RESUMEN
Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.
Asunto(s)
Caspasa 6/inmunología , Caspasa 6/metabolismo , Inflamasomas/inmunología , Animales , Apoptosis/inmunología , Muerte Celular/inmunología , Inmunidad Innata , Inflamasomas/metabolismo , Inflamasomas/fisiología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Necroptosis/inmunología , Unión Proteica , Piroptosis/inmunología , Proteínas de Unión al ARN/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2 (refs. 1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1 (ref. 4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.
Asunto(s)
Ácido Aspártico , Caspasa 6 , Infecciones por Coronavirus , Coronavirus , Cisteína , Interacciones Huésped-Patógeno , Replicación Viral , Animales , Apoptosis , Ácido Aspártico/metabolismo , Caspasa 6/metabolismo , Coronavirus/crecimiento & desarrollo , Coronavirus/patogenicidad , Infecciones por Coronavirus/enzimología , Infecciones por Coronavirus/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Cricetinae , Cisteína/metabolismo , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Humanos , Interferones/antagonistas & inhibidores , Interferones/inmunología , Pulmón/patología , Mesocricetus , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , SARS-CoV-2 , Tasa de Supervivencia , Pérdida de PesoRESUMEN
Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Ingeniería de Proteínas , Caspasa 6/metabolismo , Línea Celular , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal , Inhibidores de ProteasomaRESUMEN
Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.
Asunto(s)
Células de la Médula Ósea , Inhibidores de Caspasas , Diferenciación Celular , Osteoclastos , Ligando RANK , Animales , Ratones , Células de la Médula Ósea/metabolismo , Resorción Ósea/metabolismo , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Células Cultivadas , Ratones Endogámicos C57BL , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Fosfatasa Ácida Tartratorresistente/metabolismoRESUMEN
In Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce proteolytic cleavage and ameliorate the consequences of mutant HTT without lowering total HTT levels. To that end, we use an antisense oligonucleotide (AON) that targets HTT pre-mRNA and induces partial skipping of exon 12, which contains the critical caspase-6 cleavage site. Here, we show that AON-treatment can partially restore the phenotype of YAC128 mice, a mouse model expressing the full-length human HTT gene including 128 CAG-repeats. Wild-type and YAC128 mice were treated intracerebroventricularly with AON12.1, scrambled AON or vehicle starting at 6 months of age and followed up to 12 months of age, when MRI was performed and mice were sacrificed. AON12.1 treatment induced around 40% exon skip and protein modification. The phenotype on body weight and activity, but not rotarod, was restored by AON treatment. Genes differentially expressed in YAC128 striatum changed toward wild-type levels and striatal volume was preserved upon AON12.1 treatment. However, scrambled AON also showed a restorative effect on gene expression and appeared to generally increase brain volume.
Asunto(s)
Enfermedad de Huntington , Animales , Humanos , Ratones , Caspasa 6/genética , Caspasa 6/metabolismo , Cuerpo Estriado/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Oligonucleótidos Antisentido/farmacología , FenotipoRESUMEN
Cysteinyl aspartate specific proteinase (caspase)-6 belongs to the caspase family and plays a vital role in mediating cell death. Under certain conditions, three pathways of programmed cell death (PCD), including apoptosis, necroptosis and pyroptosis (PANoptosis), transform one way into another, with enormous therapeutic potential. Initially, scholars reported that caspase-6 is a caspase executor that mediates apoptosis. With the ceaseless exploration of the PCD types, studies have demonstrated that caspase-6 mediates pyroptosis by regulating gasdermin D and mediates necroptosis by regulating mixed lineage kinase domain-like. By regulating PANoptosis, caspase-6 plays a crucial role in tumorigenesis in humans and mediates anti-tumour immunity. Therefore, a comprehensive understanding of caspase-6 function in cancer via PANoptosis is important for the prevention and therapy of tumours. This article summarized the function of caspase-6 in PANoptosis and its impact on cancer development, providing targets and strategies for tumour treatment.
Asunto(s)
Apoptosis , Neoplasias , Humanos , Caspasa 6/metabolismo , Piroptosis , Caspasas/metabolismo , Caspasa 8/metabolismoRESUMEN
Caspases are a family of cysteine-dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human diseases. Classical chemical tools to study caspase functions lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we targeted a non-catalytic cysteine residue (C264) unique to caspase-6 (C6), an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used a structure-informed covalent ligand design to produce potent, irreversible inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over other caspase family members and high proteome selectivity. This approach and the new tools described will enable rigorous interrogation of the role of caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases.
Asunto(s)
Caspasas , Cisteína , Humanos , Caspasa 6 , Apoptosis , Inhibidores de Cisteína Proteinasa/farmacologíaRESUMEN
BACKGROUND AND AIMS: NAFLD represents an increasing health problem in association with obesity and diabetes with no effective pharmacotherapies. Growing evidence suggests that several FGFs play important roles in diverse aspects of liver pathophysiology. Here, we report a previously unappreciated role of FGF4 in the liver. APPROACH AND RESULTS: Expression of hepatic FGF4 is inversely associated with NAFLD pathological grades in both human patients and mouse models. Loss of hepatic Fgf4 aggravates hepatic steatosis and liver damage resulted from an obesogenic high-fat diet. By contrast, pharmacological administration of recombinant FGF4 mitigates hepatic steatosis, inflammation, liver damage, and fibrogenic markers in mouse livers induced to develop NAFLD and NASH under dietary challenges. Such beneficial effects of FGF4 are mediated predominantly by activating hepatic FGF receptor (FGFR) 4, which activates a downstream Ca2+ -Ca2+ /calmodulin-dependent protein kinase kinase beta-dependent AMP-activated protein kinase (AMPK)-Caspase 6 signal axis, leading to enhanced fatty acid oxidation, reduced hepatocellular apoptosis, and mitigation of liver damage. CONCLUSIONS: Our study identifies FGF4 as a stress-responsive regulator of liver pathophysiology that acts through an FGFR4-AMPK-Caspase 6 signal pathway, shedding light on strategies for treating NAFLD and associated liver pathologies.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Caspasa 6/metabolismo , Caspasa 6/farmacología , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/metabolismo , Factor 4 de Crecimiento de Fibroblastos/metabolismo , Factor 4 de Crecimiento de Fibroblastos/farmacología , Factor 4 de Crecimiento de Fibroblastos/uso terapéutico , Humanos , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/uso terapéuticoRESUMEN
BACKGROUND: Caspase 6 is an essential regulator in innate immunity, inflammasome activation and host defense. We aimed to characterize the causal mechanism of Caspase 6 in liver sterile inflammatory injury. METHODS: Human liver tissues were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. Subsequently, we created Caspase 6-knockout (Caspase 6KO) mice to analyze roles and molecular mechanisms of macrophage Caspase 6 in murine models of liver ischemia/reperfusion (IR) injury. RESULTS: In human liver biopsies, Caspase 6 expression was positively correlated with more severe histopathological injury and higher serum ALT/AST level at one day postoperatively. Moreover, Caspase 6 was mainly elevated in macrophages but not hepatocytes in ischemic livers. Unlike in controls, the Caspase 6-deficient livers were protected against IR injury, as evidenced by inhibition of inflammation, oxidative stress and iron overload. Disruption of macrophage NF-κB essential modulator (NEMO) in Caspase 6-deficient livers deteriorated liver inflammation and ferroptosis. Mechanistically, Caspase 6 deficiency spurred NEMO-mediated IκBα phosphorylation in macrophage. Then phosphorylated-inhibitor of NF-κBα (p-IκBα) co-localized with receptor-interacting serine/ threonine-protein kinase 1 (RIPK1) in the cytoplasm to degradate RIPK1 under inflammatory conditions. The disruption of RIPK1-IκBα interaction preserved RIPK1 degradation, triggering downstream apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and inciting NIMA-related kinase 7/NOD-like receptor family pyrin domain containing 3 (NEK7/NLRP3) activation in macrophages. Moreover, ablation of macrophage RIPK1 or ASK1 diminished NEK7/NLRP3-driven inflammatory response and dampened hepatocyte ferroptosis by reducing HMGB1 release from macrophages. CONCLUSIONS: Our findings underscore a novel mechanism of Caspase 6 mediated RIPK1-IκBα interaction in regulating macrophage NEK7/NLRP3 function and hepatocytes ferroptosis, which provides therapeutic targets for clinical liver IR injury. Video Abstract.
Asunto(s)
Caspasa 6 , Inmunidad Innata , Transducción de Señal , Animales , Humanos , Ratones , Caspasa 6/metabolismo , Inflamación/metabolismo , Isquemia/metabolismo , Hígado/metabolismo , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
The innate immune system acts as the first line of defense against infection. One key component of the innate immune response to gram-negative bacterial infections is inflammasome activation. The caspase-11 (CASP11)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is activated by cytosolic lipopolysaccharide, a gram-negative bacterial cell wall component, to trigger pyroptosis and host defense during infection. Although several cellular signaling pathways have been shown to regulate CASP11-NLRP3 inflammasome activation in response to lipopolysaccharide, the upstream molecules regulating CASP11 activation during infection with live pathogens remain unclear. Here, we report that the understudied caspase-6 (CASP6) contributes to the activation of the CASP11-NLRP3 inflammasome in response to infections with gram-negative bacteria. Using in vitro cellular systems with bone marrow-derived macrophages and 293T cells, we found that CASP6 can directly process CASP11 by cleaving at Asp59 and Asp285, the CASP11 auto-cleavage sites, which could contribute to the activation of CASP11 during gram-negative bacterial infection. Thus, the loss of CASP6 led to impaired CASP11-NLRP3 inflammasome activation in response to gram-negative bacteria. These results demonstrate that CASP6 potentiates activation of the CASP11-NLRP3 inflammasome to produce inflammatory cytokines during gram-negative bacterial infections.
Asunto(s)
Caspasa 6/fisiología , Caspasas Iniciadoras/metabolismo , Infecciones por Bacterias Gramnegativas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
AIM: Tau truncation (tr-tau) by active caspase-6 (aCasp-6) generates tau fragments that may be toxic. Yet the relationship between aCasp-6, different forms of tr-tau and hyperphosphorylated tau (p-tau) accumulation in human brains with Alzheimer's disease (AD) and other tauopathies remains unclear. METHODS: We generated two neoepitope monoclonal antibodies against tr-tau sites (D402 and D13) targeted by aCasp-6. Then, we used five-plex immunofluorescence to quantify the neuronal and astroglial burden of aCasp-6, tr-tau, p-tau and their co-occurrence in healthy controls, AD and primary tauopathies. RESULTS: Casp-6 activation was strongest in AD and Pick's disease (PiD) but almost absent in 4-repeat (4R) tauopathies. In neurons, the tr-tau burden was much more abundant in AD and PiD than in 4R tauopathies and disproportionally higher when normalising by p-tau pathology. Tr-tau astrogliopathy was detected in low numbers in 4R tauopathies. Unexpectedly, about half of tr-tau positive neurons in AD and PiD lacked p-tau aggregates, a finding we confirmed using several p-tau antibodies. CONCLUSIONS: Early modulation of aCasp-6 to reduce tr-tau pathology is a promising therapeutic strategy for AD and PiD but is unlikely to benefit 4R tauopathies. The large percentage of tr-tau-positive neurons lacking p-tau suggests that many vulnerable neurons to tau pathology go undetected when using conventional p-tau antibodies. Therapeutic strategies against tr-tau pathology could be necessary to modulate the extent of tau abnormalities in AD. The disproportionally higher burden of tr-tau in AD and PiD supports the development of biofluid biomarkers against tr-tau to detect AD and PiD and differentiate them from 4R tauopathies at a patient level.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Encéfalo/patología , Caspasa 6 , Humanos , Neuronas/patología , Tauopatías/diagnóstico , Tauopatías/patología , Tauopatías/terapia , Proteínas tau/metabolismoRESUMEN
INTRODUCTION: Protective effect of royal jelly (RJ) on fluoride-induced nephrotoxicity was investigated in this study. METHODS: 42 healthy male Wistar rats (n = 42, 8 weeks of age) were divided equally into 6 groups with 7 rats in each; (1) Group-1: Controls fed with standard diet; (2) Group-2: RJ [100 mg/kg] bw (body weight), by oral gavage; (3) Group-3: Fluoride [50 mg/kg] bw, in drinking water; (4) Group-4: Fluoride [100 mg/kg] bw, in drinking water; (5) Group-5: RJ [100 mg/kg] bw, by oral gavage + Fluoride [50 mg/kg] bw, in drinking water; (6) Group-6: RJ [100 mg/kg] bw, by oral gavage + Fluoride [100 mg/kg] bw, in drinking water. After 8 weeks, all rats were decapitated and their kidney tissues were removed for further analysis. The protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, VEGF, GSK-3, BDNF, COX-2 and TNF-α proteins in kidney tissue were analysed by western blotting technique. RESULTS: RJ increased Bcl-2, COX-2, GSK-3, TNF-α and VEGF protein levels and a decreased caspase-3, caspase -6, caspase-9, Bax and BDNF protein levels in fluoride-treated rats. CONCLUSION: RJ application may have a promising therapeutical potential in the treatment of many diseases in the future by reducing kidney damage.
Asunto(s)
Ácidos Grasos , Enfermedades Renales , Animales , Masculino , Ratas , Antioxidantes/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Biomarcadores , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Caspasa 6/farmacología , Caspasa 9/metabolismo , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 2/farmacología , Fluoruros/toxicidad , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/farmacología , Riñón , Enfermedades Renales/inducido químicamente , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/prevención & control , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ácidos Grasos/farmacologíaRESUMEN
Glial fibrillary acidic protein (GFAP) is the major intermediate filament III protein of astroglia cells which is upregulated in traumatic brain injury (TBI). Here we reported that GFAP is truncated at both the C- and N-terminals by cytosolic protease calpain to GFAP breakdown products (GBDP) of 46-40K then 38K following pro-necrotic (A23187) and pro-apoptotic (staurosporine) challenges to primary cultured astroglia or neuron-glia mixed cells. In addition, with another pro-apoptotic challenge (EDTA) where caspases are activated but not calpain, GFAP was fragmented internally, generating a C-terminal GBDP of 20 kDa. Following controlled cortical impact in mice, GBDP of 46-40K and 38K were formed from day 3 to 28 post-injury. Purified GFAP protein treated with calpain-1 and -2 generates (i) major N-terminal cleavage sites at A-56*A-61 and (ii) major C-terminal cleavage sites at T-383*Q-388, producing a limit fragment of 38K. Caspase-6 treated GFAP was cleaved at D-78/R-79 and D-225/A-226, where GFAP was relatively resistant to caspase-3. We also derived a GBDP-38K N-terminal-specific antibody which only labels injured astroglia cell body in both cultured astroglia and mouse cortex and hippocampus after TBI. As a clinical translation, we observed that CSF samples collected from severe human TBI have elevated levels of GBDP-38K as well as two C-terminally released GFAP peptides (DGEVIKES and DGEVIKE). Thus, in addition to intact GFAP, both the GBDP-38K as well as unique GFAP released C-terminal proteolytic peptides species might have the potential in tracking brain injury progression.
Asunto(s)
Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Animales , Astrocitos/metabolismo , Biomarcadores , Calpaína/metabolismo , Caspasa 6 , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Filamentos Intermedios/metabolismo , Ratones , Péptido Hidrolasas , PéptidosRESUMEN
Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. Temozolomide (TMZ) is the first-line chemotherapeutic drug for treating GBM. However, drug resistance is still a challenging issue in GBM therapy. Our preliminary results showed upregulation of androgen receptor (AR) gene expression in human GBM tissues. This study was designed to evaluate the effects of enzalutamide, a specific inhibitor of the AR, on killing drug-resistant and -sensitive glioblastoma cells and the possible mechanisms. Data mining from The Cancer Genome Atlas (TCGA) database revealed upregulation of AR messenger (m)RNA and protein expressions in human GBM tissues, especially in male patients, compared to normal human brains. In addition, expressions of AR mRNA and protein in human TMZ-sensitive U87 MG and -resistant U87 MG-R glioblastoma cells were elevated compared to normal human astrocytes. Exposure of human U87 MG and U87 MG-R cells to enzalutamide concentration- and time-dependently decreased cell viability. As to the mechanism, enzalutamide killed these two types of glioblastoma cells via an apoptotic mechanism. Specifically, exposure to enzalutamide augmented enzyme activities of caspase-9 rather than those of caspase-8. Moreover, enzalutamide successively triggered an elevation in levels of the proapoptotic Bax protein, a reduction in the mitochondrial membrane potential, release of cytochrome c, cascade activation of caspases-3 and -6, DNA fragmentation, and cell apoptosis in human TMZ-sensitive and -resistant glioblastoma cells. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, caused significant attenuations in enzalutamide-induced morphological shrinkage, DNA damage, and apoptotic death. Taken together, this study showed that enzalutamide could significantly induce apoptotic insults to human drug-resistant and -sensitive glioblastoma cells via an intrinsic Bax-mitochondrion-cytochrome c-caspase cascade activation pathway. Enzalutamide has the potential to be a drug candidate for treating GBM by targeting the AR signaling axis.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Apoptosis , Benzamidas , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Caspasa 6/metabolismo , Caspasa 6/farmacología , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Glioblastoma/metabolismo , Humanos , Masculino , Mitocondrias/metabolismo , Nitrilos , Feniltiohidantoína , ARN/metabolismo , ARN Mensajero/metabolismo , Receptores Androgénicos/metabolismo , Temozolomida/farmacología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.
Asunto(s)
Caspasa 3/química , Caspasa 6/química , Caspasa 9/metabolismo , Secuencia de Aminoácidos/genética , Apoptosis/fisiología , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Caspasa 8/metabolismo , Caspasa 9/fisiología , Caspasas/metabolismo , Activación Enzimática , Humanos , Transducción de Señal/genéticaRESUMEN
The main purpose of this investigation was to evaluate the effect of anticancer active compounds (I-VIII) on zebrafish development in order to select the safest molecules. Larval mortality, embryo hatchability and malformations were end-points used to assess the acute toxicity among embryos and larvae from compounds-/pemetrexed-treated and control groups. LC50 and MNLC (maximal non-lethal concentration) were determined. Lipophilicity-dependent structure-toxicity relationships were established. The results clearly indicated that the majority of test molecules are safe for zebrafish individuals and simultaneously are less toxic than an anticancer agent - pemetrexed. The subsequent aim of this study was to elucidate the molecular mechanism of antiproliferative activity of the most selective compounds. Substantially increased activation of caspase-6 and -8 in cancerous cell lines confirmed the proapoptotic action of molecules examined. Considering the safety for zebrafish individuals, the title compounds as inducers of apoptosis are promising drug candidates in the preclinical phase of drug development.
Asunto(s)
Antineoplásicos/farmacología , Embrión no Mamífero/efectos de los fármacos , Fluorocarburos/farmacología , Larva/efectos de los fármacos , Triazinas/farmacología , Células A549 , Animales , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Caspasa 6/genética , Caspasa 6/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Embrión no Mamífero/anomalías , Embrión no Mamífero/metabolismo , Fluorocarburos/síntesis química , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Larva/anatomía & histología , Larva/crecimiento & desarrollo , Larva/metabolismo , Pemetrexed/toxicidad , Relación Estructura-Actividad , Pruebas de Toxicidad , Triazinas/síntesis química , Pez Cebra/crecimiento & desarrolloRESUMEN
Caspases are cysteine-aspartic proteases involved in the regulation of programmed cell death (apoptosis) and a number of other biological processes. Despite overall similarities in structure and active-site composition, caspases show striking selectivity for particular protein substrates. Exosites are emerging as one of the mechanisms by which caspases can recruit, engage, and orient these substrates for proper hydrolysis. Following computational analyses and database searches for candidate exosites, we utilized site-directed mutagenesis to identify a new exosite in caspase-6 at the hinge between the disordered N-terminal domain (NTD), residues 23-45, and core of the caspase-6 structure. We observed that substitutions of the tri-arginine patch Arg-42-Arg-44 or the R44K cancer-associated mutation in caspase-6 markedly alter its rates of protein substrate hydrolysis. Notably, turnover of protein substrates but not of short peptide substrates was affected by these exosite alterations, underscoring the importance of this region for protein substrate recruitment. Hydrogen-deuterium exchange MS-mediated interrogation of the intrinsic dynamics of these enzymes suggested the presence of a substrate-binding platform encompassed by the NTD and the 240's region (containing residues 236-246), which serves as a general exosite for caspase-6-specific substrate recruitment. In summary, we have identified an exosite on caspase-6 that is critical for protein substrate recognition and turnover and therefore highly relevant for diseases such as cancer in which caspase-6-mediated apoptosis is often disrupted, and in neurodegeneration in which caspase-6 plays a central role.
Asunto(s)
Caspasa 6/química , Mutación Missense , Proteínas de Neoplasias/química , Neoplasias/enzimología , Enfermedades Neurodegenerativas/enzimología , Sustitución de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Caspasa 6/genética , Caspasa 6/metabolismo , Humanos , Hidrólisis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Dominios ProteicosRESUMEN
Stroke is a common cerebrovascular disease. Inflammation-induced neuronal death is one of the key factors in stroke pathology. Propofol has been shown to ameliorate neuroinflammatory injury, but the exact mechanism of its neuroprotective role remains to be fully elucidated. In the present study, we found that inflammation was activated in ischemic cortical neurons, and the expression of nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 1 (NLRP1), NLRP3 inflammasome and effectors in primary cortical neurons increased. However, we found that propofol could inhibit the increased expression of NLRP1 and NLRP3 inflammasome induced by oxygen-glucose deprivation (OGD). Furthermore, the effector molecule caspase-1 (casp1) was revealed to be the downstream target of NLRP1 and propofol repressed the activation of caspase-1 via inhibiting NLRP1 in cortical neurons. Moreover, propofol inhibits caspase-6 activation in neurons through the NLRP1-caspase-1 pathway. Once the expression of caspase6 increases, propofol reduced its neuroprotective effect in OGD-treated cortical neurons. In the stroke middle cerebral artery occlusion (MCAO) model, infusion of caspase-6 inhibitors enhanced the protective effect of propofol on infarct size and neurological function. In conclusion, our results suggest that propofol plays a neuroprotective role in stroke by inhibiting the inflammatory pathway of NLRP1-caspase-1-caspase-6. Overall, these data suggest that propofol plays a key role in the inflammatory-dependent pathway after stroke, providing an important evidence for propofol as an effective strategy for neuroprotection in stroke.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Lesiones Encefálicas/prevención & control , Caspasa 1 , Caspasa 6 , Mediadores de Inflamación/antagonistas & inhibidores , Propofol/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Caspasa 1/metabolismo , Caspasa 6/metabolismo , Células Cultivadas , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Propofol/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Caspase-6 is critical to the neurodegenerative pathways of Alzheimer's, Huntington's, and Parkinson's diseases and has been identified as a potential molecular target for treatment of neurodegeneration. Thus, understanding the global and regional changes in dynamics and conformation provides insights into the unique properties of caspase-6 that may contribute to achieving control of its function. In this work, hydrogen/deuterium exchange MS (H/DX-MS) was used to map the local changes in the conformational flexibility of procaspase-6 at the discrete states that reflect the series of cleavage events that ultimately lead to the fully active, substrate-bound state. Intramolecular self-cleavage at Asp-193 evoked higher solvent exposure in the regions of the substrate-binding loops L1, L3, and L4 and in the 130s region, the intersubunit linker region, the 26-32 region as well as in the stabilized loop 2. Additional removal of the linker allowed caspase-6 to gain more flexibility in the 130s region and in the L2 region converting caspase-6 to a competent substrate-binding state. The prodomain region was found to be intrinsically disordered independent of the activation state of caspase-6; however, its complete removal resulted in the protection of the adjacent 26-32 region, suggesting that this region may play a regulatory role. The molecular details of caspase-6 dynamics in solution provide a comprehensive scaffold for strategic design of therapeutic approaches for neurodegenerative disorders.