RESUMO
Pulmonary surfactant is a lipoprotein synthesized and secreted by alveolar type II cells in lung. We evaluated the associations between 200,139 single nucleotide polymorphisms (SNPs) of 40 surfactant-related genes and lung cancer risk using genotyped data from two independent lung cancer genome-wide association studies. Discovery data included 18,082 cases and 13,780 controls of European ancestry. Replication data included 1,914 cases and 3,065 controls of European descent. Using multivariate logistic regression, we found novel SNPs in surfactant-related genes CTSH [rs34577742 C > T, odds ratio (OR) = 0.90, 95% confidence interval (CI) = 0.89-0.93, P = 7.64 × 10-9] and SFTA2 (rs3095153 G > A, OR = 1.16, 95% CI = 1.10-1.21, P = 1.27 × 10-9) associated with overall lung cancer in the discovery data and validated in an independent replication data-CTSH (rs34577742 C > T, OR = 0.88, 95% CI = 0.80-0.96, P = 5.76 × 10-3) and SFTA2 (rs3095153 G > A, OR = 1.14, 95% CI = 1.01-1.28, P = 3.25 × 10-2). Among ever smokers, we found SNPs in CTSH (rs34577742 C > T, OR = 0.89, 95% CI = 0.85-0.92, P = 1.94 × 10-7) and SFTA2 (rs3095152 G > A, OR = 1.20, 95% CI = 1.14-1.27, P = 4.25 × 10-11) associated with overall lung cancer in the discovery data and validated in the replication data-CTSH (rs34577742 C > T, OR = 0.88, 95% CI = 0.79-0.97, P = 1.64 × 10-2) and SFTA2 (rs3095152 G > A, OR = 1.15, 95% CI = 1.01-1.30, P = 3.81 × 10-2). Subsequent transcriptome-wide association study using expression weights from a lung expression quantitative trait loci study revealed genes most strongly associated with lung cancer are CTSH (PTWAS = 2.44 × 10-4) and SFTA2 (PTWAS = 2.32 × 10-6).
Assuntos
Neoplasias Pulmonares , Surfactantes Pulmonares , Humanos , Estudo de Associação Genômica Ampla , Pulmão/metabolismo , Genótipo , Surfactantes Pulmonares/metabolismo , Tensoativos/metabolismo , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , Catepsina H/genética , Catepsina H/metabolismoRESUMO
BACKGROUND & AIMS: Improving clinical management of early stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) are important determinants of tumor progression in advanced colorectal cancer (CRC), but their role in the initial stages of CRC tumorigenesis is unknown. Therefore, we investigated the contribution of T1CAFs to early CRC progression. METHODS: Primary T1CAFs and patient-matched normal fibroblasts (NFs) were isolated from endoscopic biopsy specimens of histologically confirmed T1CRCs and normal mucosa, respectively. The impact of T1CAFs and NFs on tumor behavior was studied using 3-dimensional co-culture systems with primary T1CRC organoids and extracellular matrix (ECM) remodeling assays. Whole-transcriptome sequencing and gene silencing were used to pinpoint mediators of T1CAF functions. RESULTS: In 3-dimensional multicellular cultures, matrix invasion of T1CRC organoids was induced by T1CAFs, but not by matched NFs. Enhanced T1CRC invasion was accompanied by T1CAF-induced ECM remodeling and up-regulation of CD44 in epithelial cells. RNA sequencing of 10 NF-T1CAF pairs revealed 404 differentially expressed genes, with significant enrichment for ECM-related pathways in T1CAFs. Cathepsin H, a cysteine-type protease that was specifically up-regulated in T1CAFs but not in fibroblasts from premalignant lesions or advanced CRCs, was identified as a key factor driving matrix remodeling by T1CAFs. Finally, we showed high abundance of cathepsin H-expressing T1CAFs at the invasive front of primary T1CRC sections. CONCLUSIONS: Already in the earliest stage of CRC, cancer cell invasion is promoted by CAFs via direct interactions with epithelial cancer cells and stage-specific, cathepsin H-dependent ECM remodeling. RNA sequencing data of the 10 NF-T1CAF pairs can be found under GEO accession number GSE200660.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Colorretais , Humanos , Fibroblastos Associados a Câncer/metabolismo , Catepsina H/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fibroblastos/metabolismo , Neoplasias Colorretais/patologiaRESUMO
Despite the wide application of radiotherapy in HCC, radiotherapy efficacy is sometimes limited due to radioresistance. Although radioresistance is reported with high glycolysis, the underlying mechanism between radioresistance and cancer metabolism, as well as the role of cathepsin H (CTSH) within it, remain unclear. In this study, tumor-bearing models and HCC cell lines were used to observe the effect of CTSH on radioresistance. Proteome mass spectrometry, followed by enrichment analysis, were used to investigate the cascades and targets regulated by CTSH. Technologies such as immunofluorescence co-localization flow cytometry and Western blot were used for further detection and verification. Through these methods, we originally found CTSH knockdown (KD) perturbed aerobic glycolysis and enhanced aerobic respiration, and thus promoted apoptosis through up-regulation and the release of proapoptotic factors such as AIFM1, HTRA2, and DIABLO, consequently reducing radioresistance. We also found that CTSH, together with its regulatory targets (such as PFKL, HK2, LDH, and AIFM1), was correlated with tumorigenesis and poor prognosis. In summary, our study found that the cancer metabolic switch and apoptosis were regulated by CTSH signaling, leading to the occurrence of radioresistance in HCC cells and suggesting the potential value of HCC diagnosis and therapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/metabolismo , Catepsina H/metabolismo , Transdução de Sinais , Apoptose/genética , Regulação Neoplásica da Expressão Gênica , Glicólise , Proliferação de Células , Linhagem Celular TumoralRESUMO
Cathepsin H and Cathepsin B are two lysosomal cysteine proteases participating in various physiological processes including immune responses. In fish, the functional roles of Cathepsin H and Cathepsin B during bacterial infection are less understood. In a previous work, we characterized a Cathepsin B homologue (CsCatB) of half-smooth tongue sole (Cynoglossus semilaevis), an economically valuable fish species in China. In this report, we identified a Cathepsin H homologue (CsCatH) from C. semilaevis. In healthy tongue sole, the transcriptional expression of CsCatH was detected in nine different tissues. Laser scanning confocal microscopic analysis showed that ectopically expressed CsCatH and CsCatB were co-localized with the lysosome. Upon infection by Edwardsiella tarda, a significant fish pathogen which caused a severe fish disease termed edwardsiellosis, the expressions of CsCatH and CsCatB were remarkedly upregulated. The knockdown of CsCatH and CsCatB significantly increased the replication of E. tarda and mitigated E. tarda-induced apoptosis in tongue sole tissues. These findings revealed the importance of CsCatH and CsCatB in anti-bacterial immunity of tongue sole.
Assuntos
Infecções Bacterianas , Doenças dos Peixes , Linguados , Linguado , Animais , Catepsina B , Catepsina H/metabolismo , Edwardsiella tarda/fisiologia , Proteínas de PeixesRESUMO
Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disease, which has a high heritability of up to 79%. Exploring the genetic basis is essential for understanding the pathogenic mechanisms underlying AD development. Recent genome-wide association studies (GWASs) reported an AD-associated signal in the Cathepsin H (CTSH) gene in European populations. However, the exact functional/causal variant(s), and the genetic regulating mechanism of CTSH in AD remain to be determined. In this study, we carried out a comprehensive study to characterize the role of CTSH variants in the pathogenesis of AD. We identified rs2289702 in CTSH as the most significant functional variant that is associated with a protective effect against AD. The genetic association between rs2289702 and AD was validated in independent cohorts of the Han Chinese population. The CTSH mRNA expression level was significantly increased in AD patients and AD animal models, and the protective allele T of rs2289702 was associated with a decreased expression level of CTSH through the disruption of the binding affinity of transcription factors. Human microglia cells with CTSH knockout showed a significantly increased phagocytosis of Aß peptides. Our study identified CTSH as being involved in AD genetic susceptibility and uncovered the genetic regulating mechanism of CTSH in pathogenesis of AD.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Animais , Humanos , Doença de Alzheimer/genética , Estudo de Associação Genômica Ampla , Catepsina H/genética , Catepsina H/metabolismo , Predisposição Genética para Doença/genética , GenômicaRESUMO
BACKGROUND: Cathepsin H (CatH) is a lysosomal cysteine protease with a unique aminopeptidase activity. Its expression level is increased in activated immune cells including dendritic cells, macrophages, and microglia. We have previously reported that CatH deficiency impairs toll-like receptor 3 (TLR3)-mediated activation of interferon regulatory factor 3 (IRF3), and the subsequent secretion of interferon (IFN)-ß from dendritic cells. Furthermore, there is increasing evidence that IFN-ß secreted from microglia/macrophages has neuroprotective effects. These observations prompted further investigation into the effects of CatH deficiency on neuropathological changes. METHODS: In this study, neuropathological changes were examined using histochemical staining (both hematoxylin-eosin (H&E) and Nissl) of the hippocampus of wild-type (WT) and CatH-deficient (CatH-/-) mice after hypoxia-ischemia (HI). The density and the localization of CatH and TLR3 were examined by immunofluorescent staining. CatH processing in microglia was assayed by pulse-chase experiments, while immunoblotting was used to examine TLR3 expression and IRF3 activation in microglia/macrophages in the presence of poly(I:C). Microglial cell death was examined by fluorescence-activated cell sorting (FACS), and primary astrocyte proliferation in the presence of IFN-ß was examined using scratch wound assay. RESULTS: WT mice displayed severe atrophy in association with neuronal death and moderate astrogliosis in the hippocampus following neonatal HI. Somewhat surprisingly, CatH-/- mice showed marked neuronal death without severe atrophy in the hippocampus following HI. Furthermore, there was notable microglia/macrophages cell death and strong astrogliosis in the hippocampus. The TLR3 and phosphorylated IRF3 expression level in the hippocampus or splenocytes (mainly splenic macrophages); from CatH-/- mice was lower than in WT mice. In vitro experiments demonstrated that recombinant IFN-ß suppressed HI-induced microglial cell death and astrocyte proliferation. CONCLUSION: These observations suggest that CatH plays a critical role in the proteolytic maturation and stabilization of TLR3, which is necessary for IFN-ß production. Therefore, impaired TLR3/IFN-ß signaling resulting from CatH deficiency may induce microglial cell death after activation and astrogliosis/glial scar formation in the hippocampus following HI injury, leading to suppression of hippocampal atrophy.
Assuntos
Catepsina H/genética , Hipocampo/patologia , Hipóxia-Isquemia Encefálica/genética , Interferon beta/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Atrofia/genética , Atrofia/metabolismo , Atrofia/patologia , Catepsina H/metabolismo , Morte Celular/fisiologia , Hipocampo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Interferon beta/genética , Camundongos , Camundongos Knockout , Microglia/metabolismo , Microglia/patologia , Transdução de Sinais/fisiologia , Receptor 3 Toll-Like/genéticaRESUMO
Cathepsins have emerged out as significant targets in variety of tissue degenerative disorders such as inflammation, alzeimers, tumerogenesis including metastasis and invasion. Elevated levels of cathepsins and reduced cellular inhibitors at the site of these diseased conditions suggest the exploration of novel inhibitors of cathepsins. In the search of effective novel inhibitors as anti-cathepsin agents different natural products are also screened. One such molecule, curcumin has been reported as potential anti-cathepsin agent in recent past. Low solubility of curcumin makes it an important subject for screening effect of different pharmaceutical excipients toward enhanced solubility. In the present work we report serum protein protecting and anti-cathepsin activities of 28 different formulations of curcumin. The formulations have been prepared using four ingredients used in traditional medicinal system. Milk has been found to enhance solubility to a significant level. Cow milk fat, sucrose and piperine exhibited positive cooperation. The results have been explained on the basis of chemical behavior of different ingredients.
Assuntos
Catepsina B/antagonistas & inibidores , Catepsina H/antagonistas & inibidores , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Substâncias Protetoras/farmacologia , Soroalbumina Bovina/metabolismo , Animais , Catepsina B/metabolismo , Catepsina H/metabolismo , Bovinos , Curcumina/síntese química , Curcumina/química , Relação Dose-Resposta a Droga , Composição de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cabras , Modelos Moleculares , Estrutura Molecular , Substâncias Protetoras/síntese química , Substâncias Protetoras/química , Relação Estrutura-AtividadeRESUMO
Cathepsins have emerged as important targets in various tissues degenerative disorders due to their involvement in degradation of extracellular matrices and endogenous protein turnover. Elevated cathepsins levels vis-à-vis decreased concentration of endogenous inhibitors has been reported at different diseased sites. The design and synthesis of specific potential anti-cathepsin agents is therefore of great significance. Most of potential anti-cathepsin agents developed have peptide based structures with an active warhead. Due to oral instability and immunogenic problems related to peptidyl inhibitors drift the synthesis and evaluation of non-peptide cathepsin inhibitors in last two decades. The present work provides a detailed structure activity relationship for developing potential non-peptide anticathepsin agents based on in-vitro inhibition studies of a library of synthesized thiocarbamoyl- non-peptide inhibitors.
Assuntos
Catepsina B/antagonistas & inibidores , Catepsina H/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Tiocarbamatos/farmacologia , Catepsina B/isolamento & purificação , Catepsina B/metabolismo , Catepsina H/isolamento & purificação , Catepsina H/metabolismo , Catepsina L/isolamento & purificação , Catepsina L/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Tiocarbamatos/síntese química , Tiocarbamatos/químicaRESUMO
Chronic immune-mediated diseases of adulthood often originate in early childhood. To investigate genetic associations between neonatal immunity and disease, we map expression quantitative trait loci (eQTLs) in resting myeloid cells and CD4+ T cells from cord blood samples, as well as in response to lipopolysaccharide (LPS) or phytohemagglutinin (PHA) stimulation, respectively. Cis-eQTLs are largely specific to cell type or stimulation, and 31% and 52% of genes with cis-eQTLs have response eQTLs (reQTLs) in myeloid cells and T cells, respectively. We identified cis regulatory factors acting as mediators of trans effects. There is extensive colocalisation between condition-specific neonatal cis-eQTLs and variants associated with immune-mediated diseases, in particular CTSH had widespread colocalisation across diseases. Mendelian randomisation shows causal neonatal gene expression effects on disease risk for BTN3A2, HLA-C and others. Our study elucidates the genetics of gene expression in neonatal immune cells, and aetiological origins of autoimmune and allergic diseases.
Assuntos
Doenças Autoimunes/genética , Desenvolvimento Infantil/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Hipersensibilidade/genética , Locos de Características Quantitativas/imunologia , Doenças Autoimunes/imunologia , Butirofilinas/genética , Butirofilinas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Catepsina H/genética , Catepsina H/metabolismo , Criança , Pré-Escolar , Conjuntos de Dados como Assunto , Sangue Fetal/citologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/imunologia , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Antígenos HLA-C/genética , Antígenos HLA-C/metabolismo , Humanos , Hipersensibilidade/imunologia , Lactente , Recém-Nascido , Análise da Randomização Mendeliana , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Estudos ProspectivosRESUMO
Microglia, the resident mononuclear phagocyte population in the brain, have long been implicated in the pathology of neurodegenerative age-associated disorders. However, activated microglia have now been identified as homeostatic keepers in the brain, because they are involved in the initiation and resolution of neuropathology. The complex roles of activated microglia appear to be linked to change from inflammatory and neurotoxic to anti-inflammatory and neuroprotective phenotypes. Increased expression and secretion of various cathepsins support roles of activated microglia in chronic neuroinflammation, the neurotoxic M1-like polarization and neuronal death. Moreover, changes in expression and localization of microglial cathepsin B play a critical role in the acceleration of the brain aging. Beyond the role as brain-resident macrophages, many lines of evidence have shown that microglia have essential roles in the maturation and maintenance of neuronal circuits in the developing and adult brain. Cathepsin S secreted from microglia induces the diurnal variation of spine density of cortical neurons though proteolytic modification of peri-synaptic extracellular matrix molecules. In this review, I highlight the emerging roles of cathepsins that support the roles of microglia in both normal healthy and pathological brains. In addition, I discuss cathepsin inhibitors as potential therapeutic targets for brain disorders.
Assuntos
Catepsinas/metabolismo , Microglia/metabolismo , Animais , Encéfalo/metabolismo , Catepsina B/metabolismo , Catepsina H/metabolismo , Dor Crônica/metabolismo , Humanos , Inflamação , Microglia/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , FenótipoRESUMO
Cathepsins have been proposed as biomarkers of chemical exposure in the zebrafish embryo model but it is unclear whether they can also be used to detect sublethal stress. The present study evaluates three cathepsin types as candidate biomarkers in zebrafish embryos. In addition to other functions, cathepsins are also involved in yolk lysosomal processes for the internal nutrition of embryos of oviparous animals until external feeding starts. The baseline enzyme activity of cathepsin types H, C and L during the embryonic development of zebrafish in the first 96 h post fertilisation was studied. Secondly, the effect of leupeptin, a known cathepsin inhibitor, and four embryotoxic xenobiotic compounds with different modes of action (phenanthrene-baseline toxicity; rotenone-an inhibitor of electron transport chain in mitochondria; DNOC (Dinitro-ortho-cresol)-an inhibitor of ATP synthesis; and tebuconazole-a sterol biosynthesis inhibitor) on in vivo cathepsin H, C and L total activities have been tested. The positive control leupeptin showed effects on cathepsin L at a 20-fold lower concentration compared to the respective LC50 (0.4 mM) of the zebrafish embryo assay (FET). The observed effects on the enzyme activity of the four other xenobiotics were not or just slightly more sensitive (factor of 1.5 to 3), but the differences did not reach statistical significance. Results of this study indicate that the analysed cathepsins are not susceptible to toxins other than the known peptide-like inhibitors. However, specific cathepsin inhibitors might be identified using the zebrafish embryo.
Assuntos
Catepsina C/antagonistas & inibidores , Catepsina H/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Peixe-Zebra/embriologia , Animais , Catepsina C/metabolismo , Catepsina H/metabolismo , Catepsina L/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Leupeptinas/farmacologiaRESUMO
Background Cystatin F is a protein inhibitor of cysteine peptidases, expressed predominantly in immune cells and localised in endosomal/lysosomal compartments. In cytotoxic immune cells cystatin F inhibits both the major pro-granzyme convertases, cathepsins C and H that activate granzymes, and cathepsin L, that acts as perforin activator. Since perforin and granzymes are crucial molecules for target cell killing by cytotoxic lymphocytes, defects in the activation of either granzymes or perforin can affect their cytotoxic potential. Materials and methods Levels of cystatin F were assessed by western blot and interactions of cystatin F with cathepsins C, H and L were analysed by immunoprecipitation and confocal microscopy. In TALL-104 cells specific activities of the cathepsins and granzyme B were determined using peptide substrates. Results Two models of reduced T cell cytotoxicity of TALL-104 cell line were established, either by treatment by ionomycin or by immunosuppressive transforming growth factor beta. Reduced cytotoxicity correlated with increased levels of cystatin F and with attenuated activities of cathepsins C, H and L and of granzyme B. Co-localisation of cystatin F and cathepsins C, H and L and interactions between cystatin F and cathepsins C and H were demonstrated. Conclusions Cystatin F is designated as a possible regulator of T cell cytotoxicity, similar to its role in natural killer cells.
Assuntos
Biomarcadores Tumorais/metabolismo , Catepsina C/metabolismo , Catepsina H/metabolismo , Catepsina L/metabolismo , Cistatinas/metabolismo , Granzimas/metabolismo , Linfócitos T Citotóxicos/imunologia , Western Blotting , Catepsina C/antagonistas & inibidores , Catepsina H/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Morte Celular , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Fluoresceínas/metabolismo , Humanos , Ionomicina/farmacologia , Perforina/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Cathepsin H is a member of the papain superfamily of lysosomal cysteine proteases. It is the only known aminopeptidase in the family and is reported to be involved in cancer and other major diseases. Like many other proteases, it is synthesized as an inactive proenzyme. Although the crystal structure of mature porcine cathepsin H revealed the binding of the mini-chain and provided structural basis for the aminopeptidase activity, detailed structural and functional information on the inhibition and activation of procathepsin H has been elusive. Here we present the crystal structures of human procathepsin H at 2.00 Å and 1.66 Å resolution. These structures allow us to explore in detail the molecular basis for the inhibition of the mature domain by the prodomain. Comparison with cathepsin H structure reveals how mini-chain reorients upon activation. We further demonstrate that procathepsin H is not auto-activated but can be trans-activated by cathepsin L.
Assuntos
Catepsina H/metabolismo , Precursores Enzimáticos/metabolismo , Catepsina H/química , Catepsina H/genética , Catepsina L/química , Catepsina L/metabolismo , Cristalização , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Cathepsins have emerged as promising molecular targets in a number of diseases such as Alzeimer's, inflammation and cancer. Elevated cathepsin's levels and decreased cellular inhibitor concentrations have emphasized the search for novel inhibitors of cathepsins. The present work is focused on the design and synthesis of some acetophenone phenylhydrazone based pyrazole derivatives as novel non peptidyl inhibitors of cathepsins B, H and L. The synthesized compounds after characterization have been explored for their inhibitory potency against cathepsins B, H and L. The results show that some of the synthesized compounds exhibit anti-catheptic activity with Ki value of the order of 10-10M. Differential inhibitory effects have been observed for cathepsins B, H and L. Cathepsin L is inhibited more pronounced than cathepsin B and cathepsin H in that order.
Assuntos
Acetofenonas/química , Catepsinas/antagonistas & inibidores , Hidrazonas/química , Inibidores de Proteases/química , Pirazóis/química , Sítios de Ligação , Domínio Catalítico , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina H/antagonistas & inibidores , Catepsina H/metabolismo , Catepsina L/antagonistas & inibidores , Catepsina L/metabolismo , Catepsinas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Relação Estrutura-AtividadeRESUMO
In three-dimensional extracellular matrix, mesenchymal cells including hepatic stellate cells (HSCs) gain the ability to express matrix metalloproteinases (MMPs) on injury signals. In contrast, in myofibroblastic HSCs in fibrotic liver, many MMP genes are silenced into an epigenetically nonpermissive state. The mechanism by which the three-dimensional extracellular matrix confers the MMP genes into an epigenetically permissive state has not been well characterized. In continuation of previous work, we show here that the up-regulation of MMP genes is mediated through degradation of class IIa histone deacetylases (HDACs) by certain cysteine cathepsins (Cts). In three-dimensional extracellular matrix culture, CtsH, among other cysteine cathepsins, was up-regulated and localized as puncta in the nuclear and cytoplasmic compartments in a complex with HDAC4 for its degradation. Conversely, along with HSC trans-differentiation, CtsH and CtsL were progressively down-regulated, whereas HDAC4 was concurrently stabilized. The inhibition of cysteine cathepsins by specific proteinase inhibitors or chloroquine, which raises cellular pH, restored HDAC4. Recombinant CtsH could break down HDAC4 in the transfected cells and in vitro at acidic pH. In human cirrhotic liver, activated HSCs express high levels of class IIa HDACs but little CtsH. We propose that cysteine cathepsin-mediated degradation of class IIa HDACs plays a key role in the modulation of MMP expression/suppression and HSC functions in tissue injury and fibrosis.
Assuntos
Catepsina H/metabolismo , Epigênese Genética , Células Estreladas do Fígado/metabolismo , Histona Desacetilases/metabolismo , Cirrose Hepática/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteólise , Proteínas Repressoras/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Catepsina L/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Transdiferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Estabilidade Enzimática/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Cirrose Hepática/enzimologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Metaloproteinase 13 da Matriz/genética , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Filaggrin, which is encoded by the filaggrin gene (FLG), is an important component of the skin's barrier to the external environment, and genetic defects in FLG strongly associate with atopic dermatitis (AD). However, not all patients with AD have FLG mutations. OBJECTIVE: We hypothesized that these patients might possess other defects in filaggrin expression and processing contributing to barrier disruption and AD, and therefore we present novel therapeutic targets for this disease. RESULTS: We describe the relationship between the mechanistic target of rapamycin complex 1/2 protein subunit regulatory associated protein of the MTOR complex 1 (RAPTOR), the serine/threonine kinase V-Akt murine thymoma viral oncogene homolog 1 (AKT1), and the protease cathepsin H (CTSH), for which we establish a role in filaggrin expression and processing. Increased RAPTOR levels correlated with decreased filaggrin expression in patients with AD. In keratinocyte cell cultures RAPTOR upregulation or AKT1 short hairpin RNA knockdown reduced expression of the protease CTSH. Skin of CTSH-deficient mice and CTSH short hairpin RNA knockdown keratinocytes showed reduced filaggrin processing, and the mouse had both impaired skin barrier function and a mild proinflammatory phenotype. CONCLUSION: Our findings highlight a novel and potentially treatable signaling axis controlling filaggrin expression and processing that is defective in patients with AD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Catepsina H/metabolismo , Dermatite Atópica/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Western Blotting , Catepsina H/deficiência , Dermatite Atópica/patologia , Proteínas Filagrinas , Imunofluorescência , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Proteína Regulatória Associada a mTOR , Pele/metabolismo , Pele/patologiaRESUMO
Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tg can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways.
Assuntos
Catepsina H/metabolismo , Tireoglobulina/metabolismo , Células Epiteliais da Tireoide/metabolismo , Glândula Tireoide/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Endocitose , Regulação da Expressão Gênica , Lisossomos/metabolismo , Microscopia de Fluorescência , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
High levels of cathepsins indicated in various pathological conditions like arthritis, cancer progressions, and atherosclerosis explains the need to explore potential inhibitors of these proteases which can be of great therapeutic significance. We, in the present work, report the synthesis of some 2,5-diaryloxadiazoles from N-subsitutedbenzylidenebenzohydrazides. The synthesized compounds were screened for their inhibitory potential on cathepsins B, H and L. Structure Activity Relationship studies show that 2,5-diaryloxadiazoles were less inhibitory than their precursors. 1i and 2k have been found to be most inhibitory to cathepsins B and L. Their Ki values have been calculated as 11.38×10(-8)M and 66.4×10(-8)M for cathepsin B and 4.2×10(-9)M and 47.31×10(-9)M for cathepsin L, respectively. However, cathepsin H activity was maximally inhibited by compounds, 1e and 2c with Ki values of 4.4×10(-7)M and 5.6×10(-7)M, respectively. Enzyme kinetic studies suggest that these compounds are competitive inhibitors to the enzymes. The results have been compared with docking results obtained using iGemDock.
Assuntos
Catepsina B/antagonistas & inibidores , Catepsina H/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Oxidiazóis/farmacologia , Inibidores de Proteases/farmacologia , Animais , Catepsina B/metabolismo , Catepsina H/metabolismo , Catepsina L/metabolismo , Relação Dose-Resposta a Droga , Cabras , Fígado/enzimologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Neuroinflammation is a hallmark that leads to selective neuronal loss and/or dysfunction in neurodegenerative disorders. Microglia-derived lysosomal cathepsins are increasingly recognized as important inflammatory mediators to trigger signaling pathways that aggravate neuroinflammation. However, cathepsin H (Cat H), a cysteine protease, has been far less studied in neuroinflammation, compared to cathepsins B, D, L, and S. The expression patterns and functional roles of Cat H in the brain in neuroinflammation remain unknown. METHODS: C57BL/6J mice were intraperitoneally injected with either 0.9% saline or lipopolysaccharide (LPS, 5 mg/kg). Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to analyze expression and localization of Cat H in the brain. Nitrite assay was used to examine microglial activation in vitro; ELISA was used to determine the release of Cat H and proinflammatory cytokines (TNF-α, IL-1ß, IL-6, IFN-γ). Cat H activity was analyzed by cellular Cat H assay kit. Flow cytometry and in situ cell death detection were used to investigate neuronal death. Data were evaluated for statistical significance with one-way ANOVA and t test. RESULTS: Cat H mRNA was only present in perivascular microglia and non-parenchymal sites under normal conditions. After LPS injection, Cat H mRNA expression in activated microglia in different brain regions was increased. Twenty-four hours after LPS injection, Cat H mRNA expression was maximal in SNr; 72 h later, it peaked in cerebral cortex and hippocampus then decreased and maintained at a low level. The expression of Cat H protein exhibited the similar alterations after LPS injection. In vitro, inflammatory stimulation (LPS, TNF-α, IL-1ß, IL-6, and IFN-γ) increased the release and activity of Cat H in microglia. Conversely, addition of Cat H to microglia promoted the production and release of NO, IL-1ß, and IFN-γ which could be prevented by neutralizing antibody. Further, addition of Cat H to Neuro2a cells induced neuronal death. CONCLUSIONS: Taken together, these data indicate that the up-regulated microglial Cat H expression, release, and activity in the brain lead to neuronal death in neuroinflammation. The functional link of Cat H with microglial activation might contribute to the initiation and maintenance of microglia-driven chronic neuroinflammation.
Assuntos
Encéfalo/metabolismo , Catepsina H/metabolismo , Encefalite , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Regulação para Cima/efeitos dos fármacos , Análise de Variância , Animais , Anticorpos/uso terapêutico , Catepsina H/genética , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Encefalite/induzido quimicamente , Encefalite/metabolismo , Encefalite/patologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Nitritos , Fosfopiruvato Hidratase/metabolismo , Fatores de TempoRESUMO
A direct correlation between cathepsin expression-cancer progression and elevated levels of cathepsins due to an imbalance in cellular inhibitors-cathepsins ratio in inflammatory diseases necessitates the work on the identification of potential inhibitors to cathepsins. In the present work we report the synthesis of some 2,3-dihydroquinazolin-4(1H)-ones followed by their evaluation as cysteine protease inhibitors in general and cathepsin B and cathepsin H inhibitors in particular. 2,3-Dihydroquinazolin-4(1H)-ones, synthesized by the condensation of anthranilamide and carbonyl compound in presence of PPA-SiO2 catalyst, were characterized by spectral analysis. The designed compounds were screened as inhibitors to proteolysis on endogenous protein substrates. Further, a distinct differential pattern of inhibition was obtained for cathepsins B and H. The inhibition was more to cathepsin B with Ki values in nanomolar range. However, cathepsin H was inhibited at micromolar concentration. Maximum inhibition was shown by compounds, 1e and 1f for cathepsin B and compounds 1c and 1f for cathepsin H. The synthesized compounds were established as reversible inhibitors of cathepsins B and H. The results were also compared with the energy of interaction between enzyme active site and compounds using iGemdock software.