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1.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203974

RESUMO

In the two decades since the discovery of TNNI3K it has been implicated in multiple cardiac phenotypes and physiological processes. TNNI3K is an understudied kinase, which is mainly expressed in the heart. Human genetic variants in TNNI3K are associated with supraventricular arrhythmias, conduction disease, and cardiomyopathy. Furthermore, studies in mice implicate the gene in cardiac hypertrophy, cardiac regeneration, and recovery after ischemia/reperfusion injury. Several new papers on TNNI3K have been published since the last overview, broadening the clinical perspective of TNNI3K variants and our understanding of the underlying molecular biology. We here provide an overview of the role of TNNI3K in cardiomyopathy and arrhythmia covering both a clinical perspective and basic science advancements. In addition, we review the potential of TNNI3K as a target for clinical treatments in different cardiac diseases.


Assuntos
Cardiopatias/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Modelos Animais de Doenças , Cardiopatias/genética , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Humanos , Terapia de Alvo Molecular , Proteínas Serina-Treonina Quinases/genética , Regeneração
2.
Front Immunol ; 12: 662989, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34084167

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of current COVID-19 pandemic, and insufficient production of type I interferon (IFN-I) is associated with the severe forms of the disease. Membrane (M) protein of SARS-CoV-2 has been reported to suppress host IFN-I production, but the underlying mechanism is not completely understood. In this study, SARS-CoV-2 M protein was confirmed to suppress the expression of IFNß and interferon-stimulated genes induced by RIG-I, MDA5, IKKϵ, and TBK1, and to inhibit IRF3 phosphorylation and dimerization caused by TBK1. SARS-CoV-2 M could interact with MDA5, TRAF3, IKKϵ, and TBK1, and induce TBK1 degradation via K48-linked ubiquitination. The reduced TBK1 further impaired the formation of TRAF3-TANK-TBK1-IKKε complex that leads to inhibition of IFN-I production. Our study revealed a novel mechanism of SARS-CoV-2 M for negative regulation of IFN-I production, which would provide deeper insight into the innate immunosuppression and pathogenicity of SARS-CoV-2.


Assuntos
Interferon Tipo I/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , SARS-CoV-2/imunologia , Ubiquitina/metabolismo , Proteínas da Matriz Viral/imunologia , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Proteólise , Receptores Imunológicos/metabolismo , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo
3.
Nat Commun ; 12(1): 3392, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099666

RESUMO

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.


Assuntos
Efeito Espectador/genética , Fator de Transcrição E2F1/metabolismo , MicroRNAs/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Efeito Espectador/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Fator de Transcrição E2F1/genética , Estresse do Retículo Endoplasmático/imunologia , Endorribonucleases/metabolismo , Proteína HMGB1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Listeria monocytogenes/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Shigella flexneri/imunologia , Suínos
4.
Nat Commun ; 12(1): 3691, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140527

RESUMO

The HIV-1 accessory proteins Vif, Vpu, and Nef can promote infection by overcoming the inhibitory effects of the host cell restriction factors APOBEC3G, Tetherin, and SERINC5, respectively. However, how the HIV-1 accessory protein Vpr enhances infection in macrophages but not in CD4+ T cells remains elusive. Here, we report that Vpr counteracts lysosomal-associated transmembrane protein 5 (LAPTM5), a potent inhibitor of HIV-1 particle infectivity, to enhance HIV-1 infection in macrophages. LAPTM5 transports HIV-1 envelope glycoproteins to lysosomes for degradation, thereby inhibiting virion infectivity. Vpr counteracts the restrictive effects of LAPTM5 by triggering its degradation via DCAF1. In the absence of Vpr, the silencing of LAPTM5 precisely phenocopied the effect of Vpr on HIV-1 infection. In contrast, Vpr did not enhance HIV-1 infection in the absence of LAPTM5. Moreover, LAPTM5 was highly expressed in macrophages but not in CD4+ T lymphocytes. Re-expressing LAPTM5 reconstituted the Vpr-dependent promotion of HIV-1 infection in primary CD4+ T cells, as observed in macrophages. Herein, we demonstrate the molecular mechanism used by Vpr to overcome LAPTM5 restriction in macrophages, providing a potential strategy for anti-HIV/AIDS therapeutics.


Assuntos
Infecções por HIV/metabolismo , HIV-1/metabolismo , Interações entre Hospedeiro e Microrganismos , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Membrana/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Inativação Gênica , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , HIV-2/metabolismo , HIV-2/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/genética , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima , Vírion/metabolismo
5.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063046

RESUMO

Receptor-like kinases (RLKs) constitute a large group of cell surface receptors that play crucial roles in multiple biological processes. However, the function of most RLKs in plants has not been extensively explored, and much less for the class of cell wall associated kinases (WAKs) and WAK-like kinases (WAKLs). In this study, analyses of developmental expression patterns uncovered a putative role of AtWAKL10 in modulating leaf senescence, which was further investigated at physiological and molecular levels. The expression level of AtWAKL10 increased with the developmental progression and was rapidly upregulated in senescing leaf tissues. The promoter of AtWAKL10 contains various defense and hormone responsive elements, and its expression could be significantly induced by exogenous ABA, JA and SA. Moreover, the loss-of-function atwakl10 mutant showed earlier senescence along the course of natural development and accelerated leaf senescence under darkness and hormonal stresses, while plants overexpressing AtWAKL10 showed an opposite trend. Additionally, some defense and senescence related WRKY transcription factors could bind to the promoter of AtWAKL10. In addition, deletion and overexpression of AtWAKL10 caused several specific transcriptional alterations, including genes involved in cell extension, cell wall modification, defense response and senescence related WRKYs, which may be implicated in regulatory mechanisms adopted by AtWAKL10 in controlling leaf senescence. Taken together, these results revealed that AtWAKL10 negatively regulated leaf senescence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Parede Celular/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Parede Celular/efeitos dos fármacos , Escuridão , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação/genética , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Frações Subcelulares/metabolismo , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
6.
Cancer Sci ; 112(7): 2803-2820, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34109710

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase 2 and interaction of IRE1α with inositol 1,4,5-triphosphate receptor, which generates a condition in which an increase in intracellular Ca2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase-induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca2+ /caspases axis. Ablation of HSP47 promotes gemcitabine-induced suppression of tumor growth in PDAC cell-bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.


Assuntos
Calreticulina/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endorribonucleases/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Cálcio/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Caspases/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Técnicas de Inativação de Genes , Inativação Gênica , Proteínas de Choque Térmico HSP47/genética , Xenoenxertos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , NADPH Oxidases/metabolismo , Transplante de Neoplasias , Neoplasias Pancreáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Resposta a Proteínas não Dobradas
7.
PLoS Pathog ; 17(6): e1009644, 2021 06.
Artigo em Inglês | MEDLINE | ID: covidwho-1278205

RESUMO

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Vírus da Hepatite Murina/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator 6 Ativador da Transcrição/metabolismo , Animais , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Sistemas de Liberação de Medicamentos , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Células Vero , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Front Immunol ; 12: 662989, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1256380

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of current COVID-19 pandemic, and insufficient production of type I interferon (IFN-I) is associated with the severe forms of the disease. Membrane (M) protein of SARS-CoV-2 has been reported to suppress host IFN-I production, but the underlying mechanism is not completely understood. In this study, SARS-CoV-2 M protein was confirmed to suppress the expression of IFNß and interferon-stimulated genes induced by RIG-I, MDA5, IKKϵ, and TBK1, and to inhibit IRF3 phosphorylation and dimerization caused by TBK1. SARS-CoV-2 M could interact with MDA5, TRAF3, IKKϵ, and TBK1, and induce TBK1 degradation via K48-linked ubiquitination. The reduced TBK1 further impaired the formation of TRAF3-TANK-TBK1-IKKε complex that leads to inhibition of IFN-I production. Our study revealed a novel mechanism of SARS-CoV-2 M for negative regulation of IFN-I production, which would provide deeper insight into the innate immunosuppression and pathogenicity of SARS-CoV-2.


Assuntos
Interferon Tipo I/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , SARS-CoV-2/imunologia , Ubiquitina/metabolismo , Proteínas da Matriz Viral/imunologia , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Proteólise , Receptores Imunológicos/metabolismo , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo
9.
FASEB J ; 35(7): e21668, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34114695

RESUMO

The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell-cell contact, apical cell polarity, and mechanical signals. Miss-regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein-coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up-and-down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up- or down-regulate YAP activity through a pure Gq pathway.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Proteínas de Ciclo Celular/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Receptores Acoplados a Proteínas G/genética , Fatores de Transcrição/genética
10.
Nat Commun ; 12(1): 3856, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158470

RESUMO

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.


Assuntos
Motivos de Aminoácidos , DNA Helicases/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
11.
Nat Commun ; 12(1): 3845, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158506

RESUMO

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.


Assuntos
Axônios/metabolismo , Quinase 1 do Ponto de Checagem/genética , Canais Iônicos/genética , Regeneração Nervosa/genética , Animais , Animais Geneticamente Modificados , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Canais Iônicos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética
12.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070186

RESUMO

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Neovascularização Retiniana/etiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Eletrorretinografia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/fisiopatologia , Vasos Retinianos/patologia , Vasos Retinianos/fisiopatologia , Transdução de Sinais
13.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068565

RESUMO

Pancreatic cancer is one of the most malignant cancers with high mortality. Therefore, it is of great urgency to develop new agents that could improve the prognosis of Pancreatic cancer patients. Chinese propolis (CP), a flavonoid-rich beehive product, has been reported to have an anticancer effect. In this study, we applied CP to the human Pancreatic cancer cell line Panc-1 to verify its impact on tumor development. CP induced apoptosis in Panc-1 cells from 12.5 µg/mL in a time- and dose-dependent manner with an IC50 value of approximately 50 µg/mL. Apoptosis rate induced by CP was examined by Annexing FITC/PI assay. We found that 48 h treatment with 50 µg/mL CP resulted in 34.25 ± 3.81% apoptotic cells, as compared to 9.13 ± 1.76% in the control group. We further discovered that the Panc-1 cells tended to be arrested at G2/M phase after CP treatment, which is considered to contribute to the anti-proliferation effect of CP. Furthermore, our results demonstrated that CP suppressed Panc-1 cell migration by regulating epithelial-mesenchymal transition (EMT). Interestingly, the Hippo pathway was activated in Panc-1 cells after CP treatment, serving as a mechanism for the anti-pancreatic cancer effect of CP. These findings provide a possibility of beehive products as an alternative treatment for pancreatic cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Própole/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Padrões de Referência , Transdução de Sinais/efeitos dos fármacos
14.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072258

RESUMO

Mutations in striated preferentially expressed protein kinase (SPEG), a member of the myosin light chain kinase protein family, are associated with centronuclear myopathy (CNM), cardiomyopathy, or a combination of both. Burgeoning evidence suggests that SPEG plays critical roles in the development, maintenance, and function of skeletal and cardiac muscles. Here we review the genotype-phenotype relationships and the molecular mechanisms of SPEG-related diseases. This review will focus on the progress made toward characterizing SPEG and its interacting partners, and its multifaceted functions in muscle regeneration, triad development and maintenance, and excitation-contraction coupling. We will also discuss future directions that are yet to be investigated including understanding of its tissue-specific roles, finding additional interacting proteins and their relationships. Understanding the basic mechanisms by which SPEG regulates muscle development and function will provide critical insights into these essential processes and help identify therapeutic targets in SPEG-related disorders.


Assuntos
Suscetibilidade a Doenças , Expressão Gênica , Desenvolvimento Muscular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Acoplamento Excitação-Contração/genética , Regulação da Expressão Gênica , Humanos , Desenvolvimento Muscular/genética , Proteínas Musculares/química , Músculo Esquelético/metabolismo , Mutação , Miocárdio/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/química , Regeneração/genética , Transdução de Sinais , Relação Estrutura-Atividade
15.
Nat Commun ; 12(1): 3519, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112781

RESUMO

TLR4 signaling plays key roles in the innate immune response to microbial infection. Innate immune cells encounter different mechanical cues in both health and disease to adapt their behaviors. However, the impact of mechanical sensing signals on TLR4 signal-mediated innate immune response remains unclear. Here we show that TLR4 signalling augments macrophage bactericidal activity through the mechanical sensor Piezo1. Bacterial infection or LPS stimulation triggers assembly of the complex of Piezo1 and TLR4 to remodel F-actin organization and augment phagocytosis, mitochondrion-phagosomal ROS production and bacterial clearance and genetic deficiency of Piezo1 results in abrogation of these responses. Mechanistically, LPS stimulates TLR4 to induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for pathogen ingestion and killing. Inhibition of CaMKII or knockout of either Mst1/2 or Rac1 results in reduced macrophage bactericidal activity, phenocopying the Piezo1 deficiency. Thus, we conclude that TLR4 drives the innate immune response via Piezo1 providing critical insight for understanding macrophage mechanophysiology and the host response.


Assuntos
Infecções Bacterianas/imunologia , Imunidade Inata , Canais Iônicos/metabolismo , Macrófagos/imunologia , Fagossomos/metabolismo , Receptor 4 Toll-Like/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Infecções por Escherichia coli/imunologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Canais Iônicos/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
16.
PLoS Pathog ; 17(6): e1009644, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138976

RESUMO

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Vírus da Hepatite Murina/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator 6 Ativador da Transcrição/metabolismo , Animais , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Sistemas de Liberação de Medicamentos , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Células Vero , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
17.
Front Immunol ; 12: 618561, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936034

RESUMO

The p38 regulated/activated protein kinase (PRAK) is a protein kinase downstream of p38MAPK. The present study investigated its function in the macrophage. Myeloid-specific deletion of Prak resulted in a significant reduction in F4/80+CD11b+ peritoneal macrophages with decreased expression of MHC-II and CD80. Upon infection with Listeria monocytogenes, Prak-deficient mice demonstrated an increased mortality, which was accompanied by a higher bacterial load in multiple tissues and elevated levels of proinflammatory cytokines in the serum. While the Prak-deficient macrophage showed similar potency in phagocytosis assays, its bactericidal activity was severely impaired. Moreover, Prak deficiency was associated with defects in ROS production, inflammasome activation as well as autophagy induction. Therefore, PRAK critically contributes to the clearance of intracellular pathogens by affecting multiple aspects of the macrophage function.


Assuntos
Autofagia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Biomarcadores , Citocinas/metabolismo , Suscetibilidade a Doenças , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/microbiologia , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Listeria/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fagocitose/genética , Fagocitose/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo
18.
EMBO Rep ; 22(6): e52175, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33938130

RESUMO

Upon Mycobacterium tuberculosis (Mtb) infection, protein kinase G (PknG), a eukaryotic-type serine-threonine protein kinase (STPK), is secreted into host macrophages to promote intracellular survival of the pathogen. However, the mechanisms underlying this PknG-host interaction remain unclear. Here, we demonstrate that PknG serves both as a ubiquitin-activating enzyme (E1) and a ubiquitin ligase (E3) to trigger the ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF-ß-activated kinase 1 (TAK1), thereby inhibiting the activation of NF-κB signaling and host innate responses. PknG promotes the attachment of ubiquitin (Ub) to the ubiquitin-conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82-Ub), rather than the usual C86-Ub thiol-ester bond. PknG induces the discharge of Ub from UbcH7 by acting as an isopeptidase, before attaching Ub to its substrates. These results demonstrate that PknG acts as an unusual ubiquitinating enzyme to remove key components of the innate immunity system, thus providing a potential target for tuberculosis treatment.


Assuntos
Mycobacterium tuberculosis , Proteínas Quinases Dependentes de GMP Cíclico , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
19.
Cancer Sci ; 112(7): 2714-2727, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33939216

RESUMO

Breast cancer is one of the most commonly diagnosed malignancies worldwide, while the triple negative breast cancer (TNBC) is the most aggressive and virulent subtype in breast cancers. Compared with luminal type breast cancers, which could be well controlled by endocrine treatment, TNBC is worse in prognosis and lack of effective targeted therapy. Thus, it would be interesting and meaningful to identify novel therapeutic targets for TNBC treatments. Recent genomic data showed the activation of Hippo/YAP signaling in TNBC, indicating its critical roles in TNBC carcinogenesis and cancer progression. Hippo/YAP signaling could subject to several kinds of protein modifications, including ubiquitination and phosphorylation. Quite a few studies have demonstrated these modifications, which controlled YAP protein stability and turnover, played critical role in Hippo signaling activation In our current study, we identified ZNF213 as a negative modifier for Hippo/YAP axis. ZNF213 depletion promoted TNBC cell migration and invasion, which could be rescued by further YAP silencing. ZNF213 knocking down facilitated YAP protein stability and Hippo target gene expression, including CTGF and CYR61. Further mechanism studies demonstrated that ZNF213 associated with YAP and facilitated YAP K48-linked poly-ubiquitination at several YAP lysine sites (K252, K254, K321 and K497). Besides, the clinical data showed that ZNF213 negatively correlated with YAP protein level and Hippo target gene expression in TNBC samples. ZNF213 expression correlated with good prognosis in TNBC patients. Our data provided novel insights in YAP proteolytic regulation and TNBC progression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Linhagem Celular Tumoral , Movimento Celular , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Progressão da Doença , Feminino , Imunofluorescência , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fosforilação , Prognóstico , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição/deficiência , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ubiquitinação
20.
Mol Cell Biol ; 41(7): e0037820, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33941619

RESUMO

In response to nutrient starvation, the budding yeast Saccharomyces cerevisiae abandons mitotic proliferation and embarks on a differentiation process that leads through meiosis to the formation of haploid spores. This process is driven by cascading waves of meiosis-specific-gene expression. The early meiosis-specific genes are repressed during mitotic proliferation by the DNA-binding protein Ume6 in combination with repressors Rpd3 and Sin3. The expression of meiosis-specific transcription factor Ime1 leads to activation of the early meiosis-specific genes. We investigated the stability and promoter occupancy of Ume6 in sporulating cells and determined that it remains bound to early meiosis-specific gene promoters when those genes are activated. Furthermore, we find that the repressor Rpd3 remains associated with Ume6 after the transactivator Ime1 has joined the complex and that the Gcn5 and Tra1 components of the SAGA complex bind to the promoter of IME2 in an Ime1-dependent fashion to induce transcription of the early meiosis-specific genes. Our investigation supports a model whereby Ume6 provides a platform allowing recruitment of both activating and repressing factors to coordinate the expression of the early meiosis-specific genes in Saccharomyces cerevisiae.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Meiose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
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