RESUMEN
The receptor for advanced glycation end products (RAGE) is a highly expressed cell membrane receptor serving to anchor lung epithelia to matrix components, and it also amplifies inflammatory signaling during acute lung injury. However, mechanisms that regulate its protein concentrations in cells remain largely unknown. Here we show that RAGE exhibits an extended life span in lung epithelia (t½ 6 h), is monoubiquitinated at K374, and is degraded in lysosomes. The RAGE ligand ODN2006, a synthetic oligodeoxynucleotide resembling pathogenic hypomethylated CpG DNA, promotes rapid lysosomal RAGE degradation through activation of protein kinase Cζ (PKCζ), which phosphorylates RAGE. PKCζ overexpression enhances RAGE degradation, while PKCζ knockdown stabilizes RAGE protein levels and prevents ODN2006-mediated degradation. We identify that RAGE is targeted by the ubiquitin E3 ligase subunit F-box protein O10 (FBXO10), which associates with RAGE to mediate its ubiquitination and degradation. FBXO10 depletion in cells stabilizes RAGE and is required for ODN2006-mediated degradation. These data suggest that modulation of regulators involved in ubiquitin-mediated disposal of RAGE might serve as unique molecular inputs directing RAGE cellular concentrations and downstream responses, which are critical in an array of inflammatory disorders, including acute lung injury.-Evankovich, J., Lear, T., Mckelvey, A., Dunn, S., Londino, J., Liu, Y., Chen, B. B., Mallampalli, R. K. Receptor for advanced glycation end products is targeted by FBXO10 for ubiquitination and degradation.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Proteínas F-Box/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Proteolisis , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Ubiquitinación , Animales , Antígenos de Neoplasias/genética , Línea Celular , Islas de CpG , ADN/genética , Proteínas F-Box/genética , Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/farmacología , Proteína Quinasa C/genética , Receptor para Productos Finales de Glicación Avanzada/genéticaRESUMEN
As an α-chemokine receptor specific for stromal-derived-factor-1 (SDF-1, also called CXCL12), C-X-C chemokine receptor type 4 (CXCR4) plays a vital role in chemotactically attracting lymphocytes during inflammation. CXCR4 also regulates HIV infection due to its role as one of the chemokine coreceptors for HIV entry into CD4+ T cells. Chemokine receptors and their signaling pathways have been shown to be regulated by the process of ubiquitination, a posttranslational modification, guided by ubiquitin E3 ligases, which covalently links ubiquitin chains to lysine residues within target substrates. Here we describe a novel mechanism regulating CXCR4 protein levels and subsequent CXCR4/CXCL12 signaling pathway through the ubiquitination and degradation of the receptor in response to ligand stimulation. We identify that an uncharacterized really interesting new gene (RING) finger ubiquitin E3 ligase, RING finger protein 113A (RNF113A), directly ubiquitinates CXCR4 in cells, leading to CXCR4 degradation, and therefore disrupts the signaling cascade. We determined that the K331 residue within CXCR4 is essential for RNF113A-mediated ubiquitin conjugation. Overexpression of RNF113A significantly reduces CXCL12-induced kinase activation in HeLa cells, whereas RNF113A knockdown enhances CXCL12-induced downstream signaling. Further, RNF113A expression and silencing directly affect cell motility in a wound healing assay. These results suggest that RNF113A plays an important role in CXCR4 signaling through the ubiquitination and degradation of CXCR4. This mechanistic study might provide new understanding of HIV immunity and neutrophil activation and motility regulated by CXCR4.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Infecciones por VIH/inmunología , Células HeLa , Humanos , Estabilidad Proteica , UbiquitinaciónRESUMEN
Dysregulated cytokine signalling is a hallmark of inflammatory bowel diseases. Inflammatory responses of the colon are regulated by the suppressor of cytokine signalling (SOCS) proteins. SOCS1 is a key member of this family, and its function is critical in maintaining an appropriate inflammatory response through the JAK/STAT signalling pathway. Dysregulation of SOCS1 protein has been identified as a causal element in colonic inflammatory diseases. Despite this, it remains unclear how SOCS1 protein is regulated. Here, we identify that SOCS1 protein is targeted for degradation by the ubiquitin proteasome system, mediated by the E3 ubiquitin ligase KIAA0317 during experimental colonic inflammation. We characterize the mechanism of protein-protein interaction and ubiquitin conjugation to SOCS1 and demonstrate that the modulation of SOCS1 protein level leads to stark effects on JAK/STAT inflammatory signalling. Together, these results provide insight into the regulation of colonic inflammation through a new mechanism of ubiquitin-based control of SOCS1 protein.
Asunto(s)
Proteínas Supresoras de la Señalización de Citocinas , Ubiquitina , Humanos , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Inflamación/genética , Citocinas/metabolismo , Colon/metabolismoRESUMEN
Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation-based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson's disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.
Asunto(s)
Proteínas F-Box/antagonistas & inhibidores , Proteínas Mitocondriales/metabolismo , Fármacos Neuroprotectores/farmacología , Proteínas Quinasas/metabolismo , Proteolisis/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Animales , Línea Celular Tumoral , Estabilidad de Enzimas , Proteínas F-Box/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Ratones , Fármacos Neuroprotectores/química , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Neumonía/patologíaRESUMEN
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.
Asunto(s)
Pulmón/metabolismo , Neumonía/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Anciano , Animales , Citocinas/metabolismo , Femenino , Humanos , Inmunidad Innata , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Simulación del Acoplamiento Molecular , Neumonía/inmunología , Neumonía/patología , Unión Proteica , TranscriptomaRESUMEN
Inflammasomes regulate innate immune responses by facilitating maturation of inflammatory cytokines, interleukin (IL)-1ß and IL-18. NACHT, LRR and PYD domains-containing protein 7 (NALP7) is one inflammasome constituent, but little is known about its cellular handling. Here we show a mechanism for NALP7 protein stabilization and activation of the inflammasome by Toll-like receptor (TLR) agonism with bacterial lipopolysaccharide (LPS) and the synthetic acylated lipopeptide Pam3CSK4. NALP7 is constitutively ubiquitinated and recruited to the endolysosome for degradation. With TLR ligation, the deubiquitinase enzyme, STAM-binding protein (STAMBP) impedes NALP7 trafficking to lysosomes to increase NALP7 abundance. STAMBP deubiquitinates NALP7 and STAMBP knockdown abrogates LPS or Pam3CSK4-induced increases in NALP7 protein. A small-molecule inhibitor of STAMBP deubiquitinase activity, BC-1471, decreases NALP7 protein levels and suppresses IL-1ß release after TLR agonism. These findings describe a unique pathway of inflammasome regulation with the identification of STAMBP as a potential therapeutic target to reduce pro-inflammatory stress.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Inhibidores Enzimáticos/farmacología , Inflamasomas/genética , Leucocitos Mononucleares/efectos de los fármacos , Ubiquitina Tiolesterasa/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/inmunología , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Regulación de la Expresión Génica , Células HeLa , Humanos , Inmunidad Innata , Inflamasomas/antagonistas & inhibidores , Inflamasomas/inmunología , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Lipopéptidos/antagonistas & inhibidores , Lipopéptidos/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Simulación del Acoplamiento Molecular , Cultivo Primario de Células , Transducción de Señal , Células THP-1 , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/inmunología , Ubiquitinación/efectos de los fármacosRESUMEN
Toll-like receptor 2 (TLR2) is a pattern recognition receptor that recognizes many types of PAMPs that originate from gram-positive bacteria. Here we describe a novel mechanism regulating TLR2 protein expression and subsequent cytokine release through the ubiquitination and degradation of the receptor in response to ligand stimulation. We show a new mechanism in which an uncharacterized RING finger E3 ligase, PPP1R11, directly ubiquitinates TLR2 both in vitro and in vivo, which leads to TLR2 degradation and disruption of the signaling cascade. Lentiviral gene transfer or knockdown of PPP1R11 in mouse lungs significantly affects lung inflammation and the clearance of Staphylococcus aureus. There is a negative correlation between PPP1R11 and TLR2 levels in white blood cell samples isolated from patients with Staphylococcus aureus infections. These results suggest that PPP1R11 plays an important role in regulating innate immunity and gram-positive bacterial clearance by functioning, in part, through the ubiquitination and degradation of TLR2.
Asunto(s)
Inmunidad Innata/genética , Inflamación/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Infecciones Estafilocócicas/genética , Receptor Toll-Like 2/genética , Animales , Humanos , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Leucocitos/inmunología , Leucocitos/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Ratones , Proteolisis , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Staphylococcus aureus/patogenicidad , Receptor Toll-Like 2/inmunología , Ubiquitina-Proteína LigasasRESUMEN
The E3 small ubiquitin-like modifier (SUMO) protein ligase protein inhibitor of activated STAT 4 (PIAS4) is a pivotal protein in regulating the TGFß pathway. In this study, we discovered a new protein isoform encoded by KIAA0317, termed fibrosis-inducing E3 ligase 1 (FIEL1), which potently stimulates the TGFß signaling pathway through the site-specific ubiquitination of PIAS4. FIEL1 targets PIAS4 using a double locking mechanism that is facilitated by the kinases PKCζ and GSK3ß. Specifically, PKCζ phosphorylation of PIAS4 and GSK3ß phosphorylation of FIEL1 are both essential for the degradation of PIAS4. FIEL1 protein is highly expressed in lung tissues from patients with idiopathic pulmonary fibrosis (IPF), whereas PIAS4 protein levels are significantly reduced. FIEL1 overexpression significantly increases fibrosis in a bleomycin murine model, whereas FIEL1 knockdown attenuates fibrotic conditions. Further, we developed a first-in-class small molecule inhibitor toward FIEL1 that is highly effective in ameliorating fibrosis in mice. This study provides a basis for IPF therapeutic intervention by modulating PIAS4 protein abundance.
Asunto(s)
Pulmón/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Fibrosis Pulmonar/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Bleomicina/efectos adversos , Bleomicina/farmacología , Línea Celular , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Pulmón/patología , Ratones , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Inhibidoras de STAT Activados/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Invading pathogens may trigger overactivation of the innate immune system, which results in the release of large amounts of proinflammatory cytokines (cytokine storm) and leads to the development of pulmonary edema, multiorgan failure, and shock. PIAS1 is a multifunctional and potent anti-inflammatory protein that negatively regulates several key inflammatory pathways such as Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and nuclear factor κB (NF-κB). We discovered a ubiquitin E3 ligase, HECTD2, which ubiquitinated and mediated the degradation of PIAS1, thus increasing inflammation in an experimental pneumonia model. We found that GSK3ß phosphorylation of PIAS1 provided a phosphodegron for HECTD2 targeting. We also identified a mislocalized HECTD2 polymorphism, HECTD2(A19P), that was present in 8.5% of the population and functioned to reduce inflammation. This polymorphism prevented HECTD2/PIAS1 nuclear interaction, thus preventing PIAS1 degradation. The HECTD2(A19P) polymorphism was also protective toward acute respiratory distress syndrome (ARDS). We then developed a small-molecule inhibitor, BC-1382, that targeted HECTD2 and attenuated lipopolysaccharide (LPS)- and Pseudomonas aeruginosa-induced lung inflammation. These studies describe an unreported innate immune pathway and suggest that mutation or antagonism of the E3 ligase HECTD2 results in reduced severity of lung inflammation by selectively modulating the abundance of the anti-inflammatory protein PIAS1.
Asunto(s)
Inmunidad Innata/fisiología , Inflamación/fisiopatología , Lesión Pulmonar/inmunología , Lesión Pulmonar/fisiopatología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Técnicas de Silenciamiento del Gen , Lesión Pulmonar/microbiología , Ratones , Fosforilación , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Pseudomonas/patogenicidadRESUMEN
Cyclic adenosine monophosphate (cAMP) is a crucial intracellular second messenger molecule that converts extracellular molecules to intracellular signal transduction pathways generating cell- and stimulus-specific effects. Importantly, specific phosphodiesterase (PDE) subtypes control the amplitude and duration of cAMP-induced physiological processes and are therefore a prominent pharmacological target currently used in a variety of fields. Here we tested the extracts from traditional Chinese medicine, Forsythia suspense seeds, which have been used for more than 2000 years to relieve respiratory symptoms. Using structural-functional analysis we found its major lignin, Forsynthin, acted as an immunosuppressant by inhibiting PDE4 in inflammatory and immune cell. Moreover, several novel, selective small molecule derivatives of Forsythin were tested in vitro and in murine models of viral and bacterial pneumonia, sepsis and cytokine-driven systemic inflammation. Thus, pharmacological targeting of PDE4 may be a promising strategy for immune-related disorders characterized by amplified host inflammatory response.
Asunto(s)
Forsythia/química , Lignina/farmacología , Inhibidores de Fosfodiesterasa 4/aislamiento & purificación , Animales , Simulación por Computador , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Inflamación/tratamiento farmacológico , Lignina/aislamiento & purificación , Lesión Pulmonar/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Inhibidores de Fosfodiesterasa 4/química , Inhibidores de Fosfodiesterasa 4/farmacología , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Plantas Medicinales/química , Choque Séptico/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
Acute lung injury (ALI) is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1). Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.