microenvironment plays a pivotal role. Acid-sensing ion channel 1 (ASIC1) is upregulated in HCC tissues and facilitates tumor progression in a pH-dependent manner, while the specific mechanisms therein remain currently unclear. Herein, we aimed to investigate the underlying mechanisms by which ASIC1 contributes to the development of HCC. Using bioinformatics analysis, we found a significant association between ASIC1 expression and malignant transformation of HCC, such as poor prognosis, metastasis and recurrence. Specifically, ASIC1 enhanced the migration and invasion capabilities of Li-7 cells in the in vivo experiment using an HCC lung metastasis mouse model, as well as in the in vitro experiments such as wound healing assay and Transwell assay. Furthermore, our comprehensive gene chip and molecular biology experiments revealed that ASIC1 promoted HCC migration and invasion by activating the PRKACA/AP-1 signaling pathway. Our findings indicate that targeting ASIC1 could have therapeutic potential for inhibiting HCC progression.
Asunto(s)
Canales Iónicos Sensibles al Ácido , Carcinoma Hepatocelular , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , Invasividad Neoplásica , Transducción de Señal , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Canales Iónicos Sensibles al Ácido/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Animales , Humanos , Ratones , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/genética , Línea Celular Tumoral , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Masculino , Pronóstico , Proliferación CelularRESUMEN
INTRODUCTION: Previous studies have demonstrated that Dual-specificity phosphatase 4 (DUSP4) plays an important role in the progression of different tumor types. However, the role and mechanism of DUSP4 in colorectal cancer (CRC) remain unclear. AIMS: We investigate the role and mechanisms of DUSP4 in CRC. METHODS: Immunohistochemistry was used to investigate DUSP4 expression in CRC tissues. Cell proliferation, apoptosis and migration assays were used to validate DUSP4 function in vitro and in vivo. RNA-sequence assay was used to identify the target genes of DUSP4. Human phosphokinase array and inhibitor assays were used to explore the downstream signaling of DUSP4. RESULTS: DUSP4 expression was upregulated in CRC tissues relative to normal colorectal tissues, and DUSP4 expression showed a significant positive correlation with CRC stage. Consistently, we found that DUSP4 was highly expressed in colorectal cancer cells compared to normal cells. DUSP4 knockdown inhibits CRC cell proliferation, migration and promotes apoptosis. Furthermore, the ectopic expression of DUSP4 enhanced CRC cell proliferation, migration and diminished apoptosis in vitro and in vivo. Human phosphokinase array data showed that ectopic expression of DUSP4 promotes CREB activation. RNA-sequencing data showed that PRKACB acts as a downstream target gene of DUSP4/CREB and enhances CREB activation through PKA/cAMP signaling. In addition, xenograft model results demonstrated that DUSP4 promotes colorectal tumor progression via PRKACB/CREB activation in vivo. CONCLUSION: These findings suggest that DUSP4 promotes CRC progression. Therefore, it may be a promising therapeutic target for CRC.
Asunto(s)
Apoptosis , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Fosfatasas de Especificidad Dual , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Animales , Femenino , Masculino , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Regulación Neoplásica de la Expresión Génica , Ratones , Línea Celular Tumoral , Ratones Desnudos , Persona de Mediana Edad , Transducción de SeñalRESUMEN
Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-ß4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-ß4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-ß4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.
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Simulación de Dinámica Molecular , Regulación Alostérica , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Conformación Proteica , Unión Proteica , Nucleótidos/metabolismo , Especificidad por Sustrato , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genéticaRESUMEN
BACKGROUND: Neuroendocrine prostate cancer (NEPC), a lethal subset of prostate cancer (PCa), is characterized by loss of AR signaling and resistance to AR-targeted therapy. While it is well reported that second-generation AR blockers induce neuroendocrine (NE) trans-differentiation of castration-resistant prostate cancer (CRPC) to promote the occurrence of NEPC, and pluripotent transcription factors might be potential regulators, the underlying molecular mechanisms remain unclear. METHODS: We analyzed the data from public databsets to screen candidate genes and then focused on SOX4, a regulator of NE trans-differentiation. The expression changes of SOX4 and its relationship with tumor progression were validated in clinical tumor tissues. We evaluated malignant characteristics related to NEPC in prostate cancer cell lines with stable overexpression or knockdown of SOX4 in vitro. Tumor xenografts were analyzed after inoculating the relevant cell lines into nude mice. RNA-seq, ATAC-seq, non-targeted metabolomics analysis, as well as molecular and biochemical assays were carried out to determine the mechanism. RESULTS: We screened public datasets and identified that expression of SOX4 was significantly elevated in NEPC. Overexpressing SOX4 in C4-2B cells increased cell proliferation and migration, upregulated the expression of NE marker genes, and inhibited AR expression. Consistently, inhibition of SOX4 expression in DU-145 and PC-3 cells reduced the above malignant phenotypes and repressed the expression of NE marker genes. For the in vivo assay, we found that knockdown of SOX4 inhibited tumor growth of subcutaneous xenografts in castrated nude mice which were concomitantly treated with enzalutamide (ENZ). Mechanically, we identified that one of the key enzymes in gluconeogenesis, PCK2, was a novel target of SOX4. The activation of carbohydrate metabolism reprogramming by SOX4 could promote NE trans-differentiation via the SOX4/PCK2 pathway. CONCLUSIONS: Our findings reveal that SOX4 promotes NE trans-differentiation both in vitro and in vivo via directly enhancing PCK2 activity to activate carbohydrate metabolism reprogramming. The SOX4/PCK2 pathway and its downstream changes might be novel targets for blocking NE trans-differentiation.
Asunto(s)
Transdiferenciación Celular , Neoplasias de la Próstata Resistentes a la Castración , Factores de Transcripción SOXC , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Línea Celular Tumoral , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Ratones Desnudos , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismoRESUMEN
Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by the fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug-response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype. Significance: Alterations leading to unconstrained protein kinase A signaling, regardless of the presence or absence of PRKACA fusions, drive the phenotypes of fibrolamellar hepatocellular carcinoma, reshaping understanding of the pathogenesis of this rare liver cancer.
Asunto(s)
Carcinoma Hepatocelular , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico , Proteínas del Choque Térmico HSP40 , Neoplasias Hepáticas , Humanos , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Regulación Neoplásica de la Expresión Génica , ATPasa Intercambiadora de Sodio-PotasioRESUMEN
Post-operative cognitive dysfunction (POCD) is a common complication after surgery due to the usage of anesthetics, such as Sevoflurane, which severely impacts the life quality of patients. Currently, the pathogenesis of Sevoflurane-induced POCD has not been fully elucidated but is reportedly involved with oxidative stress (OS) injury and aggravated inflammation. Phoenixin-20 (PNX-20) is a PNX peptide consisting of 20 amino acids with promising inhibitory effects on OS and inflammation. Herein, we proposed to explore the potential protective function of PNX-20 on Sevoflurane inhalation-induced POCD in rats. Sprague-Dawley (SD) rats were treated with 100 ng/g PNX-20 for 7 days with or without pre-inhalation with 2.2% Sevoflurane. Markedly increased escape latency and decreased time in the target quadrant in the Morris water maze (MWM) test, and aggravated pathological changes and apoptosis in the hippocampus tissue were observed in Sevoflurane-treated rats, which were markedly attenuated by PNX-20. Furthermore, the aggravated inflammation and OS in the hippocampus observed in Sevoflurane-treated rats were notably abolished by PNX-20. Moreover, the brain-derived neurotrophic factor (BDNF), protein kinase A (PKA), and phospho-cAMP response element binding protein/cAMP response element binding protein (p-CREB/CREB) levels were markedly decreased in Sevoflurane-treated rats, which were memorably increased by PNX-20. Our results indicated that PNX-20 ameliorated Sevoflurane inhalation-induced POCD in rats via the activation of PKA/CREB signaling, which might supply a new treatment approach for POCD.