RESUMEN
Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.
Asunto(s)
Apoptosis/efectos de los fármacos , Homeostasis/efectos de los fármacos , Proteína de Dominio de Muerte Asociada a Receptor de TNF/antagonistas & inhibidores , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Animales , Autofagia/efectos de los fármacos , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Beclina-1/química , Beclina-1/metabolismo , Bortezomib/antagonistas & inhibidores , Bortezomib/farmacología , Línea Celular , Humanos , Proteína Huntingtina/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Masculino , Ratones , Modelos Moleculares , Ovillos Neurofibrilares/metabolismo , Proteoma/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína de Dominio de Muerte Asociada a Receptor de TNF/química , Proteína de Dominio de Muerte Asociada a Receptor de TNF/deficiencia , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitinación , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
Receptor-interacting protein kinase 1 (RIPK1) has emerged as a key regulator of cell death and inflammation, which are implicated in the pathogenesis of many inflammatory and degenerative diseases. RIPK1 is therefore a putative therapeutic target in many of these diseases. However, no pharmacological inhibitor of RIPK1-mediated cell death is currently in clinical use. Recognizing that a repurposed drug has an expedited clinical development pipeline, here we performed a high-throughput drug screen of Food and Drug Administration (FDA)-approved compounds and identified a novel use for crizotinib as an inhibitor of RIPK1-dependent cell death. Furthermore, crizotinib rescued TNF-α-induced death in mice with systemic inflammatory response syndrome. RIPK1 kinase activity was directly inhibited by crizotinib. These findings identify a new use for an established compound and are expected to accelerate drug development for RIPK1-spectrum disorders.
Asunto(s)
Apoptosis , Reposicionamiento de Medicamentos , Animales , Ratones , Crizotinib/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Muerte Celular , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Systemic inflammatory response syndrome (SIRS), at least in part driven by necroptosis, is characterized by life-threatening multiple organ failure. Blocking the progression of SIRS and consequent multiple organ dysfunction is challenging. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important cell death and inflammatory mediator, making it a potential treatment target in several diseases. Here, using a drug repurposing approach, we show that inhibiting RIPK1 is also an effective treatment for SIRS. We performed cell-based high-throughput drug screening of an US Food and Drug Administration (FDA)-approved drug library that contains 1953 drugs to identify effective inhibitors of necroptotic cell death by SYTOX green staining. Dose-response validation of the top candidate, quizartinib, was conducted in two cell lines of HT-22 and MEFs. The effect of quizartinib on necroptosis-related proteins was evaluated using western blotting, immunoprecipitation, and an in vitro RIPK1 kinase assay. The in vivo effects of quizartinib were assessed in a murine tumor necrosis factor α (TNFα)-induced SIRS model. High-throughput screening identified quizartinib as the top "hit" in the compound library that rescued cells from necroptosis in vitro. Quizartinib inhibited necroptosis by directly inhibiting RIPK1 kinase activity and blocking downstream complex IIb formation. Furthermore, quizartinib protected mice against TNFα-induced SIRS. Quizartinib, as an FDA-approved drug with proven safety and efficacy, was repurposed for targeted inhibition of RIPK1. This work provides essential preclinical data for transferring quizartinib to the treatment of RIPK1-dependent necroptosis-induced inflammatory diseases, including SIRS.
Asunto(s)
Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Factor de Necrosis Tumoral alfa , Animales , Ratones , Serina , TreoninaRESUMEN
Reduced endothelial-pericyte interactions are linked to progressive small vessel loss in pulmonary arterial hypertension (PAH), but the molecular mechanisms underlying this disease remain poorly understood. To identify relevant gene candidates associated with aberrant pericyte behavior, we performed a transcriptome analysis of patient-derived donor control and PAH lung pericytes followed by functional genomics analysis. Compared with donor control cells, PAH pericytes had significant enrichment of genes involved in various metabolic processes, the top hit being PDK4, a gene coding for an enzyme that suppresses mitochondrial activity in favor of glycolysis. Given reports that link reduced mitochondrial activity with increased PAH cell proliferation, we hypothesized that increased PDK4 is associated with PAH pericyte hyperproliferation and reduced endothelial-pericyte interactions. We found that PDK4 gene and protein expression was significantly elevated in PAH pericytes and correlated with reduced mitochondrial metabolism, higher rates of glycolysis, and hyperproliferation. Importantly, reducing PDK4 levels restored mitochondrial metabolism, reduced cell proliferation, and improved endothelial-pericyte interactions. To our knowledge, this is the first study that documents significant differences in gene expression between human donor control and PAH lung pericytes and the link between mitochondrial dysfunction and aberrant endothelial-pericyte interactions in PAH. Comprehensive characterization of these candidate genes could provide novel therapeutic targets to improve endothelial-pericyte interactions and prevent small vessel loss in PAH.
Asunto(s)
Células Endoteliales/metabolismo , Hipertensión Pulmonar/patología , Pericitos/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , TranscriptomaRESUMEN
PURPOSE: We aimed to evaluate the effects of two immune regulatory factors, interleukin-4 (IL-4) and melatonin, on several inflammatory mediators that are involved in inflammation and angiogenesis in diabetic retinopathy (DR), in high glucose or interleukin-1ß (IL-1ß) induced primary human retinal endothelial cells (RECs) and human retinal pigment epithelial (RPE) cells. METHODS: Human RECs and RPE cells were cultured in 30 mM D-glucose or 10 ng/ml IL-1ß, with or without the presence of 40 ng/ml IL-4 or 100 µM melatonin. The mRNA and protein levels of vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule-1 (ICAM-1), matrix metalloproteinases 2 (MMP2), and matrix metalloproteinases 9 (MMP9) were measured using real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: High glucose and IL-1ß induced the expression of VEGF, ICAM-1, MMP2, and MMP9 in human RECs and RPE cells. IL-4 and melatonin downregulated the expression of VEGF, ICAM-1, MMP2, and MMP9 induced by high glucose and IL-1ß. CONCLUSIONS: Our results demonstrated that IL-4 and melatonin inhibited inflammation and angiogenesis triggered by high glucose and IL-1ß, which suggests that these immune regulatory factors may be of potential therapeutic value in DR.
Asunto(s)
Células Endoteliales/patología , Células Epiteliales/patología , Glucosa/toxicidad , Inflamación/patología , Interleucina-1beta/toxicidad , Interleucina-4/farmacología , Melatonina/farmacología , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Inflamación/genética , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Epitelio Pigmentado de la Retina/patología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating fatal neurodegenerative disease with no cure. Receptor-interacting protein kinase 1 (RIPK1) has been proposed to mediate pathogenesis of ALS. Primidone has been identified as an old drug that can also inhibit RIPK1 kinase. We conducted a drug-repurposing biomarker study of primidone as a RIPK1 inhibitor using SOD1G93A mice and ALS patients. SOD1G93A mice treated with primidone showed significant delay of symptomatic onset and improved motor performance. One-hundred-sixty-two ALS participants dosed daily with primidone (62.5 mg) completed 24-week follow-up. A significant reduction was showed in serum levels of RIPK1 and IL-8, which were significantly higher in ALS patients than that of healthy controls (P < 0.0001). Serum RIPK1 levels were correlated positively with the severity of bulbar symptoms (P < 0.05). Our study suggests that serum levels of RIPK1 and IL-8 in peripheral can be used as clinical biomarkers for the activation of RIPK1 in central nervous system in human ALS patients. Repurposing primidone may provide a promising therapeutic strategy for ALS. The effect of primidone for the treatment of other inflammatory diseases may also be considered, since the activation of RIPK1 has been implicated in mediating a variety of inflammatory diseases including COVID-19-associated cytokine release syndrome (CRS). (ChiCTR2200060149).
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Biomarcadores , Interleucina-8/genética , Ratones Transgénicos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Enfermedades Neurodegenerativas/metabolismo , Primidona/metabolismo , Primidona/farmacología , Primidona/uso terapéutico , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/farmacología , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/farmacología , Superóxido Dismutasa/uso terapéutico , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/farmacologíaRESUMEN
PURPOSE: The purpose of the present study was to identify differential gene expressions (DEGs) and key pathways in neuroblastoma with MEIS2 depletion through bioinformatics. METHODS: The microarray gene expression dataset GSE56003 was downloaded from the Gene Expression Omnibus (GEO) database. DEGs were identified using Gene Level RMA sketch and Transcriptome Analysis Console. Gene ontology (GO) function and KEGG pathway enrichment analysis of DEGs were performed using the DAVID online tool. Protein-protein interaction (PPI) networks were constructed by mapping the DEGs onto Cytoscape software. MCODE algorithm was used to select the module and Centiscape was used to screen the hub genes. The Kaplan-Meier survival curves was utilized to show the correlation of specific gene expressions and the survival situation of NB patients. Resultsï¼A total of 1352 DEGs were identified in neuroblastoma with MEIS2 depletion, which were mainly enriched during the cell cycle, DNA replication, and DNA repair. CDK2, RAD51, BRCA1, and MCM3 were selected as hub genes that have the potential as novel therapeutic targets for neuroblastoma. CONCLUSION: This study revealed the hub genes and pathway involved in neuroblastoma with MEIS2 knockdown, which offered new insights into the molecular networks underlying MEIS2 depletion in neuroblastoma. Additionally, this study provided a valuable resource of potential biomarkers and therapeutic targets.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Neuroblastoma/genética , Mapeo de Interacción de Proteínas/métodos , Factores de Transcripción/metabolismo , Humanos , Neuroblastoma/patologíaRESUMEN
We investigated the effects of treating differentiated retinal pigment epithelial (RPE) cells with didanosine (ddI), which is associated with retinopathy in individuals with HIV/AIDS. We hypothesized that such treatment would cause depletion of mitochondrial DNA and provide insight into the consequences of degradation of RPE mitochondrial function in aging and disease. Treatment of differentiated ARPE-19 or human primary RPE cells with 200 µM ddI for 6-24 days was not cytotoxic but caused up to 60% depletion of mitochondrial DNA, and a similar reduction in mitochondrial membrane potential and NDUFA9 protein abundance. Mitochondrial DNA-depleted RPE cells demonstrated enhanced aerobic glycolysis by extracellular flux analysis, increased AMP kinase activation, reduced mTOR activity, and increased resistance to cell death in response to treatment with the oxidant, sodium iodate. We conclude that ddI-mediated mitochondrial DNA depletion promotes a glycolytic shift in differentiated RPE cells and enhances resistance to oxidative damage. Our use of ddI treatment to induce progressive depletion of mitochondrial DNA in differentiated human RPE cells should be widely applicable for other studies aimed at understanding RPE mitochondrial dysfunction in aging and disease.
Asunto(s)
Diferenciación Celular/genética , ADN Mitocondrial/metabolismo , Células Epiteliales/metabolismo , Epitelio Pigmentado de la Retina/citología , Adenilato Quinasa/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Didanosina/farmacología , Células Epiteliales/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
Diabetic retinopathy (DR) is a severe complication of diabetes mellitus (DM) and often causes vision loss or even blindness. Vascular endothelial growth factor (VEGF) in the retina, which is mainly derived from Müller cells, is a crucial biological factor in the development of DR. Astragalin is extracted from Astragalus membranaceus and has many pharmacological properties. Studies showed that astragalin has beneficial effects on hyperglycemia. To evaluate the effect of astragalin in preventing and treating DR and determine astragalin's mechanism of action, Müller cells were collected from rat retina, cultured in vitro and identified using immunocytochemistry. They were divided into four groups: the high glucose group (20 mmol/l), the normal control group, the astragalin group (400 mg/l) and the high glucose (20 mmol/l) + astragalin (400 mg/l) group. After 3 days of treatment, immunocytochemical and reverse transcription-polymerase chain reaction (RT-PCR) analysis of VEGF was carried out. Our results demonstrated that astragalin decreased the overexpression of VEGF in Müller cells and alleviated the effects caused by high glucose. Thus, astragalin has promising application in preventing and treating DR caused by DM.