RESUMEN
Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1ß release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1ß release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD , Inflamasomas , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/química , Caspasa 1/metabolismo , Dominio de Reclutamiento y Activación de Caspasas , Inmunoglobulina G , Apoptosis , Anticuerpos Monoclonales/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Allostery commonly refers to the mechanism that regulates protein activity through the binding of a molecule at a different, usually distal, site from the orthosteric site. The omnipresence of allosteric regulation in nature and its potential for drug design and screening render the study of allostery invaluable. Nevertheless, challenges remain as few computational methods are available to effectively predict allosteric sites, identify signalling pathways involved in allostery, or to aid with the design of suitable molecules targeting such sites. Recently, bond-to-bond propensity analysis has been shown successful at identifying allosteric sites for a large and diverse group of proteins from knowledge of the orthosteric sites and its ligands alone by using network analysis applied to energy-weighted atomistic protein graphs. To address the identification of signalling pathways, we propose here a method to compute and score paths of optimised propensity that link the orthosteric site with the identified allosteric sites, and identifies crucial residues that contribute to those paths. We showcase the approach with three well-studied allosteric proteins: h-Ras, caspase-1, and 3-phosphoinositide-dependent kinase-1 (PDK1). Key residues in both orthosteric and allosteric sites were identified and showed agreement with experimental results, and pivotal signalling residues along the pathway were also revealed, thus providing alternative targets for drug design. By using the computed path scores, we were also able to differentiate the activity of different allosteric modulators.
Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Caspasa 1 , Proteínas Proto-Oncogénicas p21(ras) , Transducción de Señal , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/química , Regulación Alostérica , Sitio Alostérico , Caspasa 1/química , Ligandos , Proteínas Proto-Oncogénicas p21(ras)/químicaRESUMEN
The inflammatory protease caspase-1 is associated with the release of cytokines. An excessive number of cytokines (a "cytokine storm") is a dangerous consequence of COVID-19 infection and has been indicated as being among the causes of death by COVID-19. The anti-inflammatory drug colchicine (which is reported in the literature to be a caspase-1 inhibitor) and the corticosteroid drugs, dexamethasone and methylprednisolone, are among the most effective active compounds for COVID-19 treatment. The SERM raloxifene has also been used as a repurposed drug in COVID-19 therapy. In this study, inhibition of caspase-1 by these four compounds was analyzed using computational methods. Our aim was to see if the inhibition of caspase-1, an important biomolecule in the inflammatory response that triggers cytokine release, could shed light on how these drugs help to alleviate excessive cytokine production. We also measured the antioxidant activities of dexamethasone and colchicine when scavenging the superoxide radical using cyclic voltammetry methods. The experimental findings are associated with caspase-1 active site affinity towards these compounds. In evaluating our computational and experimental results, we here formulate a mechanism for caspase-1 inhibition by these drugs, which involves the active site amino acid Cys285 residue and is mediated by a transfer of protons, involving His237 and Ser339. It is proposed that the molecular moiety targeted by all of these drugs is a carbonyl group which establishes a S(Cys285)-C(carbonyl) covalent bond.
Asunto(s)
Antiinflamatorios/farmacología , Tratamiento Farmacológico de COVID-19 , Caspasa 1/efectos de los fármacos , Inhibidores de Caspasas/farmacología , Proteasas 3C de Coronavirus/efectos de los fármacos , Antiinflamatorios/química , COVID-19/metabolismo , Caspasa 1/química , Caspasa 1/metabolismo , Inhibidores de Caspasas/química , Colchicina/química , Colchicina/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Dexametasona/farmacología , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Triterpenos Pentacíclicos/farmacología , Dominios y Motivos de Interacción de Proteínas , Clorhidrato de Raloxifeno/química , Clorhidrato de Raloxifeno/farmacología , Inhibidores de Proteasa Viral/química , Inhibidores de Proteasa Viral/farmacologíaRESUMEN
Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sestrin2 (SESN2), a highly evolutionarily conserved protein, is critically involved in the cellular response to various stresses and has been confirmed to maintain the homeostasis of the internal environment. However, the potential effects of SESN2 in regulating dendritic cells (DCs) pyroptosis in the context of sepsis and the related mechanisms are poorly characterized. In this study, we found that SESN2 was capable of decreasing gasdermin D (GSDMD)-dependent pyroptosis of splenic DCs by inhibiting endoplasmic reticulum (ER) stress (ERS)-related nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-mediated ASC pyroptosome formation and caspase-1 (CASP-1) activation. Furthermore, SESN2 deficiency induced NLRP3/ASC/CASP-1-dependent pyroptosis and the production of proinflammatory cytokines by exacerbating the PERK-ATF4-CHOP signaling pathway, resulting in an increase in the mortality of septic mice, which was reversed by inhibiting ERS. These findings suggest that SESN2 appears to be essential for inhibiting NLRP3 inflammasome hyperactivation, reducing CASP-1-dependent pyroptosis, and improving sepsis outcomes through stabilization of the ER. The present study might have important implications for exploration of novel potential therapeutic targets for the treatment of sepsis complications.
Asunto(s)
Caspasa 1/química , Células Dendríticas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Peroxidasas/fisiología , Sustancias Protectoras , Piroptosis , Sepsis/prevención & control , Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Caspasa 1/genética , Caspasa 1/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/patología , Estrés del Retículo Endoplásmico , Inflamasomas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Sepsis/etiología , Sepsis/metabolismo , Sepsis/patología , Transducción de SeñalRESUMEN
The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.
Asunto(s)
Apoptosis/genética , Caspasas/genética , Inmunidad Innata , Lampreas/genética , Transducción de Señal/inmunología , Animales , Apoptosis/efectos de los fármacos , Caspasa 1/química , Caspasa 1/genética , Caspasa 1/aislamiento & purificación , Caspasa 1/metabolismo , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 6/química , Caspasa 6/genética , Caspasa 6/metabolismo , Caspasa 7/química , Caspasa 7/genética , Caspasa 7/aislamiento & purificación , Caspasa 7/metabolismo , Caspasa 8/química , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/química , Caspasa 9/genética , Caspasa 9/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas/química , Caspasas/aislamiento & purificación , Caspasas/metabolismo , Evolución Molecular , Duplicación de Gen , Reordenamiento Génico , Genoma , Genómica , Células HeLa , Humanos , Inmunidad Innata/genética , Lampreas/crecimiento & desarrollo , Lampreas/inmunología , Lampreas/metabolismo , Filogenia , Proteínas Recombinantes , Alineación de Secuencia , Transducción de Señal/genética , Staphylococcus aureus/efectos de los fármacos , Regulación hacia Arriba , Vibrio/efectos de los fármacosRESUMEN
This review takes a closer look at the structural components of the molecules involved in the processes leading to caspase-1 activation. Interleukins 1ß and 18 (IL-1ß, IL-18) are well-known proinflammatory cytokines that are produced following cleavage of their respective precursor proteins by the cysteine protease caspase-1. Active caspase-1 is the final step of the NLRP3 inflammasome, a three-protein intracellular complex involved in inflammation and induction of pyroptosis (a proinflammatory cell-death process). NLRP3 activators facilitate assembly of the inflammasome complex and subsequent activation of caspase-1 by autoproteolysis. However, the definitive structural components of active caspase-1 are still unclear and new data add to the complexity of this process. This review outlines the historical and recent findings that provide supporting evidence for the structural aspects of caspase-1 autoproteolysis and activation.
Asunto(s)
Caspasa 1/metabolismo , Animales , Caspasa 1/química , Línea Celular Tumoral , Activación Enzimática/fisiología , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Multimerización de Proteína/fisiología , ProteolisisRESUMEN
The inflammasome is a three-component (sensor, adaptor, and effector) filamentous signaling platform that shields from multiple pathogenic infections by stimulating the proteolytical maturation of proinflammatory cytokines and pyroptotic cell death. The signaling process initiates with the detection of endogenous and/or external danger signals by specific sensors, followed by the nucleation and polymerization from sensor to downstream adaptor and then to the effector, caspase-1. Aberrant activation of inflammasomes promotes autoinflammatory diseases, cancer, neurodegeneration, and cardiometabolic disorders. Therefore, an equitable level of regulation is required to maintain the equilibrium between inflammasome activation and inhibition. Recent advancement in the structural and mechanistic understanding of inflammasome assembly potentiates the emergence of novel therapeutics against inflammasome-regulated diseases. In this review, we have comprehensively discussed the recent and updated insights into the structure of inflammasome components, their activation, interaction, mechanism of regulation, and finally, the formation of densely packed filamentous inflammasome complex that exists as micron-sized punctum in the cells and mediates the immune responses.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/química , Caspasa 1/metabolismo , Proteínas de Unión al ADN/química , Humanos , Inflamasomas/química , Proteína con Dominio Pirina 3 de la Familia NLR/química , Dominios Proteicos , Multimerización de ProteínaRESUMEN
Neuroinflammation is one of the most common etiology in various neurological disorders and responsible for multi-array neurotoxic manifestations such as neurodegeneration, neurotransmitters alteration and cognitive dysfunction. NR (Nerolidol) is a natural bioactive molecule which possesses significant antioxidant and anti-inflammatory potential, but suffers from glitches of low solubility, low bioavailability and fast hepatic metabolism. In the current study, we fabricated nano-engineered lipid carrier of nerolidol (NR-NLC) for its effective delivery into the brain and explored its effect on neuroinflammation, neurotransmitters level and on dysfunctional behavioral attributes induced by CYC (cyclophosphamide). The binding affinity of nerolidol with NLRP3 and TLR-4 was performed which showed stong interaction between them. NR-NLC was prepared by the ultrasonication methods and particle size was determined by Zeta-sizer. Swiss Albino mice were divided into 5 groups (n = 6), assessed for behavioral dysfunction, and sacrificed on the fifteenth day following cyclophosphamide treatment. Brains were then removed and used for biochemical, histopathological, immunohistochemical and fluorescence microscopic analysis. Biochemical analysis showed increased levels of MDA, TNF-α, IL-6, IL-1ß, acetylcholine esterase, BDNF, 5-HT and dopamine, and reduced levels of SOD, CAT, GSH, IL-10, along with significant behavioral dysfunction in cyclophosphamide-treated animals. Significant neuronal damage was also observed in the histological study. Immunohistochemical analysis demonstrated increased expression of NLRP3 and caspase-1. Fluorescence microscopic analysis showed significant availability of NR-NLC in the hippocampus and cortex region. In contrast, treatment with NR-NLC effectively mitigated the aforementioned neurotoxic manifestation as compared to NR suspension. Our results showed potent neuroprotective effect of NR-NLC via modulation of oxidative stress, NLRP3 inflammasome, caspase-1 and neurotransmitter status.
Asunto(s)
Caspasa 1/biosíntesis , Ciclofosfamida/toxicidad , Sistemas de Liberación de Medicamentos/métodos , Proteína con Dominio Pirina 3 de la Familia NLR/biosíntesis , Nanopartículas/administración & dosificación , Sesquiterpenos/administración & dosificación , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Caspasa 1/química , Ingeniería Química/métodos , Ciclofosfamida/antagonistas & inhibidores , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/síntesis química , Lípidos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/química , Nanopartículas/química , Estructura Secundaria de Proteína , Sesquiterpenos/síntesis químicaRESUMEN
The apoptosis-associated Speck-like protein containing a caspase-1 recruitment domain (ASC), present in inflammasomes, regulates inflammation events and is involved in osteogenic phenotype. Nevertheless, its function in bone repair induced by bone substitute biomaterials is unclear. This study aimed to unveil the role of ASC on osteoprogenitor and tissue response to stoichiometric-hydroxyapatite (HA), nanostructured carbonated-hydroxyapatite (CHA), and CHA containing 5% Strontium (SrCHA), characterized previously by XRD, uXRF-SR, and FTIR spectroscopy implants. Thereafter, conditioned media by the biomaterials were used later to treat pre-osteoblasts and an osteogenic stimulus was shown in response to the materials, with higher expression of Runx2, Osterix, ALP, and Collagen 1a1 genes, with significant involvement of inflammatory-related genes. Thus, to better address the involvement of inflammasome, primary cells obtained from both genotypes [Wild-Type (WT) and ASC Knockout (ASC-KO) mice] were subjected to conditioned media up to 7 days, and our data reinforces both HA and CHA induces lower levels of alkaline phosphatase (ALP) than SrCHA, considering both genotypes (p < 0.01), and ASC seems contribute with osteogenic stimulus promoted by SrCHA. Complimentarily, the biomaterials were implanted into both subcutaneous and bone defects in tibia. Histological analysis on 28 days after implantation of biomaterials into mice's subcutaneous tissue revealed moderate inflammatory response to them. Both histomorphometry and µCT analysis of tibias indicated that the biomaterials did not reverse the delay in bone repair of ASC KO, reinforcing the involvement of ASC on bone regeneration and bone de novo deposition. Also, the bone density in CHA was >2-fold higher in WT than ASC-KO samples. HA was virtually not resorbed throughout the experimental periods, in opposition to CHA in the WT group. CHA reduced to half-area after 28 days, and the bone deposition was higher in CHA for WT mice than HA. Taken together, our results show that biomaterials did not interfere with the healing pattern of the ASC KO, but CHA promoted higher bone deposition in the WT group, probably due to its greater biodegradability. These results reinforce the importance of ASC during bone de novo deposition and healing.
Asunto(s)
Materiales Biocompatibles/química , Sustitutos de Huesos/química , Caspasa 1/química , Animales , Apoptosis/efectos de los fármacos , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/uso terapéutico , Enfermedades Óseas/diagnóstico por imagen , Enfermedades Óseas/patología , Enfermedades Óseas/terapia , Sustitutos de Huesos/farmacología , Sustitutos de Huesos/uso terapéutico , Carbonatos/química , Caspasa 1/deficiencia , Caspasa 1/genética , Células Cultivadas , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Durapatita/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nanoestructuras/química , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Prótesis e Implantes , Estroncio/química , Tibia/diagnóstico por imagen , Tibia/patologíaRESUMEN
Pro-inflammatory activation of caspase-1 in the neurodegenerative pathway has been associated with age-dependent cognitive impairment and Alzheimer's disease (AD) in humans. A recent report highlighted 2,4-diaminopyrimidine ring as an essential fragment in the inhibition of human caspase-1. However, the role of the ring and its enzyme inhibitory mechanism is not thoroughly investigated at the molecular level. The purpose of this study is therefore in twofold: (1) to understand the enzyme binding mechanism of the 2,4-diaminopyrimidine ring and (2) to search for more potent caspase-1 inhibitors that contain the ring, using integrative per-residue energy decomposition (PRED) pharmacophore modeling. Ligand interaction profile of a reference compound revealed a peculiar hydrogen formation of the amino group of 2,4-diaminopyrimidine with active site residue Arg341, possibly forming the bases for its inhibitory prowess against caspase-1. A generated pharmacophore model for structure-based virtual screening identified compounds, ZINC724667, ZINC09908119, and ZINC09933770, as potential caspase-1 inhibitors that possessed desirable pharmacokinetic and physiochemical properties. Further analyses revealed active site residues, Arg179, Ser236, Cys285, Gln283, Ser339, and Arg341, as crucial to inhibitor binding by stabilizing and forming hydrogen bonds, hydrophobic, and pi-pi interactions with the 2,4-diaminopyrimidine rings. Common interaction patterns of the hits could have accounted for their selective and high-affinity ligand binding, which was characterized by notable disruptions in caspase-1 structural architecture. These compounds could further be explored as potential leads in the development of novel caspase-1 inhibitors.
Asunto(s)
Enfermedad de Alzheimer , Caspasa 1/química , Inhibidores de Caspasas/química , Pirimidinas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Inhibidores de Caspasas/uso terapéutico , Humanos , Pirimidinas/uso terapéuticoRESUMEN
The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.
Asunto(s)
Inflamasomas/ultraestructura , Complejos Multiproteicos/ultraestructura , Proteínas de Unión a Fosfato/ultraestructura , Piroptosis/genética , Animales , Caspasa 1/química , Caspasa 1/genética , Caspasa 1/ultraestructura , Caspasas Iniciadoras/química , Caspasas Iniciadoras/genética , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inflamasomas/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Proteínas de Unión a Fosfato/química , Proteínas de Unión a Fosfato/genética , Conformación Proteica en Lámina beta/genética , Dominios Proteicos/genética , Procesamiento Proteico-Postraduccional/genética , ProteolisisRESUMEN
Photothermal therapy (PTT) is recently clinically established cancer therapy that uses near-infrared light for thermal ablation of solid tumors. The biopolymer N-dihydrogalactochitosan (GC) was shown in multiple reports to act as a very effective adjunct to tumor PTT. In the present study, mouse tumor model SCCVII (squamous cell carcinoma) was used with two protocols, in situ tumor PTT and therapeutic PTT vaccine for tumors, for investigating the effects of GC. The results reveal that GC can potentiate tumoricidal action of PTT through both direct and indirect mechanisms. In addition to previously known capacity of GC for activating immune effector cells, the indirect means is shown to include reducing the populations of immunoregulatory T cells (Tregs) in PTT-treated tumors. Testing the effects of GC on PTT-treated SCCVII tumor cells in vitro uncovered the existence of a direct mechanism evident by reduced colony survival of these cells. Fluorescence microscopy demonstrated increased binding of fluorescein-labeled GC to PTT-treated compared to untreated SCCVII cells that can be blocked by pre-exposure to annexin V. The results of additional in vitro testing with specific inhibitors demonstrate that these direct mechanisms do not involve the engagement of death surface receptors that trigger extrinsic apoptosis pathway signaling but may be linked to pro-survival activity of caspase-1. Based on the latter, it can be suggested that GC-promoted killing of PTT-treated cells stems from interference of GC bound to damaged membrane components with the repair of these structures that consequently hinders cell survival.
Asunto(s)
Quitosano/química , Láseres de Semiconductores , Fototerapia/métodos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Caspasa 1/química , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quitosano/farmacología , Modelos Animales de Enfermedad , Proteína Ligando Fas/metabolismo , Fluoresceína/química , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Ratones , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Receptor fas/metabolismoRESUMEN
AIMS: Prior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice. MAIN METHODS: In order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice. KEY FINDINGS: There was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment. SIGNIFICANCE: The silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Calpaína/antagonistas & inhibidores , Caspasa 1/química , Estrés del Retículo Endoplásmico , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/patología , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Calpaína/genética , Calpaína/metabolismo , Caspasa 1/genética , Caspasa 1/metabolismo , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Inflamasomas , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Poxviruses encode many proteins that enable them to evade host anti-viral defense mechanisms. Spi-2 proteins, including Cowpox virus CrmA, suppress anti-viral immune responses and contribute to poxviral pathogenesis and lethality. These proteins are 'serpin' protease inhibitors, which function via a pseudosubstrate mechanism involving initial interactions between the protease and a cleavage site within the serpin. A conformational change within the serpin interrupts the cleavage reaction, deforming the protease active site and preventing dissociation. Spi-2 proteins like CrmA potently inhibit caspases-1, -4 and -5, which produce proinflammatory cytokines, and caspase-8, which facilitates cytotoxic lymphocyte-mediated target cell death. It is not clear whether both of these functions are equally perilous for the virus, or whether only one must be suppressed for poxviral infectivity and spread but the other is coincidently inhibited merely because these caspases are biochemically similar. We compared the caspase specificity of CrmA to three orthologs from orthopoxviruses and four from more distant chordopoxviruses. All potently blocked caspases-1, -4, -5 and -8 activity but exhibited negligible inhibition of caspases-2, -3 and -6. The orthologs differed markedly in their propensity to inhibit non-mammalian caspases. We determined the specificity of CrmA mutants bearing various residues in positions P4, P3 and P2 of the cleavage site. Almost all variants retained the ability to inhibit caspase-1, but many lacked caspase-8 inhibitory activity. The retention of Spi-2 proteins' caspase-8 specificity during chordopoxvirus evolution, despite this function being readily lost through cleavage site mutagenesis, suggests that caspase-8 inhibition is crucial for poxviral pathogenesis and spread.
Asunto(s)
Caspasa 1 , Caspasa 8 , Virus de la Viruela Vacuna , Proteolisis , Serpinas , Proteínas Virales , Caspasa 1/química , Caspasa 1/genética , Caspasa 1/metabolismo , Caspasa 8/química , Caspasa 8/genética , Caspasa 8/metabolismo , Línea Celular , Virus de la Viruela Vacuna/química , Virus de la Viruela Vacuna/genética , Virus de la Viruela Vacuna/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Serpinas/química , Serpinas/genética , Serpinas/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
BACKGROUND/AIMS: Diabetic cardiomyopathy (DCM) is a common complication of diabetes and can cause heart failure, arrhythmia and sudden death. The pathogenesis of DCM includes altered metabolism, mitochondrial dysfunction, oxidative stress, inflammation, cell death and extracellular matrix remodeling. Recently, pyroptosis, a type of programmed cell death related to inflammation, was proven to be activated in DCM. However, the molecular mechanisms underlying pyroptosis in DCM remain elusive. The long non-coding RNA (lncRNA) Kcnq1ot1 participates in many cardiovascular diseases. This study aims to clarify whether Kcnq1ot1 affects cardiac pyroptosis in DCM. METHODS: AC16 cells and primary cardiomyocytes were incubated with 5.5 and 50 mmol/L glucose. Diabetic mice were induced with streptozotocin (STZ). Kcnq1ot1 was silenced both in vitro and in vivo. qRT-PCR was used to detect the expression level of Kcnq1ot1. Immunofluorescence, qRT-PCR and western blot analyses were used to detect the degree of pyroptosis. Echocardiography, hematoxylin and eosin staining, and Masson's trichrome staining were used to detect the cardiac function and morphology in mice. Cell death and function were detected using TUNEL staining, immunofluorescence staining and Ca2+ measurements. RESULTS: The expression of Kcnq1ot1 was increased in patients with diabetes, high glucose-induced cardiomyocytes and diabetic mouse cardiac tissue. Silencing Kcnq1ot1 alleviated pyroptosis by targeting miR-214-3p and caspase-1. Furthermore, silencing Kcnq1ot1 reduced cell death, cytoskeletal structure abnormalities and calcium overload in vitro and improved cardiac function and morphology in vivo. CONCLUSION: Kcnq1ot1 is overexpressed in DCM, and silencing Kcnq1ot1 inhibits pyroptosis by influencing miR-214-3p and caspase-1 expression. We clarified for the first time that Kcnq1ot1 could be a new therapeutic target for DCM.
Asunto(s)
Cardiomiopatías Diabéticas/patología , Piroptosis , ARN Largo no Codificante/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Antagomirs/metabolismo , Caspasa 1/química , Caspasa 1/genética , Caspasa 1/metabolismo , Células Cultivadas , Citoesqueleto/química , Citoesqueleto/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Cardiomiopatías Diabéticas/etiología , Cardiomiopatías Diabéticas/genética , Glucosa/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , MicroARNs/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis/efectos de los fármacos , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes proinflammatory cytokines and Gasdermin D to induce cytokine maturation and pyroptotic cell death. Here, we present cryo-electron microscopy (cryo-EM) structures of NLRC4 CARD and ASC CARD filaments mediated by conserved three types of asymmetric interactions (types I, II, and III). We find that the CARDs of these two adaptor proteins share a similar assembly pattern, which matches that of the caspase-1 CARD filament whose structure we defined previously. These data indicate a unified mechanism for downstream caspase-1 recruitment through CARD-CARD interactions by both adaptors. Using structure modeling, we further show that full-length NLRC4 assembles via two separate symmetries at its CARD and its nucleotide-binding domain (NBD), respectively.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/química , Proteínas de Unión al Calcio/química , Caspasa 1/química , Microscopía por Crioelectrón , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasa 1/genética , Caspasa 1/metabolismo , Activación Enzimática , Humanos , Inflamasomas , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismoRESUMEN
BACKGROUND/AIMS: Alcohol abuse exerts deleterious effects on the internal organs of the body, and alcohol-related gastritis is a common disease for which prompt treatment is essential to prevent the condition from growing worse. However, the therapeutic methods have some adverse effects. Determining the pathogenic mechanisms of alcoholic gastritis is therefore essential. METHODS: The MTT assay was developed in order to determine the optimal concentration of alcohol needed to treat gastric mucosal cells. The effects of alcohol on the gastric mucosal cells were determined by qRT-PCR and western blot. The release of IL-1ß and IL-18 were determined by ELISA assay. The immunofluorescence assay was used to detect caspase-1 activation levels, while immunohistochemical assay and HE staining were performed to identify the effectiveness of the caspase-1 inhibitor on alcoholic gastritis. The TUNEL assay was used to determine DNA fragmentation. RESULTS: Here, we clarified that ethanol treatment could cause cell DNA damage, activate caspase-1, and promote the generation and release of IL-1ß and IL-18. In other words, ethanol could induce pyroptosis. Interestingly, a caspase-1 inhibitor could significantly suppress pyroptosis, decrease the release of inflammatory cytokines induced by ethanol, and cause no side effects in vivo and in vitro. CONCLUSION: Collectively, our results showed that pyroptosis is involved in the pathogenesis of alcohol-induced gastritis and that caspase-1 inhibitor Ac-yvad-cmk could effectively decrease the damage caused by alcohol, making it a potentially promising agent for the treatment of alcoholic gastritis.
Asunto(s)
Etanol/toxicidad , Gastritis/patología , Piroptosis/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Caspasa 1/química , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacología , Línea Celular , Daño del ADN/efectos de los fármacos , Gastritis/inducido químicamente , Humanos , Inmunohistoquímica , Interleucina-18/análisis , Interleucina-1beta/análisis , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease. NLRP3 inflammasome activation has been widely studied in the pathogenesis of NAFLD. Cathepsin B (CTSB) is a ubiquitous cysteine cathepsin, and the role of CTSB in the progression and development of NAFLD has received extensive concern. However, the exact roles of CTSB in the NAFLD development and NLRP3 inflammasome activation are yet to be evaluated. In the present study, we used methionine choline-deficient (MCD) diet to establish mice NASH model. CTSB inhibitor (CA-074) was used to suppress the expression of CSTB. Expressions of CTSB and caspase-1 were evaluated by immunohistochemical staining. Serum IL-1ß and IL-18 levels were also determined. Palmitic acid was used to stimulate Kupffer cells (KCs), and protein expressions of CTSB, NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), and caspase-1 in KCs were detected. The levels of IL-1ß and IL-18 in the supernatant of KCs were evaluated by enzyme-linked immunosorbent assay (ELISA). Our results showed that CTSB inhibition improved the liver function and reduced hepatic inflammation and ballooning, and the levels of pro-inflammatory cytokines IL-1ß and IL-18 were decreased. The expressions of CTSB and caspase-1 in liver tissues were increased in the NASH group. In in vitro experiments, PA stimulation could increase the expressions of CTSB and NLRP3 inflammasome in KCs, and CTSB inhibition downregulated the expression of NLRP3 inflammasome in KCs, when challenged by PA. Moreover, CTSB inhibition effectively suppressed the expression and activity of caspase-1 and subsequently secretions of IL-1ß and IL-18. Collectively, these results suggest that CTSB inhibition limits NLRP3 inflammasome-dependent NASH formation through regulating the expression and activity of caspase-1, thus providing a novel anti-inflammatory signal pathway for the therapy of NAFLD.
Asunto(s)
Caspasa 1/metabolismo , Catepsina B/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/uso terapéutico , Dipéptidos/uso terapéutico , Modelos Animales de Enfermedad , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/uso terapéutico , Caspasa 1/química , Catepsina B/metabolismo , Células Cultivadas , Inhibidores de Cisteína Proteinasa/administración & dosificación , Dipéptidos/administración & dosificación , Activación Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente Indirecta , Inmunohistoquímica , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inyecciones Intraperitoneales , Interleucina-18/antagonistas & inhibidores , Interleucina-18/sangre , Interleucina-1beta/sangre , Interleucina-1beta/metabolismo , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/agonistas , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad del Hígado Graso no Alcohólico/inmunología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Distribución AleatoriaRESUMEN
A series of caspase inhibitors containing γ-amino acid moiety have been synthesized. A systemic study on their structure-activity relationship of anti-apoptotic cellular activity is presented. These efforts led to the discovery of compound 20o as a potent caspase inhibitor, which demonstrated preclinical ameliorating total bilirubin efficacy with a significantly improved pharmacokinetic profile.