RESUMO
Genetic variation influences how the genome is interpreted in individuals and in mouse strains used to model immune responses. We developed approaches to utilize next-generation sequencing datasets to identify sequence variation in genes and enhancer elements in congenic and backcross mouse models. We defined genetic variation in the widely used B6-CD45.2 and B6.SJL-CD45.1 congenic model, identifying substantial differences in SJL genetic content retained in B6.SJL-CD45.1 strains on the basis of the vendor source of the mice. Genes encoding PD-1, CD62L, Bcl-2, cathepsin E, and Cxcr4 were within SJL genetic content in at least one vendor source of B6.SJL-CD45.1 mice. SJL genetic content affected enhancer elements, gene regulation, protein expression, and amino acid content in CD4+ T helper 1 cells, and mice infected with influenza showed reduced expression of Cxcr4 on B6.SJL-CD45.1 T follicular helper cells. These findings provide information on experimental variables and aid in creating approaches that account for genetic variables.
Assuntos
Catepsina E/metabolismo , Elementos Facilitadores Genéticos/genética , Imunidade/genética , Receptores CXCR4/metabolismo , Células Th1/imunologia , Animais , Catepsina E/genética , Comércio , Regulação da Expressão Gênica , Patrimônio Genético , Variação Genética , Centro Germinativo/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Endogamia , Antígenos Comuns de Leucócito/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Modelos Animais , Receptores CXCR4/genéticaRESUMO
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.
Assuntos
Neoplasias Ósseas , Catepsina E , MicroRNAs , Osteossarcoma , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Catepsina E/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Metástase Neoplásica , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologiaRESUMO
The role and underlying mechanism of plasma membrane-bound G protein-coupled bile acid receptor (TGR5) in regulating macrophage innate immune activation during liver ischemia and reperfusion (IR) injury remains largely unclear. Here, we demonstrated that TGR5 depletion in myeloid cells aggravated liver injury with increased macrophage infiltration and enhanced inflammation in livers post-IR. While TGR5 deficiency enhanced mobility and proinflammatory M1 polarization of macrophages, TGR5 agonist enhanced the anti-inflammatory effect of TGR5 both in vivo and in vitro. Microarray profiling revealed that TGR5-deficient macrophages exhibited enhanced proinflammatory characteristics and cathepsin E (Cat E) was the most upregulated gene. Knockdown of Cat E abolished the enhanced mobility and shift of macrophage phenotypes induced by TGR5 depletion. Moreover, Cat E knockdown attenuated liver IR injury and liver inflammation in myeloid TGR5-deficient mice. In patients undergoing partial hepatectomy, IR stress promoted TGR5 activation of CD11b+ cells in peripheral blood mononuclear cells, correlating with the shift in macrophage M2 polarization. Ursodeoxycholic acid administration enhanced TGR5 activation and the trend in macrophage M2 polarization. Our results suggest that TGR5 attenuates proinflammatory immune activation by restraining macrophage migration and facilitating macrophage M2 polarization via suppression of Cat E and thereby protects against liver IR injury.
Assuntos
Catepsina E , Fígado , Ativação de Macrófagos , Receptores Acoplados a Proteínas G , Traumatismo por Reperfusão , Animais , Humanos , Imunidade Inata , Isquemia , Leucócitos Mononucleares , Macrófagos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Catepsina E/metabolismo , Fusão de Membrana/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Suínos , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Catepsina D/antagonistas & inibidores , Catepsina E/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Catepsina D/metabolismo , Catepsina E/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Lung cancer is one of the most frequently diagnosed malignant tumors and the main reason for cancer-related death around the world, whereas nonsmall cell lung cancer that consists two subtypes: lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) is responsible for an estimated 85% of all lung cancers. The current study aimed to explore gene expression and methylation differences between LUAD and LUSC. EdgeR was used to identify differentially regulated genes between normal and cancer in the LUAD and LUSC extracted from The Cancer Genome Atlas (TCGA), respectively, whereas SAM was used to find genes with differential methylation between normal and cancer in the LUAD and LUSC, respectively. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to analyze the function which these genes enriched in. A total of 391 genes with opposite methylation patterns in LUAD and LUSC and four functional pathways were obtained (false discovery rate (FDR) < 0.1). These pathways mainly included fat digestion and absorption, phenylalanine metabolism, bile secretion, and so on, which were related to the airframe nutrition metabolic pathway. Moreover, two genes CTSE (cathepsin E) and solute carrier family 5 member 7 (SLC5A7) were also found, among which CTSE was overexpressed and hypomethylated in LUAD corresponding to normal lung tissues, whereas SLC5A7 showed the opposite in LUAD. In conclusion, this study investigated the differences between the gene expression and methylation patterns in LUAD and LUSC, and explored their different biological characteristics. Further understanding of these differences may promote the discovery and development of new, accurate strategies for the prevention, diagnosis, and treatment of lung cancer.
Assuntos
Adenocarcinoma de Pulmão/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Transcriptoma , Adenocarcinoma de Pulmão/patologia , Carcinoma de Células Escamosas/patologia , Catepsina E/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Pulmonares/patologia , Regiões Promotoras Genéticas , Transdução de Sinais/genética , Simportadores/genéticaRESUMO
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.
Assuntos
Catepsina E/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , Biomarcadores Tumorais , Catepsina E/genética , HumanosRESUMO
BACKGROUND: Despite advances in diagnostics and treatment, aortic aneurysms are an important clinical problem, mainly due to the accompanying complications that may lead to direct loss of life, also the number of diagnosed and operated aneurysms is constantly increasing. The aim of this study is to determine the relationship between the concentration of lysosomal peptidases cathepsin A, D, and E in the wall of the abdominal aortic aneurysm and the concentration of copper and zinc, and the size of the aneurysm widening in the wall of the abdominal aortic aneurysm. METHODS: The study included 27 patients with abdominal aortic aneurysm from the Department of Vascular Surgery and Transplantation of the University Clinical Hospital in Bialystok. The research material was the wall of the abdominal aortic aneurysm collected intraoperatively. The control material consisted of fragments of the abdominal aorta obtained from organ donors for transplantation. The concentration of cathepsin A, D, and E was determined using enzyme-linked immunosorbent assays. Concentrations of copper and zinc were determined by flame atomic absorption spectrometry after prior mineralization of the samples. All patients were interviewed and asked about basic demographic data, comorbidities, and risk factors for cardiovascular disease to which they were exposed in the past. The statistical analysis was performed using Statistica 10 statistical package. Mann-Whitney U-tests were used and also Spearman's r correlation assuming a significance level of P < 0.05. RESULTS: The concentration of cathepsin A, D, and E was higher in the aortic wall altered by the aneurysm than in the wall of the control aorta (P < 0.05). The analysis of the data showed that there was a positive correlation between the concentration of cathepsin A and D and the width of the aneurysmal widening (r = 0.699 and 0.750, respectively). There was no correlation between cathepsin E concentration and aneurysm width. CONCLUSIONS: The higher contents of cathepsin A, D, and E in the wall of the aortic aneurysm than in the normal aortic wall, as well as a positive correlation between the concentration of cathepsin A and D and the width of the aneurysmal widening, allow to assume the participation of these enzymes in the pathogenesis of the aneurysm.
Assuntos
Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/enzimologia , Catepsina A/análise , Catepsina D/análise , Catepsina E/análise , Cobre/análise , Zinco/análise , Idoso , Idoso de 80 Anos ou mais , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/patologia , Estudos de Casos e Controles , Dilatação Patológica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , PolôniaRESUMO
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.
Assuntos
Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/imunologia , Catepsina E/genética , Catepsina E/imunologia , Imunidade Celular/imunologia , Adulto , Neuropatias Amiloides Familiares/patologia , Animais , Catepsina E/biossíntese , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Caderinas/metabolismo , Catepsina E/metabolismo , Diferenciação Celular , Neurônios/patologia , Proteólise , Teratocarcinoma/patologia , Animais , Agregação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Complementar/genética , Immunoblotting , Camundongos , Neurônios/metabolismo , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Ratos , Teratocarcinoma/metabolismo , TransfecçãoRESUMO
Three new modified peptides named grassystatins D-F (1-3) were discovered from a marine cyanobacterium from Guam. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The hallmark structural feature in the peptides is a statine unit, which contributes to their aspartic protease inhibitory activity preferentially targeting cathepsins D and E. Grassystatin F (3) was the most potent analogue, with IC50 values of 50 and 0.5 nM against cathepsins D and E, respectively. The acidic tumor microenvironment is known to increase the activation of some of the lysosomal proteases associated with tumor metastasis such as cathepsins. Because cathepsin D is a biomarker in aggressive forms of breast cancer and linked to poor prognosis, the effects of cathepsin D inhibition by 1 and 3 on the downstream cellular substrates cystatin C and PAI-1 were investigated. Furthermore, the functional relevance of targeting cathepsin D substrates was evaluated by examining the effect of 1 and 3 on the migration of MDA-MD-231 cells. Grassystatin F (3) inhibited the cleavage of cystatin C and PAI-1, the activities of their downstream targets cysteine cathepsins and tPA, and the migration of the highly aggressive triple negative breast cancer cells, phenocopying the effect of siRNA-mediated knockdown of cathepsin D.
Assuntos
Ácido Aspártico Proteases/efeitos dos fármacos , Peptídeos/isolamento & purificação , Aminoácidos , Catepsina D/antagonistas & inibidores , Catepsina E/antagonistas & inibidores , Catepsina L/metabolismo , Cianobactérias/química , Relação Dose-Resposta a Droga , Endopeptidases/metabolismo , Feminino , Guam , Humanos , Concentração Inibidora 50 , Lisossomos/metabolismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidores de Proteases/farmacologiaRESUMO
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.
Assuntos
Catepsina B/metabolismo , Catepsina E/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Proteólise , Animais , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Catepsina E/genética , Células Cultivadas , Dipeptídeos/farmacologia , Hipocampo/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fenótipo , Inibidores de Proteases/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.
Assuntos
Catepsina E/metabolismo , Precursores Enzimáticos/metabolismo , Neoplasias Pancreáticas/patologia , Sequência de Aminoácidos , Animais , Catepsina E/antagonistas & inibidores , Catepsina E/química , Linhagem Celular Tumoral , Progressão da Doença , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica , Concentração de Íons de Hidrogênio , Camundongos , Inibidores de Proteases/farmacologia , Neoplasias PancreáticasRESUMO
In search of novel protease inhibitors with therapeutic potential, our efforts exploring the marine cyanobacterium Lyngbya sp. have led to the discovery of tasiamide F (1), which is an analogue of tasiamide B (2). The structure was elucidated using a combination of NMR spectroscopy and mass spectrometry. The key structural feature in 1 is the presence of the Phe-derived statine core, which contributes to its aspartic protease inhibitory activity. The antiproteolytic activity of 1 and 2 was evaluated in vitro against cathepsins D and E, and BACE1. Tasiamide F (1) displayed IC50 values of 57nM, 23nM, and 0.69µM, respectively, indicating greater selectivity for cathepsins over BACE1 compared with tasiamide B (2). Molecular docking experiments were carried out for compounds 1 and 2 against cathepsins D and E to rationalize their activity towards these proteases. The dysregulated activities of cathepsins D and E have been implicated in cancer and modulation of immune responses, respectively, and these proteases represent potential therapeutic targets.
Assuntos
Catepsina D/antagonistas & inibidores , Catepsina D/química , Catepsina E/antagonistas & inibidores , Catepsina E/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Animais , Cianobactérias/química , Simulação de Acoplamento Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Alimentos Marinhos/análiseRESUMO
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.
Assuntos
Catepsina E/biossíntese , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/etiologia , Escarro/metabolismo , Biomarcadores/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/fisiopatologia , Índice de Gravidade de Doença , Fumar/efeitos adversos , Escarro/citologiaRESUMO
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.
Assuntos
Catepsina D/metabolismo , Cistatina B/metabolismo , Citosol/enzimologia , Células Matadoras Naturais/enzimologia , Linfócitos/enzimologia , Macrófagos/enzimologia , Animais , Catepsina D/antagonistas & inibidores , Catepsina D/genética , Catepsina E/antagonistas & inibidores , Catepsina E/genética , Catepsina E/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cistatina B/farmacologia , Citosol/efeitos dos fármacos , Dipeptídeos/farmacologia , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Expressão Gênica , Células HEK293 , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Pepstatinas/farmacologia , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/enzimologiaRESUMO
We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.
Assuntos
Peptídeo Hidrolases/metabolismo , Especificidade por Substrato , Proteases Virais 3C , Animais , Carboxipeptidases/metabolismo , Carcinoma Ductal Pancreático/enzimologia , Catepsina E/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Cisteína Endopeptidases/metabolismo , Exopeptidases/metabolismo , Granzimas/metabolismo , Humanos , Camundongos , Elastase Pancreática/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Schistosoma mansoni , Espectrometria de Massas em Tandem , Proteínas Virais/metabolismoRESUMO
Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose function has been described only in antigen processing. We examined lung tissue sections of persons with chronic obstructive pulmonary disease, a clinical entity that includes emphysematous change. Human chronic obstructive pulmonary disease lungs had markedly increased cathepsin E protein in the lung epithelium. We generated lung epithelial-targeted transgenic cathepsin E mice and found that they develop emphysema. Overexpression of cathepsin E resulted in increased E3 ubiquitin ligase parkin, mitochondrial fission protein dynamin-related protein 1, caspase activation/apoptosis, and ultimately loss of lung parenchyma resembling emphysema. Inhibiting dynamin-related protein 1, using a small molecule inhibitor in vitro or in vivo, inhibited cathepsin E-induced apoptosis and emphysema. To the best of our knowledge, our study is the first to identify links between cathepsin E, mitochondrial fission, and caspase activation/apoptosis in the pathogenesis of pulmonary emphysema. Our data expand the current understanding of molecular mechanisms of emphysema development and may provide new therapeutic targets.
Assuntos
Catepsina E/metabolismo , Dinâmica Mitocondrial , Enfisema Pulmonar/metabolismo , Animais , Apoptose , Lavagem Broncoalveolar , Catepsina E/genética , Humanos , Pulmão/metabolismo , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/farmacologia , Enfisema Pulmonar/fisiopatologia , Quinazolinonas/farmacologia , Fumaça/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversosRESUMO
BACKGROUND: Cathepsin E (CTSE), an aspartic proteinase, is differentially expressed in the metaplasia-dysplasia-neoplasia sequence of gastric and colon cancer. We evaluated CTSE in Barrett's esophagus (BE) and cancer because increased CTSE levels are linked to improved survival in several cancers, and other cathepsins are up-regulated in BE and esophageal adenocarcinoma (EAC). METHODS: A total of 273 pretreatment tissues from 199 patients were analyzed [31 normal squamous esophagus (NE), 29 BE intestinal metaplasia, 31 BE with dysplasia (BE/D), 108 EAC]. CTSE relative mRNA expression was measured by real-time polymerase chain reaction, and protein expression was measured by immunohistochemistry. CTSE serum levels were determined by enzyme-linked immunosorbent assay. RESULTS: Median CTSE mRNA expression levels were ≥1,000-fold higher in BE/intestinal metaplasia and BE/D compared to NE. CTSE levels were significantly lower in EAC compared to BE/intestinal metaplasia and BE/D, but significantly higher than NE levels. A similar expression pattern was present in immunohistochemistry, with absent staining in NE, intense staining in intestinal metaplasia and dysplasia, and less intense EAC staining. CTSE serum analysis did not discriminate patient groups. In a uni- and multivariable Cox proportional hazards model, CTSE expression was not significantly associated with survival in patients with EAC, although CTSE expression above the 25th percentile was associated with a 41 % relative risk reduction for death (hazard ratio 0.59, 95 % confidence interval 0.27-1.26, p = 0.17). CONCLUSIONS: CTSE mRNA expression is up-regulated more than any known gene in Barrett intestinal metaplasia and dysplasia tissues. Protein expression is similarly highly intense in intestinal metaplasia and dysplasia tissues.
Assuntos
Adenocarcinoma/metabolismo , Esôfago de Barrett/metabolismo , Catepsina E/sangue , Neoplasias Esofágicas/metabolismo , Esôfago/metabolismo , Metaplasia/metabolismo , Lesões Pré-Cancerosas/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Esôfago de Barrett/mortalidade , Esôfago de Barrett/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Catepsina E/genética , Ensaio de Imunoadsorção Enzimática , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Masculino , Metaplasia/mortalidade , Metaplasia/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Lesões Pré-Cancerosas/mortalidade , Lesões Pré-Cancerosas/patologia , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.