RESUMEN
Alveolar epithelial barrier is a potential therapeutic target for acute respiratory distress syndrome (ARDS). However, an effective intervention against alveolar epithelial barrier has not been developed. Here, based on single-cell RNA and mRNA sequencing results, death receptor 3 (DR3) and its only known ligand tumor necrosis factor ligand-associated molecule 1A (TL1A) were significantly reduced in epithelium from an ARDS mice and cell models. The apparent reduction in the TL1A/DR3 axis in lungs from septic-ARDS patients was correlated with the severity of the disease. The examination of knockout (KO) and alveolar epithelium conditional KO (CKO) mice showed that TL1A deficiency exacerbated alveolar inflammation and permeability in lipopolysaccharide (LPS)-induced ARDS. Mechanistically, TL1A deficiency decreased glycocalyx syndecan-1 and tight junction-associated zonula occludens 3 by increasing cathepsin E level for strengthening cell-to-cell permeability. Additionally, DR3 deletion aggravated barrier dysfunction and pulmonary edema in LPS-induced ARDS through the above mechanisms based on the analyses of DR3 CKO mice and DR3 overexpression cells. Therefore, the TL1A/DR3 axis has a potential value as a key therapeutic signaling for the protection of alveolar epithelial barrier.
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Miembro 25 de Receptores de Factores de Necrosis Tumoral , Síndrome de Dificultad Respiratoria , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Animales , Ratones , Epitelio , Ligandos , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/genética , Factor de Necrosis Tumoral alfa , Miembro 25 de Receptores de Factores de Necrosis Tumoral/genética , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
Cancer chemotherapeutic drugs such as doxorubicin, mitomycin C, and gemcitabine, which are mostly small synthetic molecules, are still clinically useful for cancer treatment. However, despite considerable therapeutic efficacy, severe toxicity-associated problems, which are mainly caused by the non-specific mode of action such as chromosomal DNA damage and interference in the DNA replication even in normal cells, remain unresolved and a major challenge for safer and thus more widespread adoption of chemotherapy. Herein, an innovative platform is developed through beneficially integrating core peptide units into highly-ordered, stable, and flexibly guest-adaptable structure of apoferritin, which simultaneously fulfills high-capacity loading of chemotherapeutic drugs compared with the case of FDA-approved antibody-drug conjugates, efficient drug targeting to cancer cells, and cancer cell-specific drug release and activation. This approach dramatically reduces drug toxicity to normal cells, significantly enhances efficacy in in vivo cancer treatment without toxicity to normal organs of mice, and thus is expected to open up a novel clinical route to break through the limits of current cancer chemotherapy.
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Antineoplásicos , Neoplasias , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos , Ratones , Neoplasias/tratamiento farmacológico , PéptidosRESUMEN
Osteosarcoma (OS) is a malignant bone tumor characterized by poor prognosis due to its regional invasion and early metastasis. In this study, we aimed to find the role and the underlying mechanism of Cathepsin E (CTSE) in OS growth and metastasis. We found CTSE is upregulated in metastatic OS, rather than in the primary lesion, as confirmed by RT-qPCR and western blot analysis of clinical OS samples. Furthermore, both in vitro and in vivo experiments illustrated that CTSE promoted both growth and metastasis of OS cells, partially mediated through the modulation of Epithelial-Mesenchymal Transition (EMT). Bioinformatics analysis predicted that miR-185-5p downregulates CTSE via directly binding to the 3'UTR of CTSE, which was verified by luciferase reporter assay and rescue assays. This study reported for the first time that CTSE is a potential biomarker in OS tumorigenesis and metastasis, providing a promising therapeutic target for OS treatment.
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Neoplasias Óseas , Catepsina E , MicroARNs , Osteosarcoma , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Catepsina E/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Metástasis de la Neoplasia , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patologíaRESUMEN
Regulation of neuroinflammation and ß-amyloid (Aß) production are critical factors in the pathogenesis of Alzheimer's disease (AD). Cathepsin E (CatE), an aspartic protease, is widely studied as an inducer of growth arrest and apoptosis in several types of cancer cells. However, the function of CatE in AD is unknown. In this study, we demonstrated that the ablation of CatE in human amyloid precursor protein knock-in mice, called APPNL-G-F mice, significantly reduced Aß accumulation, neuroinflammation, and cognitive impairments. Mechanistically, microglial CatE is involved in the secretion of soluble TNF-related apoptosis-inducing ligand, which plays an important role in microglia-mediated NF-κB-dependent neuroinflammation and neuronal Aß production by beta-site APP cleaving enzyme 1. Furthermore, cannula-delivered CatE inhibitors improved memory function and reduced Aß accumulation and neuroinflammation in AD mice. Our findings reveal that CatE as a modulator of microglial activation and neurodegeneration in AD and suggest CatE as a therapeutic target for AD by targeting neuroinflammation and Aß pathology.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Catepsina E/genética , Catepsina E/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Enfermedades NeuroinflamatoriasRESUMEN
Aspartic proteases are important biomarkers of human disease and interesting targets for modulation of immune response via MHC class II antigen processing inhibition. The lack of inhibitors with sufficient selectivity hampers precise analysis of the role of cathepsin E and napsin A in samples containing the ubiquitous and highly abundant homolog cathepsin D. Grassystatins from marine cyanobacteria show promising selectivity for cathepsin E but contain several ester bonds that make their synthesis cumbersome and thus limit availability of the inhibitors. Herewith, we present grassystatin-derived cathepsin E inhibitors with greatly facilitated synthesis but retained selectivity profile. We demonstrate their affinity and selectivity with both enzyme kinetic assays and streptavidin-based pull-down from cells and mouse organs. Our findings suggest that grassystatin-like inhibitors are useful tools for targeted inhibition of cathepsin E and thus provide a novel approach for cancer and immunology research.
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Catepsina D/antagonistas & inhibidores , Catepsina E/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Péptidos/farmacología , Catepsina D/metabolismo , Catepsina E/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.
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Catepsina E/metabolismo , Fusión de Membrana/fisiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Proteínas del Envoltorio Viral/metabolismo , Animales , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Unión Proteica , ARN Interferente Pequeño/metabolismo , Porcinos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
We isolated and characterised a cDNA encoding the aspartic protease cathepsin E (CTSE) in Korean rose bitterling, Rhodeus uyekii. The full-length Rhodeus uyekii CTSE (RuCTSE) cDNA (1396 bp) contains an open reading frame of 1218 bp, encoding 405 amino acids. Alignment of multiple CTSE protein sequences revealed that two of the aspartyl protease active site residues and a disulphide bond were well-conserved among the other CTSE sequences. Phylogenetic analysis revealed that RuCTSE is most closely related to freshwater fish cathepsin E. RuCTSE is widely expressed in the liver, spleen, ovary, testis, brain, eye, intestine, muscle, fin, stomach, and kidney. This first report of teleost CTSE will provide important information related to the identification of other cathepsin E genes in various fish species and will serve as a useful molecular tool to help clarify biological activities in other teleosts.
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Proteasas de Ácido Aspártico/genética , Catepsina E/genética , Cyprinidae/inmunología , Proteínas de Peces/genética , Hígado/metabolismo , Ovario/metabolismo , Bazo/metabolismo , Animales , Proteasas de Ácido Aspártico/metabolismo , Catepsina E/metabolismo , Clonación Molecular , Secuencia Conservada/genética , Femenino , Proteínas de Peces/metabolismo , Especificidad de Órganos , Filogenia , Alineación de Secuencia , TranscriptomaRESUMEN
Cathepsin E (CTSE) is an intracellular, hydrolytic aspartic protease found to be expressed in cells of the immune and gastrointestinal systems, lymphoid tissues, erythrocytes, and cancer cells. The precise functions are not fully understood; however, various studies have investigated its numerous cell-type specific roles. CTSE expression has been shown to be a potential early biomarker for pancreatic ductal adenocarcinoma (PDAC). PDAC patients have low survival rates mostly due to the lack of early detection methods. CTSE-specific activity probes have been developed and tested to assist in tumor imaging and functional studies investigating the role of CTSE expression in PDAC tumors. Furthermore, a CTSE protease-specific, photodynamic therapy pro-drug was developed to explore its potential use to treat tumors that express CTSE. Since CTSE is expressed in pancreatic diseases that are risk factors for PDAC, such as pancreatic cysts and chronic pancreatitis, learning about its function in these disease types could assist in early PDAC detection and in understanding the biology of PDAC progression. Overall, CTSE expression and activity shows potential to detect PDAC and other pancreatic diseases. Further research is needed to fully understand its functions and potential translational applicability.
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Catepsina E/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , Biomarcadores de Tumor , Catepsina E/genética , HumanosRESUMEN
BACKGROUND: Increasing evidence supports a key role for inflammation in the neurodegenerative process of familial amyloidotic polyneuropathy (FAP). While there seems to be an overactivation of the neuronal interleukin-1 signaling pathway, the immune response is apparently compromised in FAP. Accordingly, little immune cell infiltration is observed around pre-fibrillar or fibrillar amyloid deposits, with the underlying mechanism for this phenomenon remaining poorly understood. Cathepsin E (CtsE) is an important intermediate for antigen presentation and chemotaxis, but its role in the pathogenesis of FAP disease remains unknown. METHODS: In this study, we used both mouse primary macrophages and in vivo studies based on transgenic models of FAP and human samples to characterize CtsE expression in different physiological systems. RESULTS: We show that CtsE is critically decreased in bone marrow-derived macrophages from a FAP mouse model, possibly contributing for cell function impairment. Compromised levels of CtsE were also found in injured nerves of transgenic mice and, most importantly, in naïve peripheral nerves, sensory ganglia, murine stomach, and sural nerve biopsies derived from FAP patients. Expression of CtsE in tissues was associated with transthyretin (TTR) deposition and differentially regulated accordingly with the physiological system under study. Preventing deposition with a TTR small interfering RNA rescued CtsE in the peripheral nervous system (PNS). In contrast, the expression of CtsE increased in splenic cells (mainly monocytes) or peritoneal macrophages, indicating a differential macrophage phenotype. CONCLUSION: Altogether, our data highlights the potential of CtsE as a novel FAP biomarker and a possible modulator for innate immune cell chemotaxis to the disease most affected tissues-the peripheral nerve and the gastrointestinal tract.
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Neuropatías Amiloides Familiares/genética , Neuropatías Amiloides Familiares/inmunología , Catepsina E/genética , Catepsina E/inmunología , Inmunidad Celular/inmunología , Adulto , Neuropatías Amiloides Familiares/patología , Animales , Catepsina E/biosíntesis , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana EdadRESUMEN
Microbial products influence immunity after allogeneic hematopoietic stem cell transplantation (allo-SCT). In this context, the role of cathepsin E (Ctse), an aspartate protease known to cleave bacterial peptides for antigen presentation in dendritic cells (DCs), has not been studied. During experimental acute graft-versus-host disease (GVHD), we found infiltration by Ctse-positive immune cells leading to higher Ctse RNA- and protein levels in target organs. In Ctse-deficient allo-SCT recipients, we found ameliorated GVHD, improved survival, and lower numbers of tissue-infiltrating DCs. Donor T cell proliferation was not different in Ctse-deficient vs. wild-type allo-SCT recipients in MHC-matched and MHC-mismatched models. Furthermore, Ctse-deficient DCs had an intact ability to induce allogeneic T cell proliferation, suggesting that its role in antigen presentation may not be the main mechanism how Ctse impacts GVHD. We found that Ctse deficiency significantly decreases DC motility in vivo, reduces adhesion to extracellular matrix (ECM), and diminishes invasion through ECM. We conclude that Ctse has a previously unrecognized role in regulating DC motility that possibly contributes to reduced DC counts and ameliorated inflammation in GVHD target organs of Ctse-deficient allo-SCT recipients. However, our data do not provide definite proof that the observed effect of Ctse-/- deficiency is exclusively mediated by DCs. A contribution of Ctse-/--mediated functions in other recipient cell types, e.g., macrophages, cannot be excluded.
RESUMEN
Cathepsin E (CatE), an aspartic protease, has a limited distribution in certain cell types such as gastric cells. CatE is not detectable in the normal brain, whereas it is increasingly expressed in damaged neurons and activated microglia of the pathological brain. Neurons expressing high levels of CatE showed apparent morphological changes, including a marked shrinkage of the cytoplasmic region and beading of neurites, suggesting neuronal damage. The intracellular level of CatE in neurons is strictly regulated at both transcriptional and translational levels. Although the up-regulation of CatE may cause pathological changes in neurons, little information is available about the precise outcome of the increased expression of CatE in neurons. In this study, we have attempted to clarify the outcome of up-regulated CatE gene expression in neurons using the P19 cell neuronal differentiation after the overexpression of CatE. We unexpectedly found that the overexpression of CatE interfered with neuronal differentiation of P19 cells through an impairment of cell aggregate formation. Pepstatin A, an aspartic protease inhibitor, restored the impaired cell aggregation of P19/CatE cells. The small number of P19 cells differentiated into neurons had abnormal morphology characterized by their fusiform cell bodies with short processes. Furthermore, CatE proteolytically cleaved the extracellular domain of N-cadherin. These observations suggest that the overexpression of CatE interferes with neuronal differentiation of P19 cells through an impairment of cell aggregate formation, possibly through proteolytic degradation of N-cadherin.
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Cadherinas/metabolismo , Catepsina E/metabolismo , Diferenciación Celular , Neuronas/patología , Proteolisis , Teratocarcinoma/patología , Animales , Agregación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , ADN Complementario/genética , Immunoblotting , Ratones , Neuronas/metabolismo , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Ratas , Teratocarcinoma/metabolismo , TransfecciónRESUMEN
Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.
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Proteasas de Ácido Aspártico/genética , Mucosa Gástrica/metabolismo , Salamandridae/metabolismo , Secuencia de Aminoácidos , Animales , Proteasas de Ácido Aspártico/clasificación , Proteasas de Ácido Aspártico/aislamiento & purificación , Catepsina E/clasificación , Catepsina E/genética , Catepsina E/aislamiento & purificación , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Pruebas de Enzimas , Precursores Enzimáticos/clasificación , Precursores Enzimáticos/genética , Precursores Enzimáticos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Pepsina A/clasificación , Pepsina A/genética , Pepsina A/aislamiento & purificación , Pepsinógenos/clasificación , Pepsinógenos/genética , Pepsinógenos/aislamiento & purificación , Pepstatinas/farmacología , Filogenia , Inhibidores de Proteasas/farmacologíaRESUMEN
BACKGROUND: It was reported that Cathepsin E (Cat E) plays a critical role in antigen processing and in the development of pulmonary emphysema. The aim of this study was to investigate the role of Cat E and airflow limitation in the pathogenesis of COPD. METHODS: Sixty-five patients with COPD, 20 smoking control subjects without COPD and 15 non-smoking healthy control subjects were enrolled. Cat E and EIC (Elastase inhibitory capacity) expressions were measured by ELISA in sputum and serum samples and compared according to different subgroups. RESULTS: Cat E concentrations were significantly higher in patients with COPD than smoking control and non-smoking control subjects (P < 0.01). The levels of CatE were inversely correlated with FEV1% predicted in COPD patients (r = -0.95, P < 0.01). The levels of EIC were inversely positively correlated with FEV1% predicted in COPD patients (r = 0.926, P < 0.01). Levels of Cat E were also inversely correlated with the levels of EIC (r = -0.922, P < 0.01). CONCLUSIONS: Cat E contributes to the severity of airflow limitation during progression of COPD.
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Catepsina E/biosíntesis , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfisema Pulmonar/etiología , Esputo/metabolismo , Biomarcadores/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/fisiopatología , Índice de Severidad de la Enfermedad , Fumar/efectos adversos , Esputo/citologíaRESUMEN
Proteinase cascades are part of the basic machinery of neuronal death pathways. Neuronal cathepsin B (CatB), a typical cysteine lysosomal protease, plays a critical role in neuronal death through lysosomal leakage or excessive autophagy. On the other hand, much attention has been paid to microglial CatB in neuronal death. We herein show the critical role of proteolytic relay through microglial CatB and CatE in the polarization of microglia/macrophages in the neurotoxic phenotype, leading to hypoxia/ischemia (HI)-induced hippocampal neuronal damage in neonatal mice. HI caused extensive brain injury in neonatal wild-type mice, but not in CatB(-/-) mice. Furthermore, HI-induced polarization of microglia/macrophages in the neurotoxic phenotype followed by the neuroprotective phenotype in wild-type mice. On the other hand, microglia/macrophages exhibited only the early and transient polarization in the neuroprotective phenotype in CatB(-/-) mice. CA-074Me, a specific CatB inhibitor, significantly inhibited the neuronal death of primary cultured hippocampal neurons induced by the conditioned medium from cultured microglia polarized in the neurotoxic phenotype. Furthermore, CA-074Me prevented the activation of nuclear factor-κB (NF-κB) in cultured microglia by inhibiting autophagic inhibitor of κBα degradation following exposure to oxygen-glucose deprivation. Rather surprisingly, CatE increased the CatB expression after HI by the liberation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from microglia through the proteasomal pathway. A significant increase in CatB and CatE levels was found exclusively in microglia/macrophages after HI. Thus, a proteolytic relay through the early CatE/TRAIL-dependent proteosomal and late CatB-dependent autophagic pathways for NF-κB activation may play a critical role in the polarization of microglia/macrophages in the neurotoxic phenotype. Significance statement: Proteinase cascades are part of the basic machinery of neuronal death pathways. Cathepsin B, a typical cysteine lysosomal protease, plays a critical role in neuronal death through lysosomal leakage or excessive autophagy in neurons. On the other hand, much attention has been also paid to the role of microglial cathepsin B in neuronal death. In this study, using in vivo and in vitro models of relevance to brain ischemia, we found a critical role of proteolytic relay through cathepsin B and cathepsin E in the neurotoxic polarization of microglia/macrophages, which is responsible for aggravation of hypoxia/ischemia-induced neuronal injury. These findings suggest orally active selective inhibitors of cathepsin B or cathepsin E as promising pharmacological agents for the treatment of ischemic brain injury.
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Catepsina B/metabolismo , Catepsina E/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Proteolisis , Animales , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Catepsina E/genética , Células Cultivadas , Dipéptidos/farmacología , Hipocampo/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , FN-kappa B/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fenotipo , Inhibidores de Proteasas/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes.
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Catepsina D/metabolismo , Cistatina B/metabolismo , Citosol/enzimología , Células Asesinas Naturales/enzimología , Linfocitos/enzimología , Macrófagos/enzimología , Animales , Catepsina D/antagonistas & inhibidores , Catepsina D/genética , Catepsina E/antagonistas & inhibidores , Catepsina E/genética , Catepsina E/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cistatina B/farmacología , Citosol/efectos de los fármacos , Dipéptidos/farmacología , Endosomas/efectos de los fármacos , Endosomas/enzimología , Expresión Génica , Células HEK293 , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Pepstatinas/farmacología , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/enzimologíaRESUMEN
Although it has been suggested that the protective effect of melatonin against seizure-induced neurotoxicity involves inhibition of neuronal lipid peroxidation, current data concerning the exact molecular mechanism are still limited. This study was undertaken to investigate the changes in neurobehavioral, cognitive and lipid metabolism-related gene expressions in both hippocampus and cerebral cortex of rats subjected to recurrent neonatal seizures, and the effects of melatonin treatment before seizure (55mg/kg, 1mg/ml). 6-day-old (P6) SD rats were randomly divided into four groups of control (CONT, the same below), melatonin treated control (Mel), recurrent neonatal seizure (RS) and melatonin and RS combination treatment (Mel+RS). Neurological behavioral parameters of brain damage (plane righting reflex, negative geotaxis reaction reflex, Cliff avoidance reflex, forelimb suspension reflex) were observed on P31. Morris water maze test was performed during P29-P35. Then the protein levels of ACAT1, Cathepsin-E and Ca(2+)/calmodulin-dependent protein kinase II (CAMK II) in hippocampus and cerebral cortex were detected by western blot method. As expected, RS group showed a significant delay or reduce of the four reflexes, as well as bad performance in the Morris water maze test. Flurothyl-induced neurobehavioral toxicology was blocked by pre-treatment with melatonin. In parallel with these behavioral changes, gene expression by western blot method demonstrated that rats pretreated with melatonin (Mel+RS) showed a significant down-regulated expression of ACAT-1, Cathepsin-E and up-regulated CAMK II in hippocampus and cerebral cortex when compared with RS group. Our findings provide support for ACAT-1/Cathepsin-E as well as CaMK II being potential targets for the treatment of neonatal seizure-induced brain damage by melatonin.
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Conducta Animal/efectos de los fármacos , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Melatonina/farmacología , Metabolismo/efectos de los fármacos , Metabolismo/genética , Convulsiones/prevención & control , Acetil-CoA C-Acetiltransferasa/metabolismo , Animales , Animales Recién Nacidos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Catepsina E/metabolismo , Corteza Cerebral/efectos de los fármacos , Femenino , Flurotilo/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratas , Reflejo/efectos de los fármacos , Convulsiones/inducido químicamenteRESUMEN
Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE(-/-)) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE(-/-) mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE(-/-) mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE(-/-) mice. In fat-induced CatE(-/-) mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE(-/-) mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia.
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Tejido Adiposo/enzimología , Tejido Adiposo/patología , Catepsina E/deficiencia , Hepatomegalia/etiología , Adipogénesis/genética , Adipogénesis/fisiología , Tejido Adiposo Pardo/enzimología , Tejido Adiposo Pardo/patología , Tejido Adiposo Blanco/enzimología , Tejido Adiposo Blanco/patología , Animales , Catepsina E/genética , Dieta Alta en Grasa/efectos adversos , Hígado Graso/etiología , Hígado Graso/patología , Hepatomegalia/enzimología , Hepatomegalia/patología , Hipercolesterolemia/etiología , Metabolismo de los Lípidos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/enzimología , Obesidad/etiología , Obesidad/patologíaRESUMEN
In a previous study, we reported that the cathepsin-cystatin system caused endometrial dysfunction in early pregnancy. Here, we investigated the existence and contribution of cathepsin E in early pregnancy in patients with recurrent miscarriage (RM). The effect of cathepsin deficiency on fertility and female reproductive organs were also analyzed in CatE(-/-) mice. Human studies were conducted in a hospital setting, with informed consent. Cervical mucus was collected from RM patients in early pregnancy (4-6 gestational weeks, n = 21), and the pregnancy outcome was compared prospectively. The cathepsin E expression in decidua of RM patients (n = 49) and normal pregnant women undergoing elective surgical abortion (n = 24) was measured using SDS-PAGE, and western blot analysis. Decidual macrophages were isolated from RM patients (n = 6) and stimulated by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Results from the mouse model showed that CatE(-/-) mice were fertile, but the litter number was significantly smaller. The uterus of CatE(-/-) mice showed granulation tissue. In human samples, protease activity of cathepsin E measured with Fluorescence-Quenching Substrate (KYS-1) in cervical mucus of patients who developed miscarriage was markedly decreased compared with patients without RM. The expression of cathepsin E in decidua, semi-quantified by SDS-PAGE, western blot analysis was significantly lower in RM patients compared with patients without RM. By double staining immunofluorescence, the staining of cathepsin E was observed in CD14 or CD68 positive cells in all deciduas. Upon stimulation with LPS and IFN-γ, the expression of cathepsin E in cell lysate of decidual macrophages was markedly reduced in RM patients compared with controls. The results suggested that decreased activity of cathepsin E produced by decidual macrophages might be responsible for the induction of miscarriages in some RM patients.
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Aborto Habitual/enzimología , Catepsina E/metabolismo , Decidua/enzimología , Macrófagos/enzimología , Aborto Habitual/genética , Aborto Habitual/patología , Animales , Estudios de Casos y Controles , Catepsina E/deficiencia , Catepsina E/genética , Células Cultivadas , Decidua/efectos de los fármacos , Decidua/patología , Regulación hacia Abajo , Femenino , Edad Gestacional , Humanos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Tamaño de la Camada , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Estudios Prospectivos , Factores de TiempoRESUMEN
Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E-inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E-activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module-finding, module-shuffling, and module-pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen.
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Aptámeros de Péptidos/química , Catepsina E/antagonistas & inhibidores , Inhibidores de Proteasas/química , Secuencia de Aminoácidos , Evolución Molecular Dirigida , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión ProteicaRESUMEN
PURPOSE: The ketogenic diet has long been used to treat epilepsy, but its mechanism is not yet clearly understood. To explore the potential mechanism, we analyzed the changes in gene expression induced by the ketogenic diet in the rat kainic acid (KA) epilepsy model. MATERIALS AND METHODS: KA-administered rats were fed the ketogenic diet or a normal diet for 4 weeks, and microarray analysis was performed with their brain tissues. The effects of the ketogenic diet on cathepsin E messenger ribonucleic acid (mRNA) expression were analyzed in KA-administered and normal saline-administered groups with semi-quantitative and real-time reverse transcription polymerase chain reaction (RT-PCR). Brain tissues were dissected into 8 regions to compare differential effects of the ketogenic diet on cathepsin E mRNA expression. Immunohistochemistry with an anti-cathepsin E antibody was performed on slides of hippocampus obtained from whole brain paraffin blocks. RESULTS: The microarray data and subsequent RT-PCR experiments showed that KA increased the mRNA expression of cathepsin E, known to be related to neuronal cell death, in most brain areas except the brain stem, and these increases of cathepsin E mRNA expression were suppressed by the ketogenic diet. The expression of cathepsin E mRNA in the control group, however, was not significantly affected by the ketogenic diet. The change in cathepsin E mRNA expression was greatest in the hippocampus. The protein level of cathepsin E in the hippocampus of KA-administered rat was elevated in immunohistochemistry and the ketogenic diet suppressed this increase. CONCLUSION: Our results showed that KA administration increased cathepsin E expression in the rat brain and its increase was suppressed by the ketogenic diet.