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1.
Br J Cancer ; 126(12): 1795-1805, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35217799

RESUMO

BACKGROUND: The lack of non-invasive methods for detection of early micro-metastasis is a major cause of the poor prognosis of non-small cell lung cancer (NSCLC) brain metastasis (BM) patients. Herein, we aimed to identify circulating biomarkers based on proteomics for the early diagnosis and monitoring of patients with NSCLC BM. METHODS: Upregulated proteins were detected by secretory proteomics in the animal-derived high brain metastatic lung cancer cell line. A well-designed study composed of three independent cohorts was then performed to verify these blood-based protein biomarkers: the serum discovery and verification cohorts (n = 80; n = 459), and the tissue verification cohort (n = 76). Logistic regression was used to develop a diagnostic biomarker panel. Model validation cohort (n = 160) was used to verify the stability of the constructed predictive model. Changes in serum Cathepsin F (CTSF) levels of patients were tracked to monitor the treatment response. Progression-free survival (PFS) and overall survival (OS) were analysed to assess their prognostic relevance. RESULTS: CTSF and Fibulin-1 (FBLN1) levels were specifically upregulated in sera and tissues of patients with NSCLC BM compared with NSCLC without BM and primary brain tumour. The combined diagnostic performance of CTSF and FBLN1 was superior to their individual ones. CTSF serum changes were found to reflect the therapeutic response of patients with NSCLC BM and the trends of progression were detected earlier than the magnetic resonance imaging changes. Elevated expression of CTSF in NSCLC BM tissues was associated with poor PFS, and was found to be an independent prognostic factor. CONCLUSIONS: We report a novel blood-based biomarker panel for early diagnosis, monitoring of therapeutic response, and prognostic evaluation of patients with NSCLC BM.


Assuntos
Neoplasias Encefálicas , Proteínas de Ligação ao Cálcio , Carcinoma Pulmonar de Células não Pequenas , Catepsina F , Neoplasias Pulmonares , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Proteínas de Ligação ao Cálcio/sangue , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Catepsina F/sangue , Catepsina F/metabolismo , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Prognóstico , Regulação para Cima
2.
Neurocase ; 28(1): 107-109, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35139754

RESUMO

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.


Assuntos
Demência Frontotemporal , Lipofuscinoses Ceroides Neuronais , Transtornos Parkinsonianos , Doença de Pick , Adulto , Catepsina F/genética , Demência Frontotemporal/diagnóstico , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , Transtornos Parkinsonianos/diagnóstico , Transtornos Parkinsonianos/genética
3.
J Biomol Struct Dyn ; 40(1): 325-336, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873185

RESUMO

The world has come to a sudden halt due to the incessant spread of a viral pneumonia dubbed COVID-19 caused by the beta-coronavirus, SARS-CoV-2. The main protease of SARS-CoV-2 plays a key role in the replication and propagation of the virus in the host cells. Inhibiting the protease blocks the replication of the virus; therefore it is considered as an attractive therapeutic target. Here we describe the screening of the DrugBank database, a public repository for small molecule therapeutics, to identify approved or experimental phase drugs that can be repurposed against the main protease of SARS-CoV-2. The initial screening was performed on more than 13,000 drug entries in the target database using an energy optimised pharmacophore hypothesis AARRR. A sub-set of the molecules selected based on the fitness score was further screened using molecular docking by sequentially filtering the molecules through the high throughput virtual screening, extra precision and standard precision docking modalities. The best hits were subjected to binding free energy estimation using the MM-GBSA method. Approved drugs viz, Cobicistat, Larotrectinib and Simeprevir were identified as potential candidates for repurposing. Drugs in the discovery phase identified as inhibitors include the known cysteine protease inhibitors, Calpain inhibitor IV and an experimental cathepsin F inhibitor. In order to analyse the stability of the binding interactions, the known cysteine protease inhibitors viz, Simeprevir, calpain inhibitor IV and the cathepsin F inhibitor in complex Mpro were subjected to molecular dynamics simulations at 100 ns. Based on the results Simeprevir was found to be a strong inhibitor of SARS-CoV-2 Mpro.Communicated by Ramaswamy H. Sarma.


Assuntos
Antivirais , Proteases 3C de Coronavírus/antagonistas & inibidores , Reposicionamento de Medicamentos , Inibidores de Proteases , SARS-CoV-2/efeitos dos fármacos , Simeprevir , Antivirais/farmacologia , COVID-19 , Catepsina F/antagonistas & inibidores , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologia , Simeprevir/farmacologia
4.
BMC Pulm Med ; 21(1): 420, 2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34923982

RESUMO

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.


Assuntos
Antígeno CTLA-4 , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Catepsina F , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Biomarcadores Tumorais , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Catepsina F/genética , Catepsina F/imunologia , Biologia Computacional , Bases de Dados Genéticas , Regulação para Baixo , Epistasia Genética , Humanos , Neoplasias Pulmonares/patologia , Prognóstico
5.
Stem Cell Res Ther ; 12(1): 447, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34372921

RESUMO

BACKGROUND: Radiation-induced dermatitis is a serious side effect of radiotherapy, and very few effective treatments are currently available for this condition. We previously demonstrated that apoptosis is an important feature of radiation-induced dermatitis and adipose-derived stem cells (ADSCs) are one of the most promising types of stem cells that have a protective effect on acute radiation-induced dermatitis. Cathepsin F (CTSF) is a recently discovered protein that plays an important role in apoptosis. In this study, we investigated whether ADSCs affect chronic radiation-induced dermatitis, and the underlying mechanisms involved. METHODS: ADSCs were isolated from male Sprague-Dawley (SD) rats and characterized. For in vivo studies, rats were randomly divided into control and ADSC-treated groups, and cultured ADSCs were transplanted into radiation-induced dermatitis model rats. The effects of ADSC transplantation were determined by skin damage scoring, histopathological analysis, electron microscopy, immunohistochemical staining, and western blotting analysis of apoptosis-related proteins. To evaluate the effects of ADSCs in vitro, radiation-induced apoptotic cells were treated with ADSC culture supernatant, and apoptosis-related protein expression was investigated by TUNEL staining, flow cytometry, and western blotting. RESULTS: In the in vivo studies, skin damage, inflammation, fibrosis, and apoptosis were reduced and hair follicle and sebaceous gland regeneration were enhanced in the ADSC group compared with the control group. Further, CTSF and downstream pro-apoptotic proteins (Bid, BAX, and caspase 9) were downregulated, while anti-apoptotic proteins (Bcl-2 and Bcl-XL) were upregulated. In vitro, ADSCs markedly attenuated radiation-induced apoptosis, downregulated CTSF and downstream pro-apoptotic proteins, and upregulated anti-apoptotic proteins. CONCLUSION: ADSCs protect against radiation-induced dermatitis by exerting an anti-apoptotic effect through inhibition of CTSF expression. ADSCs may be a good therapeutic candidate to prevent the development of radiation-induced dermatitis.


Assuntos
Células-Tronco Mesenquimais , Radiodermatite , Tecido Adiposo , Animais , Apoptose , Catepsina F , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley , Células-Tronco
6.
Am J Physiol Gastrointest Liver Physiol ; 320(5): G816-G828, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33236952

RESUMO

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.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Catepsina F/metabolismo , Proteínas de Ciclo Celular/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/metabolismo , Regulação para Cima , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais , Carcinogênese/genética , Carcinogênese/patologia , Catepsina F/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Bases de Dados Factuais , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
7.
Parasitol Res ; 119(11): 3691-3698, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33009945

RESUMO

Fascioliasis, a food- and water-borne trematodiasis, has been identified as a public health threat by the World Health Organization, with millions of people estimated to be infected or at risk of infection worldwide. We developed an immunochromatographic test (ICT) as a point-of-care (POC) tool for the rapid serodiagnosis of human fascioliasis caused by Fasciola gigantica and evaluated their diagnostic ability. Two tests were developed using antigens from adult F. gigantica excretory-secretory (ES) product and recombinant F. gigantica cathepsin L (rFgCL). Sera from 12 patients with parasitologically proven fascioliasis caused by F. gigantica, 18 with clinically suspected fascioliasis, 65 with other parasitic infections, and 30 healthy controls were used. Using a cutoff of > 0.5 for antibody detection, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the ES-based ICT method were 100%, 98.9% 96.8%, 100%, and 99.2%, respectively, and those of the rFgCL-based ICT method were 86.7%, 93.7%, 81.3%, 95.7%, and 92.0%, respectively. The concordance between the two methods was 91.2%. Tests using F. gigantica ES and rFgCL antigens can be employed quickly and easily as POC diagnostic tools. They can be used to support the clinical diagnosis of human fascioliasis gigantica and in large-scale surveys in endemic areas throughout tropical regions without necessitating additional facilities or ancillary supplies.


Assuntos
Antígenos de Helmintos/imunologia , Catepsina L/imunologia , Fasciola/isolamento & purificação , Fasciolíase/diagnóstico , Animais , Anticorpos Anti-Helmínticos/sangue , Catepsina F/sangue , Cromatografia de Afinidade , Fasciola/imunologia , Humanos , Testes Imediatos , Sensibilidade e Especificidade , Testes Sorológicos/métodos
8.
Stem Cell Reports ; 13(2): 380-393, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31378672

RESUMO

Here, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes.


Assuntos
Doença de Fabry/patologia , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Receptores Depuradores/metabolismo , Potenciais de Ação , Biomarcadores/metabolismo , Catepsina F/metabolismo , Edição de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/fisiologia , Mutação Puntual , Mapas de Interação de Proteínas , Proteômica , Vacúolos/metabolismo , alfa-Galactosidase/genética
9.
Fish Shellfish Immunol ; 80: 141-147, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29879509

RESUMO

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 3 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.


Assuntos
Catepsina F , Infecções por Bactérias Gram-Positivas/imunologia , Pectinidae/genética , Pectinidae/imunologia , Vibrioses/imunologia , Sequência de Aminoácidos , Animais , Catepsina F/química , Catepsina F/genética , Catepsina F/imunologia , Infecções por Bactérias Gram-Positivas/veterinária , Hemócitos/imunologia , Hepatopâncreas/imunologia , Micrococcus luteus , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Vibrio , Vibrioses/veterinária
10.
Biochem Biophys Res Commun ; 498(1): 9-17, 2018 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-29499196

RESUMO

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel ß-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.


Assuntos
Clonorchis sinensis/química , Cistatinas/química , Inibidores de Cisteína Proteinase/química , Sequência de Aminoácidos , Animais , Catepsina F/antagonistas & inibidores , Catepsina F/metabolismo , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica
11.
Oncol Res ; 26(1): 83-93, 2018 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-28474574

RESUMO

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.


Assuntos
Apoptose/genética , Catepsina F/metabolismo , Proliferação de Células/genética , Genes Supressores de Tumor , Neoplasias Gástricas/patologia , Biomarcadores Tumorais/genética , Catepsina F/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Gástricas/genética , Transcriptoma
12.
Int J Neurosci ; 128(6): 573-576, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29120254

RESUMO

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.


Assuntos
Catepsina F/genética , Lipofuscinoses Ceroides Neuronais/diagnóstico , Lipofuscinoses Ceroides Neuronais/genética , Adulto , Códon sem Sentido , Feminino , Humanos , Mutação de Sentido Incorreto , Lipofuscinoses Ceroides Neuronais/patologia , Lipofuscinoses Ceroides Neuronais/fisiopatologia
13.
Fish Shellfish Immunol ; 66: 270-279, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28501446

RESUMO

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.


Assuntos
Catepsina F/genética , Catepsina F/imunologia , Doenças dos Peixes/imunologia , Linguados , Imunidade nas Mucosas/genética , Infecções Estreptocócicas/veterinária , Vibrioses/veterinária , Sequência de Aminoácidos , Animais , Catepsina F/química , DNA Complementar/genética , DNA Complementar/metabolismo , Doenças dos Peixes/genética , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Linguados/classificação , Linguados/genética , Linguados/imunologia , Conformação Molecular , Membrana Mucosa/imunologia , Filogenia , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/imunologia , Streptococcus iniae/fisiologia , Vibrio/fisiologia , Vibrioses/genética , Vibrioses/imunologia
14.
Mol Cells ; 40(2): 100-108, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28196413

RESUMO

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.


Assuntos
Elementos Alu , Catepsina F/genética , Macaca fascicularis/genética , Macaca mulatta/genética , Processamento Alternativo , Animais , Evolução Biológica , Humanos , Masculino
15.
Meat Sci ; 126: 73-81, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28064046

RESUMO

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).


Assuntos
Manipulação de Alimentos , Produtos da Carne , Polimorfismo de Nucleotídeo Único , Carne Vermelha , Suínos/genética , Proteínas Quinases Ativadas por AMP/genética , Animais , Catepsina B/genética , Catepsina F/genética , Conectina/genética , Conectina/metabolismo , Proteínas do Citoesqueleto/genética , Qualidade dos Alimentos , Frequência do Gene , Marcadores Genéticos , Técnicas de Genotipagem , Concentração de Íons de Hidrogênio , Proteínas Musculares/genética , Músculo Esquelético/química , Fenótipo , Gordura Subcutânea/química
16.
Parasitol Int ; 66(4): 443-447, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27140305

RESUMO

Diagnosis of Opisthorchis viverrini infection by conventional stool examination is increasingly difficult due to the low intensity of the infection after several rounds of control programmes in endemic regions as well as coinfections with intestinal flukes. Therefore sensitive and specific diagnostic test is needed. In this study, a coproantigen sandwich ELISA using recombinant O. viverrini cathepsin F (rOv-CF) was developed. This sandwich ELISA employing chicken IgY raised against rOv-CF in combination with rabbit IgG antibody to the somatic O. viverrini antigens showed a lower detection limit (LLD) of 70ng native O. viverrini somatic antigens by spiking the parasite antigens into control feces. When applied to the diagnosis, the IgY-based sandwich ELISA exhibited sensitivity and specificity of 93.3% and 76.7%, respectively, in an investigation of 90 human cases positive or negative for opisthorchiasis. The positive predictive value (PPV) and negative predictive value (NPV) for this coproantigen detection were 66.7% and 95.2%, respectively. This IgY-based sandwich ELISA using parasite cathepsin F detection shows a promising immunodiagnostic alternative for human opisthorchiasis in endemic regions.


Assuntos
Antígenos de Helmintos/metabolismo , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Imunoglobulinas/metabolismo , Opistorquíase/diagnóstico , Opisthorchis/isolamento & purificação , Animais , Catepsina F/metabolismo , Galinhas , Proteínas de Helminto/metabolismo , Humanos , Opistorquíase/parasitologia , Coelhos , Sensibilidade e Especificidade
17.
Neurobiol Aging ; 46: 236.e1-6, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27524508

RESUMO

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.


Assuntos
Doença de Alzheimer/genética , Catepsina F/genética , Consanguinidade , Exoma/genética , Estudo de Associação Genômica Ampla , Homozigoto , Mutação/genética , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Lipofuscinoses Ceroides Neuronais/genética , Análise de Sequência , Irmãos
18.
J Vis Exp ; (110): e54058, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27077448

RESUMO

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.


Assuntos
Adenoviridae/genética , Doenças da Córnea/terapia , Neuropatias Diabéticas/terapia , Epitélio Corneano/citologia , Terapia Genética , Limbo da Córnea/citologia , Células-Tronco/metabolismo , Cicatrização/fisiologia , Biomarcadores/metabolismo , Catepsina F/genética , Contagem de Células , Epitélio Corneano/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Humanos , Limbo da Córnea/metabolismo , Metaloproteinase 10 da Matriz/genética , Técnicas de Cultura de Órgãos , Proteínas Proto-Oncogênicas c-met/genética
19.
Parasit Vectors ; 8: 652, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26692208

RESUMO

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.


Assuntos
Catepsina F/genética , Catepsina F/metabolismo , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Animais , Clonagem Molecular , Inibidores de Cisteína Proteinase/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Leucina/análogos & derivados , Leucina/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Trichinella spiralis/crescimento & desenvolvimento
20.
Parasitol Res ; 114(12): 4571-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26344868

RESUMO

Opisthorchis viverrini, a food-borne trematode parasite endemic in the lower Mekong countries, is conventionally diagnosed by stool examination. However, parasitological stool-based diagnosis can be unreliable in light infections. The goal of this study was to develop the immunodiagnosis of opisthorchiasis using cathepsin F cysteine protease of O. viverrini in both indirect and sandwich ELISA assays. A recombinant O. viverrini cathepsin F (rOv-CF) of 40 kDa was expressed in E. coli strain BL21 (DE3), affinity purified, and deployed in ELISA assays. Human sera from 272 cases were investigated by indirect rOv-CF-based ELISA. Positive antibody response to rOv-CF was found in 137 out of 272 cases (50.37 %) using a cutoff OD (0.400) determined by ROC analysis. In comparison to parasitological stool examined for fluke eggs, the gold standard, the rOv-CF indirect ELISA showed a sensitivity and specificity of 62.1 and 84.05 %, respectively. Serum antibody levels correlated well with egg counts per gram feces (EPG) (P < 0.001). In addition, chicken IgY antibody raised against rOv-CF was tested in a sandwich ELISA for detection of coproantigen in the feces of experimentally infected hamsters. The sandwich ELISA using this chicken IgY in combination with rabbit antibody to O. viverrini somatic antigens showed sensitivity and specificity of 93.3 and 78.57 %, respectively. Together, these findings indicated the potential of rOv-CF for diagnosis of opisthorchiasis, including for uses with chicken IgY for detection of coproantigens of O. viverrini.


Assuntos
Catepsina F/análise , Ensaio de Imunoadsorção Enzimática/métodos , Opistorquíase/diagnóstico , Opisthorchis/imunologia , Animais , Catepsina F/imunologia , Cricetinae , Fezes/parasitologia , Humanos , Testes Imunológicos , Masculino , Opistorquíase/imunologia , Opistorquíase/parasitologia , Opisthorchis/enzimologia , Opisthorchis/isolamento & purificação , Curva ROC , Coelhos , Sensibilidade e Especificidade
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