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1.
Front Immunol ; 15: 1397629, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39161760

RESUMEN

Introduction: The acute respiratory distress syndrome (ARDS) is a common complication of severe COVID-19 and contributes to patient morbidity and mortality. ARDS is a heterogeneous syndrome caused by various insults, and results in acute hypoxemic respiratory failure. Patients with ARDS from COVID-19 may represent a subgroup of ARDS patients with distinct molecular profiles that drive disease outcomes. Here, we hypothesized that longitudinal transcriptomic analysis may identify distinct dynamic pathobiological pathways during COVID-19 ARDS. Methods: We identified a patient cohort from an existing ICU biorepository and established three groups for comparison: 1) patients with COVID-19 ARDS that survived hospitalization (COVID survivors, n = 4), 2) patients with COVID-19 ARDS that did not survive hospitalization (COVID non-survivors, n = 5), and 3) patients with ARDS from other causes as a control group (ARDS controls, n = 4). RNA was isolated from peripheral blood mononuclear cells (PBMCs) at 4 time points (Days 1, 3, 7, and 10 following ICU admission) and analyzed by bulk RNA sequencing. Results: We first compared transcriptomes between groups at individual timepoints and observed significant heterogeneity in differentially expressed genes (DEGs). Next, we utilized the likelihood ratio test to identify genes that exhibit different patterns of change over time between the 3 groups and identified 341 DEGs across time, including hemoglobin subunit alpha 2 (HBA1, HBA2), hemoglobin subunit beta (HBB), von Willebrand factor C and EGF domains (VWCE), and carbonic anhydrase 1 (CA1), which all demonstrated persistent upregulation in the COVID non-survivors compared to COVID survivors. Of the 341 DEGs, 314 demonstrated a similar pattern of persistent increased gene expression in COVID non-survivors compared to survivors, associated with canonical pathways of iron homeostasis signaling, erythrocyte interaction with oxygen and carbon dioxide, erythropoietin signaling, heme biosynthesis, metabolism of porphyrins, and iron uptake and transport. Discussion: These findings describe significant differences in gene regulation during patient ICU course between survivors and non-survivors of COVID-19 ARDS. We identified multiple pathways that suggest heme and red blood cell metabolism contribute to disease outcomes. This approach is generalizable to larger cohorts and supports an approach of longitudinal sampling in ARDS molecular profiling studies, which may identify novel targetable pathways of injury and resolution.


Asunto(s)
COVID-19 , Eritrocitos , Perfilación de la Expresión Génica , Homeostasis , Hierro , Síndrome de Dificultad Respiratoria , SARS-CoV-2 , Transcriptoma , Humanos , COVID-19/genética , COVID-19/sangre , Masculino , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/sangre , Persona de Mediana Edad , SARS-CoV-2/fisiología , Femenino , Hierro/metabolismo , Eritrocitos/metabolismo , Anciano , Estudios Longitudinales
2.
Nat Commun ; 15(1): 6172, 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-39039092

RESUMEN

The severity of bacterial pneumonia can be worsened by impaired innate immunity resulting in ineffective pathogen clearance. We describe a mitochondrial protein, aspartyl-tRNA synthetase (DARS2), which is released in circulation during bacterial pneumonia in humans and displays intrinsic innate immune properties and cellular repair properties. DARS2 interacts with a bacterial-induced ubiquitin E3 ligase subunit, FBXO24, which targets the synthetase for ubiquitylation and degradation, a process that is inhibited by DARS2 acetylation. During experimental pneumonia, Fbxo24 knockout mice exhibit elevated DARS2 levels with an increase in pulmonary cellular and cytokine levels. In silico modeling identified an FBXO24 inhibitory compound with immunostimulatory properties which extended DARS2 lifespan in cells. Here, we show a unique biological role for an extracellular, mitochondrially derived enzyme and its molecular control by the ubiquitin apparatus, which may serve as a mechanistic platform to enhance protective host immunity through small molecule discovery.


Asunto(s)
Aspartato-ARNt Ligasa , Inmunidad Innata , Ratones Noqueados , Mitocondrias , Ubiquitinación , Animales , Aspartato-ARNt Ligasa/metabolismo , Aspartato-ARNt Ligasa/genética , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Ratones Endogámicos C57BL , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteolisis , Femenino , Masculino , Citocinas/metabolismo , Células HEK293 , Acetilación , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética
3.
Am J Respir Cell Mol Biol ; 68(5): 566-576, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36730646

RESUMEN

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a significant public health burden with limited treatment options. Many ß-coronaviruses, including SARS-CoV-2, gain entry to host cells through the interaction of SARS-CoV-2 spike protein with membrane-bound ACE2 (angiotensin-converting enzyme 2). Given its necessity for SARS-CoV-2 infection, ACE2 represents a potential therapeutic target in COVID-19. However, early attempts focusing on ACE2 in COVID-19 have not validated it as a druggable target nor identified other ACE2-related novel proteins for therapeutic intervention. Here, we identify a mechanism for ACE2 protein modulation by the deubiquitinase (DUB) enzyme UCHL1 (ubiquitin carboxyl-terminal hydrolase isozyme L1). ACE2 is constitutively ubiquitinated and degraded by the proteasome in lung epithelia. SARS-CoV-2 spike protein cellular internalization increased ACE2 protein abundance by decreasing its degradation. Using an siRNA library targeting 96 human DUBs, we identified UCHL1 as a putative regulator of ACE2 function as a viral receptor. Overexpressed UCHL1 preserved ACE2 protein abundance, whereas silencing of the DUB in cells destabilized ACE2 through increased polyubiquitination. A commercially available small molecule inhibitor of UCHL1 DUB activity decreased ACE2 protein concentrations coupled with inhibition of SARS-CoV-2 infection in epithelial cells. These findings describe a unique pathway of ACE2 regulation uncovering UCHL1 as a potential therapeutic target to modulate COVID-19 viral entry as a platform for future small molecule design and testing.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica
4.
J Biol Chem ; 298(12): 102698, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36379255

RESUMEN

Influenza remains a major public health challenge, as the viral infection activates multiple biological networks linked to altered host innate immunity. Following infection, IFN-λ, a ligand crucial for the resolution of viral infections, is known to bind to its cognate receptor, IFNLR1, in lung epithelia. However, little is known regarding the molecular expression and regulation of IFNLR1. Here, we show that IFNLR1 is a labile protein in human airway epithelia that is rapidly degraded after influenza infection. Using an unbiased proximal ligation biotin screen, we first identified that the Skp-Cullin-F box E3 ligase subunit, FBXO45, binds to IFNLR1. We demonstrate that FBXO45, induced in response to influenza infection, mediates IFNLR1 protein polyubiquitination and degradation through the ubiquitin-proteasome system by docking with its intracellular receptor domain. Furthermore, we found ectopically expressed FBXO45 and its silencing in cells differentially regulated both IFNLR1 protein stability and interferon-stimulated gene expression. Mutagenesis studies also indicated that expression of a K319R/K320R IFNLR1 variant in cells exhibited reduced polyubiquitination, yet greater stability and proteolytic resistance to FBXO45 and influenza-mediated receptor degradation. These results indicate that the IFN-λ-IFNLR1 receptor axis is tightly regulated by the Skp-Cullin-F box ubiquitin machinery, a pathway that may be exploited by influenza infection as a means to limit antiviral responses.


Asunto(s)
Gripe Humana , Humanos , Proteínas Cullin/inmunología , Gripe Humana/inmunología , Interferón lambda , Interferones/inmunología , Receptores de Interferón/inmunología , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Unión Proteica
5.
Transl Res ; 240: 1-16, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34740873

RESUMEN

The acute respiratory distress syndrome (ARDS) is a common complication of severe COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection. Knowledge of molecular mechanisms driving host responses to SARS-CoV-2 is limited by the lack of reliable preclinical models of COVID-19 that recapitulate human illness. Further, existing COVID-19 animal models are not characterized as models of experimental acute lung injury (ALI) or ARDS. Acknowledging differences in experimental lung injury in animal models and human ARDS, here we systematically evaluate a model of experimental acute lung injury as a result of SARS-CoV-2 infection in Syrian golden hamsters. Following intranasal inoculation, hamsters demonstrate acute SARS-CoV-2 infection, viral pneumonia, and systemic illness but survive infection with clearance of virus. Hamsters exposed to SARS-CoV-2 exhibited key features of experimental ALI, including histologic evidence of lung injury, increased pulmonary permeability, acute inflammation, and hypoxemia. RNA sequencing of lungs indicated upregulation of inflammatory mediators that persisted after infection clearance. Lipidomic analysis demonstrated significant differences in hamster phospholipidome with SARS-CoV-2 infection. Lungs infected with SARS-CoV-2 showed increased apoptosis and ferroptosis. Thus, SARS-CoV-2 infected hamsters exhibit key features of experimental lung injury supporting their use as a preclinical model of COVID-19 ARDS.


Asunto(s)
COVID-19/patología , Modelos Animales de Enfermedad , Pulmón/patología , Neumonía Viral/patología , SARS-CoV-2/patogenicidad , Animales , COVID-19/virología , Cricetinae , Masculino , Mesocricetus , Neumonía Viral/virología , SARS-CoV-2/aislamiento & purificación
6.
Cell Signal ; 79: 109859, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33253913

RESUMEN

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a multimeric, cytoplasmic, protein complex that regulates maturation and secretion of interleukin (IL)-1ß, a potent pro-inflammatory cytokine. Critical to host defense against pathogens, IL-1ß amplifies early innate immune responses by activating transcription of numerous other cytokines and chemokines. Excessive IL-1ß is associated with poor outcomes in inflammatory illnesses, such as sepsis and the acute respiratory distress syndrome (ARDS). Tight regulation of this signaling axis is vital, but little is known about mechanisms to limit excessive inflammasome activity. Here we identify the deubiquitinase STAM-binding protein (STAMBP) as a negative regulator of the NLRP3 inflammasome. In monocytes, knockout of STAMBP by CRISPR/Cas9 gene editing increased expression of numerous cytokines and chemokines in response to Toll-like receptor (TLR) agonists or bacterial lipopolysaccharide (LPS). This exaggerated inflammatory response was dependent on IL-1ß signaling, and STAMBP knockout directly increased release of IL-1ß with TLR ligation. While STAMBP does not modulate NLRP3 protein abundance, cellular depletion of the deubiquitinase increased NLRP3 K63 chain polyubiquitination resulting in increased NLRP3 inflammasome activation. These findings describe a unique mechanism of non-degradative ubiquitination of NLRP3 by STAMBP to limit excessive inflammasome activation and to reduce injurious IL-1ß signaling.


Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Transducción de Señal/inmunología , Ubiquitina Tiolesterasa/inmunología , Ubiquitinación/inmunología , Células HEK293 , Humanos , Células THP-1
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