RESUMEN
Abnormalities in the extracellular matrix (ECM) play important roles in the regulation and progression of clear cell renal cell carcinoma (ccRCC). The cysteine cathepsin is one of the major proteases involved in ECM remodeling and has been shown to be aberrantly expressed in multiple cancer types. However, the clinical significance and biological function of distinct cysteine cathepsins in ccRCC remain poorly understood. In this study, several bioinformatics databases, including UALCAN, TIMER, GEPIA and the Human Protein Atlas datasets, were used to analyze the expression and prognostic value of different cysteine cathepsin family members in ccRCC. We found that the expression level of CTSF was downregulated in tumor tissues and closely related to the poor survival of ccRCC patients. Further in vitro experiments suggested that CTSF overexpression suppressed the proliferation and migration of ccRCC cells. Moreover, the expression of CTSF was shown to be associated with several immune-infiltrating cells and immunomodulators in ccRCC. These results indicated that CTSF might be a promising diagnostic and prognostic marker in ccRCC.
Asunto(s)
Carcinoma de Células Renales , Catepsina F , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales , Humanos , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/mortalidad , Pronóstico , Neoplasias Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Femenino , Masculino , Catepsina F/metabolismo , Catepsina F/genética , Línea Celular Tumoral , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Movimiento Celular/genética , Persona de Mediana Edad , Regulación hacia AbajoRESUMEN
BACKGROUND: Thyroid cancer is one of the most common cancers in the world. Genetic factors are important in the occurrence and development of thyroid cancer, and genetic diagnosis has become an important basis for the prognosis of benign and malignant nodules. We identify a family of six siblings with inherited thyroid cancer susceptibility. All six members of this generation have been definitely diagnosed with papillary thyroid carcinoma. This work aims at confirming the relevant causative genes for thyroid cancer in this pedigree. METHODS: We extract DNA from the peripheral blood of six individuals and perform whole genome sequencing. Sanger sequencing and immunohistochemistry further testify the cathepsin F (CTSF) mutation and expression. RESULTS: We identify 57 single nucleotide variations (SNVs) out of at least 4 affected family members via certain filter criteria. The CTSF gene found in five of the six family members is here considered the most promising candidate gene mutation for familial thyroid cancer. Besides, our research also proves several known genes including CTSB, TEKT4, ESR1, MSH6, DIRC3, GNAS, and BANCR that act as probable oncogenic drivers in this family. The Sanger sequencing identifies the existence and veracity of CTSF somatic mutations. The CTSF immunohistochemistry of thyroid cancer tissue specimens displays that higher CTSF expression in mutated patients than that in wild-type patient as well as pericarcinomatous tissue. CONCLUSIONS: We conclude that the evaluation of CTSF gene mutations of patients in thyroid cancer families may be predictive and valuable for the familial heredity of thyroid cancer.
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Catepsina F , Neoplasias de la Tiroides , Catepsina F/genética , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad , Humanos , Mutación , Nucleótidos , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patologíaRESUMEN
Adult-onset neuronal ceroid lipofuscinoses (ANCLs, Kufs disease-KD) are rare, inherited, progressive, neurodegenerative, lysosomal storage diseases. Mutations in cathepsin F (CTSF) were linked to KD type B. Conversely, Frontotemporal dementia (FTD) is the second most common type of presenile dementia and Parkinsonism is a mostly common accompanying feature. Due to pronounced behavioral, cognitive, and motor features in the patients with KD type B, mutations in CTSF may resemble FTD-parkinsonism. Here, we present a case of KD type B with a novel homozygous frameshift pathogenic variant (p.Gly439Alafs*36) in the Cathepsin F (CTSF) gene presenting behavioral changes, cognitive disturbances and parkinsonism with a family history mimicking FTD-parkinsonism.
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Demencia Frontotemporal , Lipofuscinosis Ceroideas Neuronales , Trastornos Parkinsonianos , Enfermedad de Pick , Adulto , Catepsina F/genética , Demencia Frontotemporal/diagnóstico , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Humanos , Mutación , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/patología , Trastornos Parkinsonianos/diagnóstico , Trastornos Parkinsonianos/genéticaRESUMEN
BACKGROUND: In recent years, immunotherapies and targeted therapies contribute to population-level improvement in NSCLC cancer-specific survival, however, the two novel therapeutic options have mainly benefit patients containing mutated driven genes. Thus, to explore other potential genes related with immunity or targeted therapies may provide novel options to improve survival of lung cancer patients without mutated driven genes. CTSF is unique in human cysteine proteinases. Presently, CTSF has been detected in several cell lines of lung cancer, but its role in progression and prognosis of lung cancer remains unclear. METHODS: CTSF expression and clinical datasets of lung cancer patients were obtained from GTEx, TIMER, CCLE, THPA, and TCGA, respectively. Association of CTSF expression with clinicopathological parameters and prognosis of lung cancer patients was analyzed using UALCAN and Kaplan-Meier Plotter, respectively. LinkedOmics were used to analyze correlation between CTSF and CTSF co-expressed genes. Protein-protein interaction and gene-gene interaction were analyzed using STRING and GeneMANIA, respectively. Association of CTSF with molecular markers of immune cells and immunomodulators was analyzed with Immunedeconv and TISIDB, respectively. RESULTS: CTSF expression was currently only available for patients with NSCLC. Compared to normal tissues, CTSF was downregulated in NSCLC samples and high expressed CTSF was correlated with favorable prognosis of NSCLC. Additionally, CTSF expression was correlated with that of immune cell molecular markers and immunomodulators both in LUAD and LUSC. Noticeably, high expression of CTSF-related CTLA-4 was found to be associated with better OS of LUAD patients. Increased expression of CTSF-related LAG-3 was related with poor prognosis of LUAD patients while there was no association between CTSF-related PD-1/PD-L1 and prognosis of LUAD patients. Moreover, increased expression of CTSF-related CD27 was related with poor prognosis of LUAD patients while favorable prognosis of LUSC patients. CONCLUSIONS: CTSF might play an anti-tumor effect via regulating immune response of NSCLC.
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Antígeno CTLA-4 , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Catepsina F , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Biomarcadores de Tumor , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Catepsina F/genética , Catepsina F/inmunología , Biología Computacional , Bases de Datos Genéticas , Regulación hacia Abajo , Epistasis Genética , Humanos , Neoplasias Pulmonares/patología , PronósticoRESUMEN
Upregulating the expression of long noncoding RNA LINC00982 controlled cell proliferation in gastric cancer, but the regulatory molecular mechanisms are yet to be expounded. We here aimed to elaborate how LINC00982 regulated the malignancy of gastric cancer cells. RT-qPCR and Western blot analysis were used to detect the expression of LINC00982 and cathepsin F (CTSF) in gastric cancer tissues and cells. Modulatory effect of LINC00982 on gastric cancer cells was assessed by CCK-8, colony formation, Transwell migration, and invasion assays. The relationship between LINC00982, YRPW motif 1 (HEY1), and CTSF was examined by RNA-binding protein immunoprecipitation, luciferase assay, and chromatin immunoprecipitation, and their interaction in the regulation of gastric cancer cellular functions was analyzed by performing gain-of-function and rescue assays. The nude mouse model of tumor formation was developed to examine the effects of LINC00982 on tumorigenesis. LINC00982 was lowly expressed in gastric cancer tissues, whereas its overexpression impaired the proliferative, migratory, and invasive properties of gastric cancer cells. Furthermore, LINC00982 could bind to transcription factor HEY1 and inhibited its expression. Through blocking the binding of HEY1 to CTSF promoter, LINC00982 promoted the expression of CTSF. Overexpression of HEY1 or inhibition of CTSF could reverse the antitumor effects of LINC00982 on gastric cancer, which were further demonstrated in vivo. All these taken together, LINC00982 acted as a tumor suppressor in gastric cancer, which is therefore suggested to be a potential antitumor target for gastric cancer.NEW & NOTEWORTHY We identified LINC00982 as a promising antitumor target for the treatment of patients with gastric cancer. We also determined a regulatory network involved in the pathophysiology of gastric cancer wherein LINC00982 could bind to HEY1 to impair its binding to cathepsin F (CTSF) promoter and hence promote CTSF expression, which aids in better understanding of molecular mechanisms related to gastric tumorigenesis.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Catepsina F/metabolismo , Proteínas de Ciclo Celular/metabolismo , ARN Largo no Codificante/metabolismo , Neoplasias Gástricas/metabolismo , Regulación hacia Arriba , Anciano , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores de Tumor , Carcinogénesis/genética , Carcinogénesis/patología , Catepsina F/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Bases de Datos Factuales , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Largo no Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
Cathepsin F is a unique papain cysteine proteinase with highly conserved structures: catalytic triad and a cystatin domain contained in the elongated N-terminal pro-region. It has been reported that cathepsin F is associated with the establishment of innate immune in several vertebrate including fish in aquaculture, but not known in bivalves. In this study, we firstly identified and characterized cathepsin F in the Yesso scallop (Patinopecten yessoensis). The protein structural and phylogenetic analyses were then conducted to determine its identity and evolutionary position. We've also investigated the expression levels of cathepsin F gene at different embryonic developmental stages, in healthy adult tissues and especially in the hemocytes and hepatopancreas after Gram-positive (Micrococcus luteus) and negative (Vibrio anguillarum) challenges using quantitative real-time PCR (qPCR). Cathepsin F was significantly up-regulated 3â¯h after infection of V. anguillarum in hemocytes, suggesting its participation in immune response. Our findings have provided strong evidence that cathepsin F may be a good target for enhancing the immune activity in Yesso scallop.
Asunto(s)
Catepsina F , Infecciones por Bacterias Grampositivas/inmunología , Pectinidae/genética , Pectinidae/inmunología , Vibriosis/inmunología , Secuencia de Aminoácidos , Animales , Catepsina F/química , Catepsina F/genética , Catepsina F/inmunología , Infecciones por Bacterias Grampositivas/veterinaria , Hemocitos/inmunología , Hepatopáncreas/inmunología , Micrococcus luteus , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/genética , Vibrio , Vibriosis/veterinariaRESUMEN
Gastric cancer (GC) is one of the most common cancers in the world. The cathepsin F (CTSF) gene has recently been found to participate in the progression of several types of cancer. However, the clinical characteristics and function of CTSF in GC as well as its molecular mechanisms are not clear. Six GC cell lines and 44 paired adjacent noncancerous and GC tissue samples were used to assess CTSF expression by quantitative polymerase chain reaction (qPCR). We used lentivirus-mediated small hairpin RNA (Lenti-shRNA) against CTSF to knock down the expression of CTSF in GC cells. Western blot and qPCR were used to analyze the mRNA and related protein expression. The biological phenotypes of gastric cells were examined by cell proliferation and apoptosis assays. Microarray-based mRNA expression profile screening was also performed to evaluate the potential molecular pathways in which CTSF may be involved. The CTSF mRNA level was associated with tumor differentiation, depth of tumor invasion, and lymph node metastasis. Downregulation of CTSF expression efficiently inhibited apoptosis and promoted the proliferation of GC cells. Moreover, a total of 1,117 upregulated mRNAs and 1,143 downregulated mRNAs were identified as differentially expressed genes (DEGs). Further analysis identified the involvement of these mRNAs in cancer-related pathways and various other biological processes. Nine DEGs in cancer-related pathways and three downstream genes in the apoptosis pathway were validated by Western blot, which was mainly in agreement with the microarray data. To our knowledge, this is the first report investigating the effect of CTSF on the growth and apoptosis in GC cells and its clinical significance. The CTSF gene may function as a tumor suppressor in GC and may be a potential therapeutic target in the treatment of GC.
Asunto(s)
Apoptosis/genética , Catepsina F/metabolismo , Proliferación Celular/genética , Genes Supresores de Tumor , Neoplasias Gástricas/patología , Biomarcadores de Tumor/genética , Catepsina F/genética , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Gástricas/genética , TranscriptomaRESUMEN
BACKGROUND: Kufs disease type B (also termed CLN13), an adult-onset form of neuronal ceroid lipofuscinosis (NCL), is genetically heterogeneous and challenging to diagnose. Recently, mutations in cathepsin-F have been identified as the causative gene for autosomal recessive Kufs disease type B. RESULTS: Here, we report a sporadic case of Kufs disease type B with novel compound heterozygous mutations, a novel missense mutation c.977G>T (p.C326F) and a novel nonsense mutation c.416C>A (p.S139X), in the cathepsin-F gene. The magnetic resonance imaging findings were consistent with those demonstrated in adult neuronal ceroid lipofuscinosis: diffuse cortical atrophy, mild hyperintensity and reduction of the deep white matter on T2-weighted images. A skin biopsy was negative for abnormalities. CONCLUSIONS: Altogether, our findings broaden the mutation database in relation to the neuronal ceroid lipofuscinosis, and the clinical diagnosis of Kufs disease type B was confirmed.
Asunto(s)
Catepsina F/genética , Lipofuscinosis Ceroideas Neuronales/diagnóstico , Lipofuscinosis Ceroideas Neuronales/genética , Adulto , Codón sin Sentido , Femenino , Humanos , Mutación Missense , Lipofuscinosis Ceroideas Neuronales/patología , Lipofuscinosis Ceroideas Neuronales/fisiopatologíaRESUMEN
Cathepsin F (CTSF) is a recently described papain-like cysteine protease and unique among cathepsins due to an elongated N-terminal pro-region, which contains a cystatin domain. CTSF likely plays a regulatory role in processing the invariant chain which is associated with the major histocompatibility complex (MHC) class II. In this regard, we identified the CTSF gene of turbot as well as its protein structure, phylogenetic relationships, and expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. We also determined the expression patterns of CTSF in mucosal tissues after vaccinated with the formalin-inactivated V. vulnificus whole-cell vaccine. Briefly, turbot CTSF gene showed the closest relationship with that of Paralichthys olivaceus in phylogenetic analysis. And CTSF was ubiquitously expressed in all tested tissues with the highest expression level in gill. In addition, CTSF gene showed different expression patterns following different bacterial challenge. The significant quick regulation of CTSF in mucosal surfaces against infection indicated its roles in mucosal immunity. Functional studies should further characterize avail utilization of CTSF function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infection and to facilitate selection of the disease resistant family/strain in turbot.
Asunto(s)
Catepsina F/genética , Catepsina F/inmunología , Enfermedades de los Peces/inmunología , Peces Planos , Inmunidad Mucosa/genética , Infecciones Estreptocócicas/veterinaria , Vibriosis/veterinaria , Secuencia de Aminoácidos , Animales , Catepsina F/química , ADN Complementario/genética , ADN Complementario/metabolismo , Enfermedades de los Peces/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/metabolismo , Peces Planos/clasificación , Peces Planos/genética , Peces Planos/inmunología , Conformación Molecular , Membrana Mucosa/inmunología , Filogenia , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/inmunología , Streptococcus iniae/fisiología , Vibrio/fisiología , Vibriosis/genética , Vibriosis/inmunologíaRESUMEN
Cathepsin F, which is encoded by CTSF, is a cysteine proteinase ubiquitously expressed in several tissues. In a previous study, novel transcripts of the CTSF gene were identified in the crab-eating monkey deriving from the integration of an Alu element-AluYRa1. The occurrence of AluYRa1-derived alternative transcripts and the mechanism of exonization events in the CTSF gene of human, rhesus monkey, and crab-eating monkey were investigated using PCR and reverse transcription PCR on the genomic DNA and cDNA isolated from several tissues. Results demonstrated that AluYRa1 was only integrated into the genome of Macaca species and this lineage-specific integration led to exonization events by producing a conserved 3' splice site. Six transcript variants (V1-V6) were generated by alternative splicing (AS) events, including intron retention and alternative 5' splice sites in the 5' and 3' flanking regions of CTSF_AluYRa1. Among them, V3-V5 transcripts were ubiquitously expressed in all tissues of rhesus monkey and crab-eating monkey, whereas AluYRa1-exonized V1 was dominantly expressed in the testis of the crab-eating monkey, and V2 was only expressed in the testis of the two monkeys. These five transcript variants also had different amino acid sequences in the C-terminal region of CTSF, as compared to reference sequences. Thus, species-specific Alu-derived exonization by lineage-specific integration of Alu elements and AS events seems to have played an important role during primate evolution by producing transcript variants and gene diversification.
Asunto(s)
Elementos Alu , Catepsina F/genética , Macaca fascicularis/genética , Macaca mulatta/genética , Empalme Alternativo , Animales , Evolución Biológica , Humanos , MasculinoRESUMEN
Single nucleotide polymorphisms (SNPs) of six genes (TTN, PRKAG3, CAST, CTSB, CTSF, and MYPN), known for associations with carcass and meat quality traits, post mortem proteolysis, were screened in a commercial crossed population of 368 heavy pigs (Large White x Landrace)×Duroc, reared according to the rules of Italian Protected Designation of Origin (PDO) dry-cured ham. Carcass, longissimus thoracis et lumborum muscle (LTL), and green ham traits were obtained after slaughtering, main weight losses of dry-cured hams were collected during processing. The results showed the impact of CAST variants on carcass weight, of CTSF on LTL tenderness, ham weight and fatness, of PRKAG3 and TTN on ultimate pH, hamweight. This study, while confirming significant associations between SNPs of genes and qualitative traits of carcass, longissimus and ham, supports CTSF as candidate gene suitable for fresh consumption purpose (tenderness of longissimus at 24h post mortem), and for dry-cured ham processing (higher thickness of ham subcutaneous fat).
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Manipulación de Alimentos , Productos de la Carne , Polimorfismo de Nucleótido Simple , Carne Roja , Porcinos/genética , Proteínas Quinasas Activadas por AMP/genética , Animales , Catepsina B/genética , Catepsina F/genética , Conectina/genética , Conectina/metabolismo , Proteínas del Citoesqueleto/genética , Calidad de los Alimentos , Frecuencia de los Genes , Marcadores Genéticos , Técnicas de Genotipaje , Concentración de Iones de Hidrógeno , Proteínas Musculares/genética , Músculo Esquelético/química , Fenotipo , Grasa Subcutánea/químicaRESUMEN
We have previously reported the whole genome genotyping analysis of 2 consanguineous siblings clinically diagnosed with early onset Alzheimer's disease (AD). In this analysis, we identified several large regions of homozygosity shared between both affected siblings, which we suggested could be candidate loci for a recessive genetic lesion underlying the early onset AD in these cases. We have now performed exome sequencing in one of these siblings and identified the potential cause of disease: the CTSF c.1243G>A:p.Gly415Arg mutation in homozygosity. Biallelic mutations in this gene have been shown to cause Type B Kufs disease, an adult-onset neuronal ceroid lipofuscinosis with some cases resembling the impairment seen in AD.
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Enfermedad de Alzheimer/genética , Catepsina F/genética , Consanguinidad , Exoma/genética , Estudio de Asociación del Genoma Completo , Homocigoto , Mutación/genética , Anciano , Humanos , Masculino , Persona de Mediana Edad , Lipofuscinosis Ceroideas Neuronales/genética , Análisis de Secuencia , HermanosRESUMEN
The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.
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Adenoviridae/genética , Enfermedades de la Córnea/terapia , Neuropatías Diabéticas/terapia , Epitelio Corneal/citología , Terapia Genética , Limbo de la Córnea/citología , Células Madre/metabolismo , Cicatrización de Heridas/fisiología , Biomarcadores/metabolismo , Catepsina F/genética , Recuento de Células , Epitelio Corneal/metabolismo , Terapia Genética/métodos , Vectores Genéticos , Humanos , Limbo de la Córnea/metabolismo , Metaloproteinasa 10 de la Matriz/genética , Técnicas de Cultivo de Órganos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genéticaRESUMEN
BACKGROUND: Trichinellosis is a re-emerging infectious disease, caused by Trichinella spp. Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths, and it may be a potential anti-helminth drug target and vaccine candidate. The aim of this study was to clone, express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics. METHODS: The full-length cDNA encoding a putative cathepsin F-like protease in T. spiralis, TsCF1, was cloned and its biochemical characterization and expression profile were analyzed. Transcription of TsCF1 at different developmental stages of T. spiralis was observed by RT-PCR. The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1 (rTsCF1) was analyzed by western blotting. And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique. Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed. Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate. RESULTS: Sequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46. The cysteine protease conserved active site of Cys173, His309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1. Sequence alignment analysis revealed a higher than 40 % identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode. RT-PCR revealed the gene was expressed in muscle larvae, newborn larvae and adult stages. SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa. The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae. After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 µM and Vmax = 6.12 RFU/s µM at pH 5.5, and the IC50 value of E64 was 135.50 ± 16.90 nM. CONCLUSION: TsCF1 was expressed in all stages of T. spiralis and localized in the cuticle and stichosome. TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection.
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Catepsina F/genética , Catepsina F/metabolismo , Trichinella spiralis/enzimología , Trichinella spiralis/genética , Animales , Clonación Molecular , Inhibidores de Cisteína Proteinasa/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Leucina/análogos & derivados , Leucina/metabolismo , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Trichinella spiralis/crecimiento & desarrolloRESUMEN
The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase ß-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.
Asunto(s)
Catepsina F/genética , Catepsina F/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Receptores Depuradores/metabolismo , Animales , Antígenos CD36/química , Antígenos CD36/genética , Antígenos CD36/metabolismo , Línea Celular , Células HEK293 , Humanos , Proteínas de Membrana de los Lisosomas/química , Proteínas de Membrana de los Lisosomas/genética , Lisosomas/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Proteolisis , Receptores Depuradores/química , Receptores Depuradores/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Paragonimiasis, caused by the lung fluke Paragonimus, is a major food-borne helminthic disease. Differential diagnosis of paragonimiasis from tuberculosis and other infectious granulomas in the lung is a prerequisite to proper management of patients. Cysteine proteases of Paragonimus westermani (PwCPs) invoke specific antibody responses against patient sera, while antibody capturing activity of different PwCPs has not been comparatively analyzed. In this study, we observed the expressional regulation of 11 species of different PwCPs (PwCP1-11). We expressed recombinant PwCPs and assessed diagnostic reliability employing sera from patients with P. westermani (n=138), other trematodiases (n=80), cestodiases (n=60) and pulmonary tuberculosis (n=20), and those of normal controls (n=20). PwCPs formed a monophyletic clade into cathepsin F and showed differential expression patterns along with developmental stages of worm. Bacterially expressed recombinant PwCPs (rPwCPs) exhibited variable sensitivity of 38.4-84.5% and specificity of 87.2-100% in diagnosing homologous infection. rPwCPs recognized specific antibodies of experimental cat sera as early as 3 or 6weeks after infection. Patient sera of fascioliasis, Schistosomiasis japonicum and clonorchiasis demonstrated weak cross-reactions. Our results demonstrate that diverse PwCPs of the cathepsin F family participate in inducing specific antibody responses. Most P. westermani cathepsin F, except for PwCP2 (AAF21461), which showed negligible antibody responses, might be applicable for paragonimiasis serodiagnosis.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Catepsina F/inmunología , Paragonimiasis/diagnóstico , Paragonimiasis/inmunología , Paragonimus westermani/inmunología , Animales , Antígenos Helmínticos/inmunología , Catepsina F/genética , Gatos , Clonorquiasis/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Fascioliasis/inmunología , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Paragonimus westermani/enzimología , Filogenia , Proteínas Recombinantes/inmunología , Esquistosomiasis Japónica/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/inmunologíaAsunto(s)
Catepsina F/genética , Mutación/genética , Lipofuscinosis Ceroideas Neuronales/genética , Adulto , Salud de la Familia , Femenino , Humanos , Masculino , Lipofuscinosis Ceroideas Neuronales/patología , Presenilina-1/genética , Piel/patología , Piel/ultraestructura , Ubiquitina C/metabolismoRESUMEN
PURPOSE: Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression. METHODS: Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections. RESULTS: Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α3ß1 integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain. CONCLUSIONS: Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
Asunto(s)
Catepsina F/genética , Enfermedades de la Córnea/genética , Diabetes Mellitus/genética , Silenciador del Gen , Metaloproteinasa 10 de la Matriz/genética , Células Madre/metabolismo , Cicatrización de Heridas/genética , Anciano , Anciano de 80 o más Años , Catepsina F/biosíntesis , Células Cultivadas , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Epitelio Corneal/metabolismo , Femenino , Terapia Genética/métodos , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 10 de la Matriz/biosíntesis , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Células Madre/citologíaRESUMEN
The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. We predicted that the wild-type human cathepsin F contains three natively disordered regions within the enzyme's propeptide and various amino acid stretches with high fibrillation propensity. Wild-type human cathepsin F and its N-terminally truncated forms, Ala(20)-Asp(484) (Δ(19)CatF), Pro(126)-Asp(484) (Δ(125)CatF), and Met(147)-Asp(484) (Δ(146)CatF) were cloned into the pcDNA3 vector and overexpressed in HEK 293T cells. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides (Δ(19)CatF), results in protein aggregation. Noteworthy, they concentrated large perinuclear-juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2-3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Caspasas/metabolismo , Catepsina F/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencia de Aminoácidos , Apoptosis , Autofagia , Western Blotting , Catepsina F/genética , Células Cultivadas , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Glicosilación , Humanos , Técnicas para Inmunoenzimas , Proteína 2 de la Membrana Asociada a los Lisosomas , Datos de Secuencia Molecular , Plásmidos , Multimerización de Proteína , Proteína Sequestosoma-1 , Fracciones SubcelularesRESUMEN
Kufs disease, an adult-onset neuronal ceroid lipofuscinosis, is challenging to diagnose and genetically heterogeneous. Mutations in CLN6 were recently identified in recessive Kufs disease presenting as progressive myoclonus epilepsy (Type A), whereas the molecular basis of cases presenting with dementia and motor features (Type B) is unknown. We performed genome-wide linkage mapping of two families with recessive Type B Kufs disease and identified a single region on chromosome 11 to which both families showed linkage. Exome sequencing of five samples from the two families identified homozygous and compound heterozygous missense mutations in CTSF within this linkage region. We subsequently sequenced CTSF in 22 unrelated individuals with suspected recessive Kufs disease, and identified an additional patient with compound heterozygous mutations. CTSF encodes cathepsin F, a lysosomal cysteine protease, dysfunction of which is a highly plausible candidate mechanism for a storage disorder like ceroid lipofuscinosis. In silico modeling suggested the missense mutations would alter protein structure and function. Moreover, re-examination of a previously published mouse knockout of Ctsf shows that it recapitulates the light and electron-microscopic pathological features of Kufs disease. Although CTSF mutations account for a minority of cases of type B Kufs, CTSF screening should be considered in cases with early-onset dementia and may avoid the need for invasive biopsies.