RESUMEN
The main protease of severe acute respiratory syndrome coronavirus 2, Mpro, is a key viral protein essential for viral infection and replication. Mpro has been the target of many pharmacological efforts; however, the host-specific regulation of Mpro protein remains unclear. Here, we report the ubiquitin-proteasome-dependent degradation of Mpro protein in human cells, facilitated by the human E3 ubiquitin ligase ZBTB25. We demonstrate that Mpro has a short half-life that is prolonged via proteasomal inhibition, with its Lys-100 residue serving as a potential ubiquitin acceptor. Using in vitro binding assays, we observed ZBTB25 and Mpro bind to each other in vitro, and using progressive deletional mapping, we further uncovered the required domains for this interaction. Finally, we used an orthologous beta-coronavirus infection model and observed that genetic ablation of ZBTB25 resulted in a more highly infective virus, an effect lost upon reconstitution of ZBTB25 to deleted cells. In conclusion, these data suggest a new mechanism of Mpro protein regulation as well as identify ZBTB25 as an anticoronaviral E3 ubiquitin ligase.
Asunto(s)
Proteasas 3C de Coronavirus , Proteínas de Unión al ADN , SARS-CoV-2 , Humanos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteasas Virales/genética , Proteasas Virales/metabolismo , Proteínas Virales/metabolismo , SARS-CoV-2/fisiología , Proteasas 3C de Coronavirus/metabolismo , COVID-19/virologíaRESUMEN
Systemic scleroderma (SSc) is an autoimmune disease that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive profibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three-quarters of individuals with SSc develop pulmonary fibrosis within 5 years, the main cause of SSc mortality. No approved medicines to manage lung SSc currently exist. Recent research suggests that profibrotic signaling by transforming growth factor ß (TGF-ß) is directly tied to SSc. Previous studies have also shown that ubiquitin E3 ligases potently control TGF-ß signaling through targeted degradation of key regulatory proteins; however, the roles of these ligases in SSc-TGF-ß signaling remain unclear. Here we utilized primary SSc patient lung cells for high-throughput screening of TGF-ß signaling via high-content imaging of nuclear translocation of the profibrotic transcription factor SMAD family member 2/3 (SMAD2/3). We screened an RNAi library targeting ubiquitin E3 ligases and observed that knockdown of the E3 ligase Kelch-like protein 42 (KLHL42) impairs TGF-ß-dependent profibrotic signaling. KLHL42 knockdown reduced fibrotic tissue production and decreased TGF-ß-mediated SMAD activation. Using unbiased ubiquitin proteomics, we identified phosphatase 2 regulatory subunit B'ϵ (PPP2R5ϵ) as a KLHL42 substrate. Mechanistic experiments validated ubiquitin-mediated control of PPP2R5ϵ stability through KLHL42. PPP2R5ϵ knockdown exacerbated TGF-ß-mediated profibrotic signaling, indicating a role of PPP2R5ϵ in SSc. Our findings indicate that the KLHL42-PPP2R5ϵ axis controls profibrotic signaling in SSc lung fibroblasts. We propose that future studies could investigate whether chemical inhibition of KLHL42 may ameliorate profibrotic signaling in SSc.
Asunto(s)
Proteína Fosfatasa 2/genética , Esclerodermia Sistémica/genética , Proteína Smad2/genética , Factor de Crecimiento Transformador beta/genética , Ubiquitina-Proteína Ligasas/genética , Fibroblastos/metabolismo , Fibrosis/genética , Fibrosis/patología , Humanos , Pulmón/citología , Pulmón/metabolismo , Proteolisis , Proteómica , Esclerodermia Sistémica/patología , Transducción de Señal/genéticaRESUMEN
Nutrient sensing is a critical cellular process controlling metabolism and signaling. mTOR complex 1 (mTORC1) is the primary signaling hub for nutrient sensing and, when activated, stimulates anabolic processes while decreasing autophagic flux. mTORC1 receives nutrient status signals from intracellular amino acid sensors. One of these sensors, Sestrin-2, functions as an intracellular sensor of cytosolic leucine and inhibitor of mTORC1 activity. Genetic studies of Sestrin-2 have confirmed its critical role in regulating mTORC1 activity, especially in the case of leucine starvation. Sestrin-2 is known to be transcriptionally controlled by several mechanisms; however, the post-translational proteolytic regulation of Sestrin-2 remains unclear. Here, we explored how Sestrin-2 is regulated through the ubiquitin proteasome system. Using an unbiased screening approach of an siRNA library targeting ubiquitin E3 ligases, we identified a RING-type E3 ligase, ring finger protein 186 (RNF186), that critically mediates the Sestrin-2 ubiquitination and degradation. We observed that RNF186 and Sestrin-2 bind each other through distinct C-terminal motifs and that Lys-13 in Sestrin-2 is a putative ubiquitin acceptor site. RNF186 knockdown increased Sestrin-2 protein levels and decreased mTORC1 activation. These results reveal a new mechanism of E3 ligase control of mTORC1 activity through the RNF186-Sestrin-2 axis, suggesting that RNF186 inhibition may be a potential strategy to increase levels of the mTORC1 inhibitor Sestrin-2.
Asunto(s)
Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Línea Celular , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Cicloheximida/farmacología , Humanos , Leupeptinas/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Nucleares/química , Unión Proteica , Estabilidad Proteica/efectos de los fármacos , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Critical illness can significantly alter the composition and function of the human microbiome, but few studies have examined these changes over time. Here, we conduct a comprehensive analysis of the oral, lung, and gut microbiota in 479 mechanically ventilated patients (223 females, 256 males) with acute respiratory failure. We use advanced DNA sequencing technologies, including Illumina amplicon sequencing (utilizing 16S and ITS rRNA genes for bacteria and fungi, respectively, in all sample types) and Nanopore metagenomics for lung microbiota. Our results reveal a progressive dysbiosis in all three body compartments, characterized by a reduction in microbial diversity, a decrease in beneficial anaerobes, and an increase in pathogens. We find that clinical factors, such as chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, are associated with specific patterns of dysbiosis. Interestingly, unsupervised clustering of lung microbiota diversity and composition by 16S independently predicted survival and performed better than traditional clinical and host-response predictors. These observations are validated in two separate cohorts of COVID-19 patients, highlighting the potential of lung microbiota as valuable prognostic biomarkers in critical care. Understanding these microbiome changes during critical illness points to new opportunities for microbiota-targeted precision medicine interventions.
Asunto(s)
COVID-19 , Disbiosis , Microbioma Gastrointestinal , Pulmón , Microbiota , Humanos , Femenino , Masculino , Disbiosis/microbiología , Persona de Mediana Edad , Pulmón/microbiología , COVID-19/microbiología , COVID-19/virología , Anciano , Microbiota/genética , Microbioma Gastrointestinal/genética , Interacciones Microbiota-Huesped/genética , Estudios Longitudinales , ARN Ribosómico 16S/genética , Insuficiencia Respiratoria/microbiología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Adulto , Respiración Artificial , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificación , Enfermedad Crítica , Metagenómica/métodosRESUMEN
Background: Lung hyperinflation with elevated residual volume (RV) is associated with poor prognosis in adults with chronic obstructive pulmonary disease (COPD) and is a critical criterion for lung volume reduction selection. Here, we proposed that patterns within spirometric measures could represent the degree of hyperinflation. Methods: Fractional polynomial multivariate regression was used to develop a prediction model based on age, biological sex, forced expiratory volume in 1 second (FEV1), and forced vital capacity (FVC) to estimate plethysmography measured RV in patients in the Pittsburgh Specialized Center for Clinically Oriented Research (SCCOR) cohort (n=450). Receiver operating characteristic area under the curve (ROC-AUC) and optimal cut-points from the model were identified. The model was validated in a separate cohort (n=793). Results: The best fit model: RV %est=[FVC %predicted] x 3.46-[FEV1/FVC] x 179.80- [FVC % (sqrt)] x 79.53-[age] x 0.98- [sex] x 10.88 + 737.06, where [sex], m=1. R2 of observed versus %predicted RV was 0.71. The optimal cut-point to predict an RV % >175% was 161. At this cut-point, ROC-AUC was 0.95, with a sensitivity 0.95, specificity 0.86, positive predictive value (PPV) of 97%, negative predictive value (NPV) of 76%, positive likelihood ratio (LR) of 6.6, and negative LR of 0.06. In a validation cohort of COPD patients (n=793), the model performed similarly, with a sensitivity of 0.82, specificity of 0.83, PPV of 85%, NPV of 79%, positive LR of 4.7, and negative LR of 0.21. Conclusion: In patients with COPD, a model using only spirometry, age, and biological sex can estimate elevated RV. This tool could facilitate the identification of candidates for lung volume reduction procedures and can be integrated into existing epidemiologic databases to investigate the clinical impact of hyperinflation.
RESUMEN
Critical illness can disrupt the composition and function of the microbiome, yet comprehensive longitudinal studies are lacking. We conducted a longitudinal analysis of oral, lung, and gut microbiota in a large cohort of 479 mechanically ventilated patients with acute respiratory failure. Progressive dysbiosis emerged in all three body compartments, characterized by reduced alpha diversity, depletion of obligate anaerobe bacteria, and pathogen enrichment. Clinical variables, including chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, shaped dysbiosis. Notably, of the three body compartments, unsupervised clusters of lung microbiota diversity and composition independently predicted survival, transcending clinical predictors, organ dysfunction severity, and host-response sub-phenotypes. These independent associations of lung microbiota may serve as valuable biomarkers for prognostication and treatment decisions in critically ill patients. Insights into the dynamics of the microbiome during critical illness highlight the potential for microbiota-targeted interventions in precision medicine.
RESUMEN
Critical illness can disrupt the composition and function of the microbiome, yet comprehensive longitudinal studies are lacking. We conducted a longitudinal analysis of oral, lung, and gut microbiota in a large cohort of 479 mechanically ventilated patients with acute respiratory failure. Progressive dysbiosis emerged in all three body compartments, characterized by reduced alpha diversity, depletion of obligate anaerobe bacteria, and pathogen enrichment. Clinical variables, including chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, shaped dysbiosis. Notably, of the three body compartments, unsupervised clusters of lung microbiota diversity and composition independently predicted survival, transcending clinical predictors, organ dysfunction severity, and host-response sub-phenotypes. These independent associations of lung microbiota may serve as valuable biomarkers for prognostication and treatment decisions in critically ill patients. Insights into the dynamics of the microbiome during critical illness highlight the potential for microbiota-targeted interventions in precision medicine.
RESUMEN
SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Homoharringtonina/farmacología , Piperidinas/farmacología , Quinazolinonas/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Internalización del Virus/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , COVID-19/patología , COVID-19/virología , Células Cultivadas , Chlorocebus aethiops , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Ratones , Inhibidores de la Síntesis de la Proteína/farmacología , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
BACKGROUNDThe fungal cell wall constituent 1,3-ß-d-glucan (BDG) is a pathogen-associated molecular pattern that can stimulate innate immunity. We hypothesized that BDG from colonizing fungi in critically ill patients may translocate into the systemic circulation and be associated with host inflammation and outcomes.METHODSWe enrolled 453 mechanically ventilated patients with acute respiratory failure (ARF) without invasive fungal infection and measured BDG, innate immunity, and epithelial permeability biomarkers in serially collected plasma samples.RESULTSCompared with healthy controls, patients with ARF had significantly higher BDG levels (median [IQR], 26 pg/mL [15-49 pg/mL], P < 0.001), whereas patients with ARF with high BDG levels (≥40 pg/mL, 31%) had higher odds for assignment to the prognostically adverse hyperinflammatory subphenotype (OR [CI], 2.88 [1.83-4.54], P < 0.001). Baseline BDG levels were predictive of fewer ventilator-free days and worse 30-day survival (adjusted P < 0.05). Integrative analyses of fungal colonization and epithelial barrier disruption suggested that BDG may translocate from either the lung or gut compartment. We validated the associations between plasma BDG and host inflammatory responses in 97 hospitalized patients with COVID-19.CONCLUSIONBDG measurements offered prognostic information in critically ill patients without fungal infections. Further research in the mechanisms of translocation and innate immunity recognition and stimulation may offer new therapeutic opportunities in critical illness.FUNDINGUniversity of Pittsburgh Clinical and Translational Science Institute, COVID-19 Pilot Award and NIH grants (K23 HL139987, U01 HL098962, P01 HL114453, R01 HL097376, K24 HL123342, U01 HL137159, R01 LM012087, K08HK144820, F32 HL142172, K23 GM122069).
Asunto(s)
COVID-19 , Candida , Inmunidad Innata/inmunología , Respiración Artificial , beta-Glucanos/sangre , Biomarcadores/sangre , COVID-19/inmunología , COVID-19/terapia , Candida/inmunología , Candida/aislamiento & purificación , Permeabilidad Capilar/inmunología , Enfermedad Crítica/terapia , Femenino , Microbioma Gastrointestinal/inmunología , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Respiración Artificial/efectos adversos , Respiración Artificial/métodos , Insuficiencia Respiratoria/etiología , Insuficiencia Respiratoria/terapia , Sistema Respiratorio/inmunología , Sistema Respiratorio/microbiología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Análisis de SupervivenciaRESUMEN
NRF2 is a master regulator of cellular anti-oxidant and anti-inflammatory responses, and strategies to augment NRF2-dependent responses may beneficial in many diseases. Basal NRF2 protein level is constrained by constitutive KEAP1-mediated degradation, but in the presence of electrophiles, NRF2 ubiquitination is inhibited. Impeded NRF2 degradation increases NRF2 protein, resulting in up-regulation of anti-oxidant gene transcription, and decreased inflammation. KEAP1-independent mechanisms regulating NRF2 stability have also been reported. Here we employed an HTS approach and identified a small molecule, BC-1901S, that stabilized NRF2 and increased its activity. BC-1901S activated NRF2 by inhibiting NRF2 ubiquitination in a KEAP1-independent manner. It further increased NRF2-dependent anti-oxidant gene transcription, and exhibited anti-inflammatory effects in vitro and in vivo. Further, we identified a new NRF2-interacting partner, DDB1 and CUL4 Associated Factor 1 (DCAF1), an E3 ligase that targeted NRF2 for proteasomal degradation. Mechanistically, BC-1901S directly bound to DCAF1 and disrupted NRF2/DCAF1 interaction, thus activating NRF2. These findings provide new insights in NRF2 biology and NRF2 based anti-inflammatory therapy.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Ubiquitina-Proteína Ligasas , Humanos , Inflamación/tratamiento farmacológico , Inflamación/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identified small molecules that can reduce surface expression of TMPRSS2 using a 2,700 FDA-approved or current clinical trial compounds. Among these, homoharringtonine and halofuginone appear the most potent agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrated marked resistance to SARS-CoV-2 pseudoviral infection. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat COVID-19 infection.
RESUMEN
PURPOSE: To assess the longitudinal evolution of radiographic edema using chest X-rays (CXR) in patients with Acute Respiratory Distress Syndrome (ARDS) and to examine its association with prognostic biomarkers, ARDS subphenotypes and outcomes. MATERIALS AND METHODS: We quantified radiographic edema on CXRs from patients with ARDS or cardiogenic pulmonary edema (controls) using the Radiographic Assessment of Lung Edema (RALE) score on day of intubation and up to 10 days after. We measured baseline plasma biomarkers and recorded clinical variables. RESULTS: The RALE score had good inter-rater agreement (r = 0.83, p < 0.0001) applied on 488 CXRs from 129 patients, with higher RALE scores in patients with ARDS (n = 108) compared to controls (n = 21, p = 0.01). Baseline RALE scores were positively correlated with levels of the receptor for end-glycation end products (RAGE) in ARDS patients (p < 0.05). Baseline RALE scores were not predictive of 30- or 90-day survival. Persistently elevated RALE scores were associated with prolonged need for mechanical ventilation (p = 0.002). CONCLUSIONS: The RALE score is easily implementable with high inter-rater reliability. Longitudinal RALE scoring appears to be a reproducible approach to track the evolution of radiographic edema in patients with ARDS and can potentially predict prolonged need for mechanical ventilation.
Asunto(s)
Pulmón/fisiopatología , Edema Pulmonar/complicaciones , Respiración Artificial , Síndrome de Dificultad Respiratoria/complicaciones , Adulto , Anciano , Biomarcadores , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Fenotipo , Pronóstico , Estudios Prospectivos , Edema Pulmonar/diagnóstico por imagen , Edema Pulmonar/terapia , Radiografía Torácica , Reproducibilidad de los Resultados , Síndrome de Dificultad Respiratoria/terapia , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Dysregulated proinflammatory cytokine release has been implicated in the pathogenesis of several life-threatening acute lung illnesses such as pneumonia, sepsis, and acute respiratory distress syndrome. Suppressors of cytokine signaling proteins, particularly SOCS2, have recently been described as antiinflammatory mediators. However, the regulation of SOCS2 protein has not been described. Here we describe a mechanism of SOCS2 regulation by the action of the ubiquitin E3 ligase KIAA0317. KIAA0317-mediated degradation of SOCS2 exacerbated inflammation in vitro, and depletion of KIAA0317 in vivo ameliorated pulmonary inflammation. KIAA0317-knockout mice exhibited resistance to LPS-induced pulmonary inflammation, while KIAA03017 reexpression mitigated this effect. We uncovered a small molecule inhibitor of KIAA0317 protein (BC-1365) that prevented SOCS2 degradation and attenuated LPS- and P. aeruginosa-induced lung inflammation in vivo. These studies show KIAA0317 to be a critical mediator of pulmonary inflammation through its degradation of SOCS2 and a potential candidate target for therapeutic inhibition.