RESUMO
Activation of the epidermal growth factor receptor (EGFR) is crucial for development, tissue homeostasis, and immunity. Dysregulation of EGFR signaling is associated with numerous diseases. EGFR ubiquitination and endosomal trafficking are key events that regulate the termination of EGFR signaling, but their underlying mechanisms remain obscure. Here, we reveal that ZNRF1, an E3 ubiquitin ligase, controls ligand-induced EGFR signaling via mediating receptor ubiquitination. Deletion of ZNRF1 inhibits endosome-to-lysosome sorting of EGFR, resulting in delayed receptor degradation and prolonged downstream signaling. We further demonstrate that ZNRF1 and Casitas B-lineage lymphoma (CBL), another E3 ubiquitin ligase responsible for EGFR ubiquitination, mediate ubiquitination at distinct lysine residues on EGFR. Furthermore, loss of ZNRF1 results in increased susceptibility to herpes simplex virus 1 (HSV-1) infection due to enhanced EGFR-dependent viral entry. Our findings identify ZNRF1 as a novel regulator of EGFR signaling, which together with CBL controls ligand-induced EGFR ubiquitination and lysosomal trafficking.
RESUMO
Caveolin-1 (CAV1), the major constituent of caveolae, plays a pivotal role in various cellular biological functions, including cancer and inflammation. The ubiquitin/proteasomal pathway is known to contribute to the regulation of CAV1 expression, but the ubiquitin ligase responsible for CAV1 protein stability remains unidentified. Here we reveal that E3 ubiquitin ligase ZNRF1 modulates CAV1 protein stability to regulate Toll-like receptor (TLR) 4-triggered immune responses. We demonstrate that ZNRF1 physically interacts with CAV1 in response to lipopolysaccharide and mediates ubiquitination and degradation of CAV1. The ZNRF1-CAV1 axis regulates Akt-GSK3ß activity upon TLR4 activation, resulting in enhanced production of pro-inflammatory cytokines and inhibition of anti-inflammatory cytokine IL-10. Mice with deletion of ZNRF1 in their hematopoietic cells display increased resistance to endotoxic and polymicrobial septic shock due to attenuated inflammation. Our study defines ZNRF1 as a regulator of TLR4-induced inflammatory responses and reveals another mechanism for the regulation of TLR4 signalling through CAV1.
Assuntos
Caveolina 1/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Animais , Caveolina 1/química , Ceco/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Deleção de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Mediadores da Inflamação/metabolismo , Ligadura , Lipopolissacarídeos , Lisina/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Punções , Células RAW 264.7 , Choque Séptico/imunologia , Choque Séptico/metabolismo , Choque Séptico/patologia , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
RATIONALE: Conventional mass spectrometry is encumbered by laborious and inconvenient sample pretreatment. Ambient thermal desorption electrospray ionization mass spectrometry (TD-ESI-MS) is most noted for its rapid, simple, and sensitive detection capabilities. In this study, TD-ESI-MS was used to rapidly characterize residual pesticides on the surfaces of fruits and vegetables. METHODS: A direct sampling probe was used to obtain analytes from sample surfaces. MS and MS/MS analyses were performed on fruits and vegetables via TD-ESI-MS. External calibration curves and reproducibility tests were performed using liquid pesticide standards. Pesticide decay and distribution on samples was studied, as well as the removal of residual pesticides via soaking in water or detergent baths. RESULTS: Since sample pretreatment was unnecessary, an analysis was completed in approximately 15 s or less, with no visible sample damage. Mass spectra were obtained for 22 pesticides. Linear calibrations (R(2) from 0.9414-0.999) had limits of detection as low as 0.5 µg·L(-1), with satisfactory reproducibilities for liquids and solids. Pesticides on sample surfaces decayed over 2 weeks under ambient conditions. Residual pesticides localized at the fruit peel. Detergent baths removed more pesticide than water baths. CONCLUSIONS: TD-ESI-MS was used to rapidly screen residual pesticides in liquids and solids. Pesticides were found on fruits and vegetables, where the decay, distribution, and removal of pesticides on samples were also explored. Due to short analysis times, the technique allows for high-throughput analyses for applications in food and environmental safety.
Assuntos
Frutas/química , Resíduos de Praguicidas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Verduras/química , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , ÁguaRESUMO
The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex, triggers caspase-1 activation and maturation of the proinflammatory cytokines IL-1ß and IL-18 upon sensing a wide range of pathogen- and damage-associated molecules. Dysregulation of NLRP3 inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)-dependent caspase-1 activation and IL-1ß secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabilized the autophagic protein Beclin 1 to promote autophagy, resulting in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and pro-IL-1ß processing. Likewise, overexpressing Beclin 1 in PAI-2-deficient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inflammasome activation and reveal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1ß-driven inflammation.