RESUMO
The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.
Assuntos
Alelos , Influenza Humana , Macrófagos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Transativadores , Humanos , Transativadores/genética , Transativadores/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Macrófagos/metabolismo , Células U937 , Influenza Humana/genética , Sítios de Ligação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Regulação da Expressão GênicaRESUMO
This study is to elucidate the effect of the LINC00663/EBF1/NR2F1 axis on inflammation and angiogenesis in bladder cancer (BC) and related molecular mechanisms. After transfection, functional experiments were conducted to test cell proliferation and invasion, tube formation ability, and content of inflammatory factors, Snail, E-cadherin, and VEGFA. Meanwhile, the relationships among LINC00663, EBF1, and NR2F1 were predicted and verified. In addition, xenograft experiments in nude mice were performed to observe the oncogenicity of 5637 BC cells in vivo. In BC tissues and cells, LINC00663 and NR2F1 were upregulated. Silencing NR2F1 or LINC00663 repressed cell proliferation and invasion, weakened vascular mimicry in vitro, decreased inflammatory factor, Snail, and VEGFA levels, and increased expression of E-cadherin. LINC00663 positively regulated NR2F1 expression through EBF1. Additionally, in vivo experiments showed that NR2F1 upregulation reversed the suppression effects of LINC00663 silencing on tumour growth, inflammation, and angiogenesis. Silencing LINC00663 decreased NR2F1 expression by mediating EBF1, thereby inhibiting BC inflammation and angiogenesis.
Assuntos
Fator I de Transcrição COUP , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inflamação , Neovascularização Patológica , RNA Longo não Codificante , Transativadores , Neoplasias da Bexiga Urinária , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Animais , Camundongos , Linhagem Celular Tumoral , Fator I de Transcrição COUP/metabolismo , Fator I de Transcrição COUP/genética , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , Transativadores/metabolismo , Transativadores/genética , Feminino , Masculino , Camundongos Nus , Inativação Gênica , Movimento Celular , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , AngiogêneseRESUMO
Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.
Assuntos
Linfócitos T CD8-Positivos , Citoesqueleto , Interleucina-2 , Camundongos Endogâmicos C57BL , Fator de Resposta Sérica , Transativadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/metabolismo , Interleucina-2/genética , Animais , Transativadores/metabolismo , Transativadores/genética , Citoesqueleto/metabolismo , Camundongos , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/genética , Listeriose/microbiologia , Actinas/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Camundongos Knockout , Proliferação de Células , Ativação LinfocitáriaRESUMO
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
Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.
Assuntos
Proteínas de Bactérias , Infecções Estafilocócicas , Staphylococcus aureus , Testosterona , Masculino , Testosterona/farmacologia , Testosterona/metabolismo , Animais , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência , Infecções Estafilocócicas/microbiologia , Transativadores/genética , Transativadores/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Simulação de Acoplamento Molecular , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Abscesso/microbiologia , Hemólise , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genéticaRESUMO
Rationale: Regulatory processes of transcription factors (TFs) shape heart development and influence the adult heart's response to stress, contributing to cardiac disorders. Despite their significance, the precise mechanisms underpinning TF-mediated regulation remain elusive. Here, we identify that EBF1, as a TF, is highly expressed in human heart tissues. EBF1 is reported to be associated with human cardiovascular disease, but its roles are unclear in heart. In this study, we investigated EBF1 function in cardiac system. Methods: RNA-seq was utilized to profile EBF1 expression patterns. CRISPR/Cas9 was utilized to knock out EBF1 to investigate its effects. Human pluripotent stem cells (hPSCs) differentiated into cardiac lineages were used to mimic cardiac development. Cardiac function was evaluated on mouse model with Ebf1 knockout by using techniques such as echocardiography. RNA-seq was conducted to analyze transcriptional perturbations. ChIP-seq was employed to elucidate EBF1-bound genes and the underlying regulatory mechanisms. Results: EBF1 was expressed in some human and mouse cardiomyocyte. Knockout of EBF1 inhibited cardiac development. ChIP-seq indicated EBF1's binding on promoters of cardiogenic TFs pivotal to cardiac development, facilitating their transcriptional expression and promoting cardiac development. In mouse, Ebf1 depletion triggered transcriptional perturbations of genes, resulting in cardiac remodeling. Mechanistically, we found that EBF1 directly bound to upstream chromatin regions of cardiac hypertrophy-inducing genes, contributing to cardiac hypertrophy. Conclusions: We uncover the mechanisms underlying EBF1-mediated regulatory processes, shedding light on cardiac development, and the pathogenesis of cardiac remodeling. These findings emphasize EBF1's critical role in orchestrating diverse aspects of cardiac processes and provide a promising therapeutic intervention for cardiomyopathy.
Assuntos
Perfilação da Expressão Gênica , Miócitos Cardíacos , Transativadores , Animais , Humanos , Camundongos , Transativadores/genética , Transativadores/metabolismo , Miócitos Cardíacos/metabolismo , Diferenciação Celular/genética , Coração/fisiopatologia , Camundongos Knockout , Células-Tronco Pluripotentes/metabolismo , Transcriptoma/genética , Sistemas CRISPR-Cas/genéticaRESUMO
Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.
Assuntos
Proteínas de Bactérias , Percepção de Quorum , Proteínas Recombinantes , Streptococcus thermophilus , Percepção de Quorum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Camundongos , Regulação Bacteriana da Expressão Gênica , Transativadores/genética , Transativadores/metabolismo , Feromônios/metabolismo , Feromônios/genéticaRESUMO
Selective elimination of cancer cells without causing deleterious effects on normal cells is an ideal anti-cancer strategy. Here, using Drosophila cancer model, we performed an in vivo RNAi screen for anti-cancer targets that selectively eliminate tumors without affecting normal tissue growth. In Drosophila imaginal epithelium, clones of cells expressing oncogenic Ras with simultaneous mutations in the cell polarity gene scribble (RasV12/scrib-/-) develop into malignant tumors. We found that knockdown of Crk, the Drosophila ortholog of human CRK (CT10 regulatory kinase) and CRKL (Crk-like) adapter proteins, significantly suppresses growth of RasV12/scrib-/- tumors by inducing c-Jun N-terminal kinase (JNK)-mediated apoptosis, while it does not affect growth of normal epithelium. Mechanistically, Crk inhibition blocks Yorkie (Yki)/YAP activity by impairing F-actin accumulation, an upstream event of Yki/YAP activation in tumors. Inhibition of Yki/YAP in tumors causes intracellular JNK signaling to be used for apoptosis induction. Given that molecules and signaling pathways identified in Drosophila are highly conserved and activated in human cancers, our findings would provide a novel, to the best of our knowledge, anti-cancer strategy against YAP-activated cancers.
Assuntos
Apoptose , Proteínas de Drosophila , Transativadores , Proteínas de Sinalização YAP , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Transativadores/metabolismo , Transativadores/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Drosophila melanogaster/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Drosophila/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas , Staphylococcus aureus , Transativadores , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/imunologia , Staphylococcus aureus/genética , Camundongos , Animais , Transativadores/genética , Transativadores/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/imunologia , Pele/microbiologia , Pele/patologia , Pele/imunologia , Fatores de Virulência/genética , Humanos , Infecções dos Tecidos Moles/microbiologia , Infecções dos Tecidos Moles/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Citocinas/imunologia , Citocinas/genéticaRESUMO
As an essential transcriptional activator, PDX1 plays a crucial role in pancreatic development and ß-cell function. Mutations in the PDX1 gene may lead to type 4 maturity-onset diabetes of the young (MODY4) and neonatal diabetes mellitus. However, the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models. In this study, three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology, all of which succumbed shortly postpartum, exhibiting pancreatic agenesis. Notably, one tri-allelic PDX1-mutant cynomolgus macaque (designated as M4) developed a pancreas, whereas the two mono-allelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation. RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions, indicating developmental delay and PDX1 haploinsufficiency. A marked change in m6A methylation was identified in the M4 pancreas, confirmed through cultured PDX1-mutant islet organoids. Notably, overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids. This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.
Assuntos
Proteínas de Homeodomínio , Macaca fascicularis , Pâncreas , Transativadores , Animais , Macaca fascicularis/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mutação , Metilação , Feminino , Pancreatopatias/genética , Pancreatopatias/veterinária , Masculino , Doenças dos Macacos/genéticaRESUMO
The calmodulin-binding transcriptional activator (CAMTA) is a small, conserved gene family in plants that plays a crucial role in regulating growth, development, and responses to various abiotic stress. Given the significance of the CAMTA gene family, various studies have been dedicated to uncovering its functional characteristics. In this study, genome-wide identification and bioinformatics analysis were conducted to explore CAMTAs in Phoebe bournei. A total of 17 CAMTA genes, each containing at least one domain from CG-1, TIG, ANK, or IQ, were identified in the P. bournei genome. The diversity of PbCAMTAs could be varied depending on their subcellular localization. An analysis of protein motifs, domains, and gene structure revealed that members within the same subgroup exhibited similar organization, supporting the results of the phylogenetic analysis. Gene duplications occurred among members of the PbCAMTA gene family. According to the cis-regulatory element prediction and protein-protein interaction network analysis, eight genes were subjected to qRT-PCR under drought, heat, and light stresses. The expression profiles indicated that PbCAMTAs, particularly PbCAMTA2, PbCAMTA12, and PbCAMTA16, were induced by abiotic stress. This study provides profound insights into the functions of CAMTAs in P. bournei.
Assuntos
Secas , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas , Estresse Fisiológico , Estresse Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Luz , Transativadores/genética , Transativadores/metabolismo , Temperatura AltaRESUMO
Epithelioid hemangioendothelioma (EHE) is a rare malignant vascular tumor arising from vascular endothelial cells. This study delves into the molecular mechanisms underlying EHE, with a specific focus on the Hippo-YAP/TAZ pathway. EHE is characterized molecularly by transcriptional co-activator with a PDZ-motif (TAZ)-calmodulin binding transcription activator 1 (CAMTA1) or Yes-associated protein (YAP)-transcription factor E3 (TFE3) fusions. YAP/TAZ, a transcription co-activator, binds to transcription factors and regulates gene expression. The YAP/TAZ and its upstream Hippo pathway are involved in cell proliferation and cell contact inhibition, regulating organ size and carcinogenesis. In addition to oncogenic effects, dysfunction or gene duplication of the Hippo pathway results in a poor prognosis due to epithelial-mesenchymal transformation of epithelial cells, stem cell transformation, and increased drug resistance. Notably, the TAZ-CAMTA1 fusion is specific to EHE, and genetic alterations in the Hippo pathway other than this fusion gene are absent in EHE. The TAZ-CAMTA1 fusion is a promising therapeutic target. This review summarizes recent advances in EHE, focusing on the role of the Hippo-YAP/TAZ pathway in EHE and its potential as a therapeutic target for drug development.
Assuntos
Hemangioendotelioma Epitelioide , Via de Sinalização Hippo , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Humanos , Hemangioendotelioma Epitelioide/metabolismo , Hemangioendotelioma Epitelioide/patologia , Hemangioendotelioma Epitelioide/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Transativadores/metabolismo , Transativadores/genética , Proteínas de Sinalização YAP/metabolismo , Terapia de Alvo Molecular , AnimaisRESUMO
Mammalian Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) and Drosophila Yorkie (Yki) are transcription cofactors of the highly conserved Hippo signaling pathway. It has been long assumed that the YAP/TAZ/Yki signaling drives cell proliferation during organ growth. However, its instructive role in regulating developmentally programmed organ growth, if any, remains elusive. Out-of-context gain of YAP/TAZ/Yki signaling often turns oncogenic. Paradoxically, mechanically strained, and differentiated squamous epithelia display developmentally programmed constitutive nuclear YAP/TAZ/Yki signaling. The unknown, therefore, is how a growth-promoting YAP/TAZ/Yki signaling restricts proliferation in differentiated squamous epithelia. Here, we show that reminiscent of a tumor suppressor, Yki negatively regulates the cell growth-promoting PI3K/Akt/TOR signaling in the squamous epithelia of Drosophila tubular organs. Thus, downregulation of Yki signaling in the squamous epithelium of the adult male accessory gland (MAG) up-regulates PI3K/Akt/TOR signaling, inducing cell hypertrophy, exit from their cell cycle arrest, and, finally, culminating in squamous cell carcinoma (SCC). Thus, blocking PI3K/Akt/TOR signaling arrests Yki loss-induced MAG-SCC. Further, MAG-SCCs, like other lethal carcinomas, secrete a cachectin, Impl2-the Drosophila homolog of mammalian IGFBP7-inducing cachexia and shortening the lifespan of adult males. Moreover, in the squamous epithelium of other tubular organs, like the dorsal trunk of larval tracheal airways or adult Malpighian tubules, downregulation of Yki signaling triggers PI3K/Akt/TOR-induced cell hypertrophy. Our results reveal that Yki signaling plays an instructive, antiproliferative role in the squamous epithelia of tubular organs.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Proteínas Nucleares , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Transativadores , Proteínas de Sinalização YAP , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Transativadores/metabolismo , Transativadores/genética , Masculino , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Epitélio/metabolismo , Proliferação de Células , Fosfatidilinositol 3-Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Hepatitis B virus (HBV) infection remains a major public health concern worldwide, with approximately 296 million individuals chronically infected. The HBV-encoded X protein (HBx) is a regulatory protein of 17 kDa, reportedly responsible for a broad range of functions, including viral replication and oncogenic processes. In this review, we summarize the state of knowledge on the mechanisms underlying HBx functions in viral replication, the antiviral effect of therapeutics directed against HBx, and the role of HBx in liver cancer development (including a hypothetical model of hepatocarcinogenesis). We conclude by highlighting major unanswered questions in the field and the implications of their answers.
Assuntos
Vírus da Hepatite B , Neoplasias Hepáticas , Transativadores , Proteínas Virais Reguladoras e Acessórias , Replicação Viral , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Humanos , Transativadores/metabolismo , Transativadores/genética , Vírus da Hepatite B/fisiologia , Vírus da Hepatite B/genética , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/metabolismo , Hepatite B/virologia , Hepatite B/complicações , Hepatite B Crônica/virologia , Hepatite B Crônica/complicaçõesRESUMO
During embryonic development, Wnt signaling influences both proliferation and sensory formation in the cochlea. How this dual nature of Wnt signaling is coordinated is unknown. In this study, we define a novel role for a Wnt-regulated gene, Mybl2, which was already known to be important for proliferation, in determining the size and patterning of the sensory epithelium in the murine cochlea. Using a quantitative spatial analysis approach and analyzing Mybl2 loss-of-function, we show that Mybl2 promoted proliferation in the inner sulcus domain but limited the size of the sensory domain by influencing their adjoining boundary position via Jag1 regulation during development. Mybl2 loss-of-function simultaneously decreased proliferation in the inner sulcus and increased the size of the sensory domain, resulting in a wider sensory epithelium with ectopic inner hair cell formation during late embryonic stages. These data suggest that progenitor cells in the inner sulcus determine boundary formation and pattern the sensory epithelium via MYBL2.
Assuntos
Proliferação de Células , Cóclea , Proteína Jagged-1 , Células-Tronco , Animais , Cóclea/embriologia , Cóclea/citologia , Cóclea/metabolismo , Camundongos , Epitélio/embriologia , Epitélio/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Regulação da Expressão Gênica no Desenvolvimento , Via de Sinalização Wnt , Padronização Corporal/genética , Transativadores/metabolismo , Transativadores/genética , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/citologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Fold-switching enables metamorphic proteins to reversibly interconvert between two highly dissimilar native states to regulate their protein functions. While about 100 proteins have been identified to undergo fold-switching, unveiling the key residues behind this mechanism for each protein remains challenging. Reasoning that fold-switching in proteins is driven by dynamic changes in local energetic frustration, we combined fold-switching simulations generated using simplified structure-based models with frustration analysis to identify key residues involved in this process based on the change in the density of minimally frustrated contacts during refolding. Using this approach to analyze the fold-switch of the bacterial transcription factor RfaH, we identified 20 residues that significantly change their frustration during its fold-switch, some of which have been experimentally and computationally reported in previous works. Our approach, which we developed as an additional module for the FrustratometeR package, highlights the role of local frustration dynamics in protein fold-switching and offers a robust tool to enhance our understanding of other proteins with significant conformational shifts.
Assuntos
Proteínas de Escherichia coli , Dobramento de Proteína , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transativadores/química , Transativadores/metabolismo , Transativadores/genética , Simulação de Dinâmica Molecular , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Modelos Moleculares , Conformação Proteica , TermodinâmicaRESUMO
Esophageal fibrosis can develop due to caustic or radiation injuries. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are known to mitigate fibrosis in various organs. However, the potential effects of UC-MSCs on human esophageal fibrosis remain underexplored. This study investigated the anti-fibrogenic properties and mechanisms of UC-MSC-derived conditioned media (UC-MSC-CM) on human esophageal fibroblasts (HEFs). HEFs were treated with TGF-ß1 and then cultured with UC-MSC-CM, and the expression levels of extracellular matrix (ECM) components, RhoA, myocardin related transcription factor A (MRTF-A), serum response factor (SRF), Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) were measured. UC-MSC-CM suppressed TGF-ß1-induced fibrogenic activation in HEFs, as evidenced by the downregulation of ECM. UC-MSC-CM diminished the expression of RhoA, MRTF-A, and SRF triggered by TGF-ß1. In TGF-ß1-stimulated HEFs, UC-MSC-CM decreased the nuclear localization of MRTF-A and YAP. Additionally, UC-MSC-CM diminished the TGF-ß1-induced nuclear expressions of YAP and TAZ, while concurrently enhancing the cytoplasmic presence of phosphorylated YAP. Furthermore, UC-MSC-CM reduced TGF-ß1-induced phosphorylation of Smad2. These findings suggest that UC-MSC-CM may inhibit TGF-ß1-induced fibrogenic activation in HEFs by targeting the Rho-mediated MRTF/SRF and YAP/TAZ pathways, as well as the Smad2 pathway. This indicates its potential as a stem cell therapy for esophageal fibrosis.
Assuntos
Esôfago , Fibroblastos , Fibrose , Células-Tronco Mesenquimais , Transativadores , Fatores de Transcrição , Fator de Crescimento Transformador beta1 , Proteína rhoA de Ligação ao GTP , Humanos , Células-Tronco Mesenquimais/metabolismo , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Esôfago/metabolismo , Esôfago/citologia , Fibroblastos/metabolismo , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Cordão Umbilical/citologia , Proteínas de Sinalização YAP/metabolismo , Fator de Resposta Sérica/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transdução de Sinais , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Proteína Smad2/metabolismoRESUMO
Background: Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in ß-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with ß-thalassemia/HbE. However, the expression and function of miR-155, especially in ß-thalassemia, have not yet been explored. Methods: To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from ß IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. Results: miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in ß-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in ß-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in ß-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. Conclusion: miR-155 regulates IE in ß-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.
Assuntos
Eritropoese , MicroRNAs , Talassemia beta , MicroRNAs/genética , MicroRNAs/metabolismo , Eritropoese/genética , Animais , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia , Camundongos , Humanos , Masculino , Diferenciação Celular , Feminino , Eritroblastos/metabolismo , Eritroblastos/patologia , Transativadores/genética , Transativadores/metabolismo , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Adulto , Adolescente , Proliferação de Células , Proteínas Proto-Oncogênicas , Fatores de Transcrição de Zíper de Leucina BásicaRESUMO
AIMS: Cardiac fibrosis is characterized by aberrant collagen deposition in the heart. Macrophage polarization or infiltration is the main reason to accelerate the collagen deposition. We attempted to investigate the involvement of MKL1 in macrophages during the development of cardiac fibrosis. MATERIALS AND METHODS: Cardiac fibrosis is induced by myocardial infarction (MI). The MKL1f/f mice were crossed to the Lyz2-cre mice to generate macrophage conditional MKL1 knockout mice (MKL1ΔMφ). In addition, macrophage conditional MKL1 overexpression mice (MKL1MÏ-OE) were constructed by crossing Lyz2-cre mice to MKL1ΔN200-Rosa26 mice. KEY FINDINGS: MKL1 expression was significantly increased in macrophages of both ischemic cardiomyopathy (ICM) patients and mice induced to develop myocardial infarction. Deletion of MKL1 in macrophages improved the heart function after MI-induced cardiac fibrosis. Consistently, MKL1MÏ-OE mice displayed more severe cardiac fibrosis and worsened heart function than the control mice after MI. Moreover, administration of a small-molecule MKL1 inhibitor CCG-1423 also decreased the collagen deposition after MI. SIGNIFICANCE: Our data demonstrate that MKL1 in macrophages contributes to cardiac fibrosis pathogenesis and reinforce the notion that targeting MKL1 may yield effective antifibrotic therapeutics in the heart.
Assuntos
Modelos Animais de Doenças , Fibrose , Macrófagos , Infarto do Miocárdio , Transativadores , Animais , Humanos , Masculino , Camundongos , Colágeno/metabolismo , Fibrose/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/metabolismo , Transativadores/metabolismo , Transativadores/genéticaRESUMO
Diabetes mellitus, a chronic and non-transmissible disease, triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ß-like cells (PßLCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways, directing the differentiation of iPSCs into pancreatic ß cells. Despite significant progress and improved protocols, the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here, we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into PßLCs. Our protocol has shown to be effective, with the resulting cells exhibiting key functional properties of pancreatic ß cells, including the expression of crucial molecular markers (pdx1, nkx6.1, ngn3) and the capability to secrete insulin in response to glucose. Furthermore, the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ß cell genes.