RESUMEN
Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.
Asunto(s)
Catepsina B , Quirópteros , Cistatina A , Animales , Humanos , Catepsina B/metabolismo , Catepsina B/química , Catepsina B/genética , Catepsina B/antagonistas & inhibidores , Cistatina A/metabolismo , Cistatina A/química , Cistatina A/genética , Simulación del Acoplamiento Molecular , Secuencia de Aminoácidos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Catepsina L/metabolismo , Catepsina L/química , Catepsina L/genética , Catepsina L/antagonistas & inhibidores , Cinética , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/metabolismoRESUMEN
Cystatin A (CyA), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyA and its potential molecular mechanism during virus infection in fish remain unknown. In our study, we cloned the open reading frame (ORF) of CyA homology from orange-spotted grouper (Ec-CyA) consisting of 303 nucleotides and encoding a 101-amino acid protein. Ec-CyA included two conserved sequences containing one N-terminal glycine fragment and one QXVXG sequence (48aa-52aa) without the signal peptide. Tissue distribution analysis showed that Ec-CyA was highly expressed in spleen and head kidney. Moreover, further analysis indicated that the expression of Ec-CyA increased during SGIV simulation in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyA was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyA promoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was facilitated, as well as the activation of caspase-3/7, caspase-9. In addition, Ec-CyA overexpression down-regulated the expression of interferon (IFN) related molecules including ISG15, IFN, IRF3, MAVS, MyD88, TRAF6 and up-regulated proinflammatory factors such as IL-1ß, IL-8 and TNF-α. At the same time, Ec-CyA-overexpressing inhibited the activity of IFN and ISRE promoter, but induced NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyA was involved in innate immune response and played a key role in DNA virus infection.
Asunto(s)
Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Animales , Secuencia de Bases , Clonación Molecular , Cistatina A/genética , Proteínas de Peces/metabolismo , Inmunidad Innata , FilogeniaRESUMEN
The cysteine protease Cathepsin B (CtsB) plays a critical role in multiple signaling pathways, intracellular protein degradation, and processing. Endogenous inhibitors regulate its enzymatic activity, including stefins and other cystatins. Recent data proved that CtsB is implicated in tumor extracellular matrix remodeling, cell invasion, and metastasis: a misbalance between cathepsins and their natural inhibitors is often considered a sign of disease progression. In the present study, we investigated CtsB and stefin A (StfA) expression in renal cell carcinoma (RCC). mRNA analysis unveiled a significant CTSB and STFA increase in RCC tissues compared to adjacent non-cancerogenic tissues and a higher CtsB expression in malignant tumors than in benign renal neoplasms. Further analysis highlighted a positive correlation between CtsB and StfA expression as a function of patient sex, age, tumor size, grade, lymph node invasion, metastasis occurrence, and survival. Alternative overexpression and silencing of CtsB and StfA confirmed the correlation expression between these proteins in human RCC-derived cells through protein analysis and fluorescent microscopy. Finally, the ectopic expression of CtsB and StfA increased RCC cell proliferation. Our data strongly indicated that CtsB and StfA expression play an important role in RCC development by mutually stimulating their expression in RCC progression.
Asunto(s)
Carcinoma de Células Renales , Catepsina B/metabolismo , Cistatina A/metabolismo , Cistatinas , Neoplasias Renales , Carcinoma de Células Renales/genética , Catepsina B/genética , Cistatina A/genética , Cistatinas/metabolismo , Femenino , Humanos , Neoplasias Renales/genética , Masculino , ARN Mensajero/genéticaRESUMEN
To fully understand the properties of piscine stefins (family I cystatins), the 294-bp stefinA gene from grass carp (Ctenopharyngodon idella, Ci) was cloned and expressed in E. coli BL21 (DE3). After purification by Ni2+-NTA agarose affinity chromatography, the CiStefin A protein was tested to have a molecular weight of 11.48 kDa and an isoelectric point of 8.7. The typical motif of the cystatins superfamily was characterized from CiStefin A (QVVQG). CiStefin A specifically inhibited the activity of papain and cathepsin B/L. The Ki value of CiStefin A against papain was 6.5 × 10-11 M. CiStefin A showed excellent heat and acid-base tolerance. StefinA gene transcription occurred in all tested tissues of grass carp, with the highest level in the hepatopancreas. Immunolocalization staining with an anti-CiStefinA antibody revealed the CiStefinA protein distribution in all tested tissues at various levels. Overall, these results clarified the physical and biochemical properties of grass carp stefin A.
Asunto(s)
Carpas/genética , Cistatina A/genética , Cistatina A/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Animales , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Filogenia , Transporte de ProteínasRESUMEN
Lung cancer is one of the most common cancer types worldwide and causes more than one million deaths annually. Lung adenocarcinoma (AC) and lung squamous cell cancer (SCC) are two major lung cancer subtypes and have different characteristics in several aspects. Identifying their differentially expressed genes and different gene expression patterns can deepen our understanding of these two subtypes at the transcriptomic level. In this work, we used several machine learning algorithms to investigate the gene expression profiles of lung AC and lung SCC samples retrieved from Gene Expression Omnibus. First, the profiles were analyzed by using a powerful feature selection method, namely, Monte Carlo feature selection. A feature list, ranking all features according to their importance, and some informative features were obtained. Then, the feature list was used in the incremental feature selection method to extract optimal features, which can allow the support vector machine (SVM) to yield the best performance for classifying lung AC and lung SCC samples. Some top genes (CSTA, TP63, SERPINB13, CLCA2, BICD2, PERP, FAT2, BNC1, ATP11B, FAM83B, KRT5, PARD6G, PKP1) were extensively analyzed to prove that they can be differentially expressed genes between lung AC and lung SCC. Meanwhile, a rule learning procedure was applied on informative features to construct the classification rules. These rules provide a clear procedure of classification and show some different gene expression patterns between lung AC and lung SCC.
Asunto(s)
Adenocarcinoma del Pulmón/genética , Carcinoma de Células Escamosas/genética , Biología Computacional/métodos , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Aprendizaje Automático/estadística & datos numéricos , Adenocarcinoma del Pulmón/diagnóstico , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Cistatina A/genética , Cistatina A/metabolismo , Conjuntos de Datos como Asunto , Diagnóstico Diferencial , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Método de Montecarlo , Serpinas/genética , Serpinas/metabolismo , Terminología como Asunto , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Despite advances in early detection and treatment, invasion and metastasis of breast tumors remains a major hurdle. Cystatin A (CSTA, also called stefin A), an estrogen-regulated gene in breast cancer cells, is an inhibitor of cysteine cathepsins, and a purported tumor suppressor. Loss of CSTA expression in breast tumors evidently shifts the balance in favor of cysteine cathepsins, thereby promoting extracellular matrix remodeling, tumor invasion and metastasis. However, the underlying mechanism behind the loss of CSTA expression in breast tumors is not known. Here, we have analyzed CSTA expression, and methylation of upstream and intron-2 CpG sites within the CSTA locus in human breast cancer cell lines and breast tumors of the TCGA cohort. Results showed an inverse relationship between expression and methylation. Sequence analysis revealed a potential estrogen response element (ERE) in the intron-2. Analysis of ChIP-seq data (ERP000380) and our own ChIP experiments showed that 17ß-estradiol (E2) enhanced ERα binding to this ERE in MCF-7 cells. This ERE was located amidst the differentially methylated intron-2 CpG sites, which provoked us to examine the possible conflict between estrogen-regulation of CSTA and DNA methylation in the intron-2. We analyzed the expression of CSTA and its regulation by E2 in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza significantly demethylated intron-2 CpGs, and enhanced estrogen-induced ERα occupancy at the intron-2 ERE, leading to restoration of estrogen-regulation. Taken together, our results indicate that DNA methylation-dependent silencing could play a significant role in the loss of CSTA expression in breast tumors. The potential of DNA methylation as an indicator of CSTA expression or as a marker of tumor progression can be explored in future investigations. Furthermore, our results indicate the convergence of ERα-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ERα signaling axis in breast tumor invasion and metastasis.
Asunto(s)
Neoplasias de la Mama/genética , Cistatina A/genética , Cistatina A/metabolismo , Metilación de ADN , Receptor alfa de Estrógeno/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Metilación de ADN/efectos de los fármacos , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Células Tumorales CultivadasRESUMEN
We previously conducted transcriptome analysis of a paired specimen of normal and esophageal squamous cell carcinoma (ESCC) tissues and found that mRNA expression of cystatin A (CSTA), a member of the cystatin superfamily, was perturbed in tumors compared with that in the background mucosa. However, little is known about the significance of CSTA expression in ESCC.The mRNA expression of CSTA was evaluated by qRT-PCR using 28 paired frozen samples of tumor and nontumor mucosae. The protein expression of CSTA was evaluated by the immunostaining of formalin-fixed, paraffin-embedded sections of ESCC samples from 59 patients who underwent surgery, and its relationship with clinical features was analyzed.The mRNA expression of CSTA was significantly decreased in ESCC compared with that in matched normal mucosa (Pâ<â.0001). The protein expression of CSTA was limited in stratum granulosum and stratum spinosum but not in stratum basal in normal esophageal mucosa. It was reduced in all ESCC tissue samples compared with normal tissues; however, CSTA expression levels in tumors showed considerable variation. Of the 59 samples, 20 did not express CSTA, whereas 39 clearly expressed it. The expression of CSTA in tumors was significantly associated with pT classification (deeper tumor invasions) (Pâ=â.0118) and advanced TNM stages (Pâ=â.0497). In CSTA-positive tumor samples, CSTA-expressing cancer cells often expressed Ki67, a proliferation marker, which was in sharp contrast to normal mucosa, where Ki67-expressing cells were limited to the basal layer and did not express CSTA. Furthermore, CSTA expression was observed in all 22 lymph node metastases analyzed.Relatively high levels of CSTA expression in tumors were correlated with tumor progression and advanced cancer stage, including lymph node metastasis.
Asunto(s)
Carcinoma de Células Escamosas , Cistatina A , Mucosa Esofágica , Neoplasias Esofágicas , Anciano , Biomarcadores de Tumor , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Cistatina A/genética , Cistatina A/metabolismo , Progresión de la Enfermedad , Mucosa Esofágica/metabolismo , Mucosa Esofágica/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Japón , Antígeno Ki-67/análisis , Metástasis Linfática/genética , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , ARN Mensajero/análisis , Estadística como AsuntoRESUMEN
Cystatins are a large family of the proteins that function as reversible and tight-binding inhibitors of cysteine proteases, which consequently regulate multiple physiological activities including apoptosis and innate immunity. In the present study, we cloned a gene from Crassostrea gigas encoding cystatin, which is related to cystatin A superfamily. CgCytA was comprised of a cystatin-like domain with two conserved glycine residues (GG) near the N-terminal and a highly conserved glutamine-valine-glycine (Q-X-V-X-G) motif in the form of QVVAG loop. Transcription analysis of CgCytA indicated its constitutive expression in all tissues including mantle, gill, digestive tract, hemocytes, heart, adductor muscle, and gonads. Immune challenge with Vibrio alginolyticus, resulted in significant down-regulation of CgCytA expression at the initial stages of infection (till 12â¯h post infection) and the expression of cystatin increased 48â¯h post infection. Protease assay demonstrated the concentration of cystatin needed to inhibit half of the maximum biological response of cysteine protease is 14.4⯵g/L (IC50). Furthermore, RNAi of CgCytA resulted in increase of apoptotic cell population in hemocytes of C. gigas, suggesting protection role of CgCytA from hemocytes apoptosis. Unexpectedly, knockdown of CgCytA leaded to enhancement of bacterial clearance in vivo, implying that CgCytA may negatively regulate immune defense by suppressing endogenous cysteine protease. Therefore, CgCytA plays dual roles in protection of host hemocytes from apoptosis and control of bacterial clearance, which may server as one of key endogenous balancer between apoptosis and innate immunity in oyster.
Asunto(s)
Crassostrea/genética , Crassostrea/inmunología , Cistatina A/genética , Cistatina A/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cistatina A/química , Perfilación de la Expresión Génica , Filogenia , Interferencia de ARN , Alineación de Secuencia , Vibrio alginolyticusRESUMEN
Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Movimiento Celular , Cistatina A/metabolismo , Células Epiteliales/metabolismo , Glándulas Mamarias Humanas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/tratamiento farmacológico , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/patología , Catepsina B/antagonistas & inhibidores , Catepsina B/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Técnicas de Cocultivo , Cistatina A/genética , Inhibidores de Cisteína Proteinasa/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Humanos , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/patología , Ratones , Invasividad Neoplásica , Interferencia de ARN , Transducción de Señal , Transfección , Microambiente Tumoral , Proteínas Supresoras de Tumor/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy known, with an extremely poor prognosis due to the lack of an efficient diagnostic scheme and no radical treatment option, except surgery. Therefore, understanding the pathophysiology of, and finding a novel biomarker to detect, PDAC should be prioritized. We observed an increase in mRNA expression of the cysteine protease inhibitor cystatin A (CSTA) in CD4+ T cells in peripheral blood cells of nine patients with PDAC, compared with the expression in seven healthy volunteers. Moreover, we confirmed significantly higher CSTA mRNA expression in a larger cohort of 41 patients with PDAC compared with that in 20 healthy volunteers. Correspondingly, the serum CSTA concentrations in 36 patients with PDAC were higher than those in 37 healthy volunteers, and this increase was correlated with PDAC clinical stage. Furthermore, the expression of CSTA and cathepsin B, which is a lysosomal cysteine protease inhibited by CSTA, was observed in tumor tissues and tumor-infiltrating immune cells in 20 surgically resected PDAC tissues by immunohistochemical staining. Expression of CSTA was detected in some tumor tissues and many tumor-infiltrating immune cells. Cathepsin B expression was also observed in most tumor tissues and tumor-infiltrating immune cells. In conclusion, CSTA and its substrate cathepsin B are involved in PDAC-related inflammation. The increment of CSTA expression in peripheral blood of patients with PDAC may have a potential role as a PDAC immunopathologic biomarker.
Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Catepsina B/genética , Cistatina A/genética , Adenocarcinoma/patología , Anciano , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Carcinoma Ductal Pancreático/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/patología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Pronóstico , ARN Mensajero/genéticaRESUMEN
Pulmonary surfactant is broadly known to keep the lung dry, clean and open by lowering the surface tension of the fluid-film that lines the alveoli. The surfactant's protein component, the so called surfactant proteins (SPs), make up a multifunctional protein family. In addition to the four "classical" surfactant proteins (SP-A, SP-B, SP-C and SP-D), which possess immunologic as well as surfactant regulatory properties, two novel putative surfactant proteins (SFTA2 and SFTA3) have recently been described. Neither of them shows sequential nor structural similarity with the already known surfactant proteins. However, bioinformatic analyses as well as first molecular-biological studies reveal properties that have already been described for known surfactant proteins. In our present work we introduce a technique to synthesize, purify and stabilize recombinant SFTA3 derived from the human embryonic kidney cell line HEK 293T. This will provide investigators with a valuable source of further examination and characterization of this fascinating novel member of the surfactant protein family.
Asunto(s)
Cistatina A/genética , Cistatina A/metabolismo , Células HEK293/fisiología , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Clonación Molecular/métodos , Cistatina A/química , Humanos , Proteínas Recombinantes/químicaRESUMEN
Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.
Asunto(s)
Cistatina A/genética , Proteínas de Unión al ADN/genética , Expresión Génica/efectos de los fármacos , Mutágenos/toxicidad , Proteína p53 Supresora de Tumor/genética , Células Hep G2 , Humanos , Mediciones Luminiscentes , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Nagashima-type palmoplantar keratosis (NPPK) is an autosomal recessive form of palmoplantar keratoderma (PPK), which is caused by mutations in the SERPINB7 gene. NPPK has only been reported in Japanese and Chinese populations. The present study was conducted on 12 unrelated Chinese patients who were clinically predicted to suffer from NPPK. Mutation screening was performed by direct sequencing of the entire coding regions of SERPINB7, SLURP1, AQP5, CSTA, KRT1 and KRT9 genes. Direct sequencing of SERPINB7 revealed five homozygous founder mutations (c.796C>T) and four compound heterozygous mutations in nine patients, including one novel mutation (c.122_127delTGGTCC). Nine out of the 12 patients were diagnosed with NPPK due to SERPINB7 pathogenic mutations, and the results expanded the known mutation spectrum of NPPK. Taking the other seven reported Chinese patients, who had been definitively diagnosed with NPPK by genetic testing, into account, the present study further demonstrated that NPPK is a common entity in Mainland China, and c.796C>T is the most prevalent mutation and exerts a founder effect. Furthermore, the NPPK cases described in the current study presented a consistently mild phenotype, as compared with the degrees of phenotypic variability associated with other types of relatively severe PPK, including Mal de Meleda and Olmsted syndrome.
Asunto(s)
Pruebas Genéticas , Queratodermia Palmoplantar/genética , Serpinas/genética , Adolescente , Adulto , Antígenos Ly/genética , Acuaporina 5/genética , Pueblo Asiatico , Niño , Preescolar , China , Cistatina A/genética , Femenino , Efecto Fundador , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Lactante , Queratina-1/genética , Queratina-9/genética , Queratodermia Palmoplantar/patología , Masculino , Persona de Mediana Edad , Mutación , Linaje , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Activating transcription factor 4 (ATF4) is a transcription factor with an important biological activity. ATF4 is induced by various stresses, such as endoplasmic reticulum stress, through the phosphorylation of eukaryotic translation initiation factor 2α. ATF4 is also involved in lipid metabolism. In the present study, we performed a microarray experiment to identify new ATF4 target genes, particularly those involved in lipid metabolism, and identified C12orf39, CSTA, and CALCB as novel ATF4 target genes. An amino acid response element (AARE) as an ATF4-binding site is present in the promoter regions of these genes. In a detailed analysis using luciferase assay, we showed that ATF4 activated C12orf39 promoter activity and that this activation was diminished by deletion or mutation of the AARE sequence in the promoter region. Our results suggest that C12orf39, CSTA, and CALCB are novel ATF4 target genes and that C12orf39 promoter activity is activated by ATF4 through AARE.
Asunto(s)
Factor de Transcripción Activador 4/genética , Péptido Relacionado con Gen de Calcitonina/genética , Cistatina A/genética , Regulación Neoplásica de la Expresión Génica , Hepatocitos/metabolismo , Hormonas Peptídicas/genética , Factor de Transcripción Activador 4/metabolismo , Sitios de Unión , Péptido Relacionado con Gen de Calcitonina/metabolismo , Línea Celular Tumoral , Cistatina A/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Perfilación de la Expresión Génica , Genes Reporteros , Hepatocitos/patología , Humanos , Metabolismo de los Lípidos/genética , Luciferasas/genética , Luciferasas/metabolismo , Análisis por Micromatrices , Mutación , Hormonas Peptídicas/metabolismo , Unión Proteica , Elementos de Respuesta , Transducción de SeñalRESUMEN
Stefins have been reported to be associated with the progression and metastasis of various malignant tumors. However, the expressions of stefins in hepatocellular carcinoma (HCC) have not been well-defined. In this study, the protein levels of stefin A and stefin B were assessed by immunohistochemical staining, and the mRNA levels were quantified by real-time polymerase chain reaction in 85 primary HCC tissues, 85 surrounding non-cancerous tissues, and 9 normal hepatic tissues. The immunohistochemical staining of cathepsin B and cathepsin D, and the ratio of cathepsins to stefins were assessed. The mRNA expressions of stefin A and stefin B in HCC tissues were significantly higher than surrounding noncancerous tissues and normal hepatic tissues, respectively. A significant positive relationship of stefin A and stefin B was found with node metastasis, tumor size, and Edmondson grade for HCC. Univariate and multivariate analyses revealed that Edmondson grade and stefin B expression were independent factors associated with the risk of lymph node metastasis in HCC. The ratios of cathepsin B to stefin A, cathepsin D to stefin A, cathepsin B to stefin B and cathepsin D to stefin B of the HCC group were significantly higher than that of the surrounding noncancerous group. A significant positive correlation between the ratio of cathepsins to stefins (cathepsin B/stefin A, cathepsin B/stefin B and cathepsin D/stefin B) and node metastasis was demonstrated. We concluded that high expressions of stefin A and stefin B may be an important factor contributing to the development and metastasis of HCC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Catepsinas/metabolismo , Cistatina A/metabolismo , Cistatina B/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundario , Estudios de Casos y Controles , Catepsinas/genética , Cistatina A/genética , Cistatina B/genética , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease.
Asunto(s)
Cistatina A/genética , Cistatina A/aislamiento & purificación , Cistatina B/genética , Cistatina B/aislamiento & purificación , Cistatina C/genética , Cistatina C/aislamiento & purificación , Catepsina L/antagonistas & inhibidores , Catepsina L/química , Cistatina A/química , Cistatina A/metabolismo , Cistatina B/química , Cistatina B/metabolismo , Cistatina C/química , Cistatina C/metabolismo , Endopeptidasas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Acral peeling skin syndrome (APSS) is a rare autosomal recessive condition, characterized by asymptomatic peeling of the skin of the hands and feet, often linked to mutations in the gene TGM5. However, more recently recessive loss of function mutations in CSTA, encoding cystatin A, have been linked with APSS and exfoliative ichthyosis. We describe the clinical features in two sisters with APSS, associated with a novel large homozygous deletion encompassing exon 1 of CSTA.
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Cistatina A/genética , Cistatina A/metabolismo , Mutación/genética , Enfermedades de la Piel/congénito , Piel/patología , Piel/fisiopatología , Niño , Preescolar , Cistatina A/fisiología , Análisis Mutacional de ADN , Eritema/patología , Femenino , Pie/patología , Mano/patología , Homocigoto , Humanos , Hiperopía/congénito , Ictiosis/etiología , Ictiosis/genética , Linaje , Enfermedades de la Piel/genética , Enfermedades de la Piel/patología , Enfermedades de la Piel/fisiopatología , Población BlancaRESUMEN
Cell-cell adhesion is paramount in providing and maintaining multicellular structure and signal transmission between cells. In the skin, disruption to desmosomal regulated intercellular connectivity may lead to disorders of keratinization and hyperproliferative disease including cancer. Recently we showed transgenic mice overexpressing desmoglein 2 (Dsg2) in the epidermis develop hyperplasia. Following microarray and gene network analysis, we demonstrate that Dsg2 caused a profound change in the transcriptome of keratinocytes in vivo and altered a number of genes important in epithelial dysplasia including: calcium-binding proteins (S100A8 and S100A9), members of the cyclin protein family, and the cysteine protease inhibitor cystatin A (CSTA). CSTA is deregulated in several skin cancers, including squamous cell carcinomas (SCC) and loss of function mutations lead to recessive skin fragility disorders. The microarray results were confirmed by qPCR, immunoblotting, and immunohistochemistry. CSTA was detected at high level throughout the newborn mouse epidermis but dramatically decreased with development and was detected predominantly in the differentiated layers. In human keratinocytes, knockdown of Dsg2 by siRNA or shRNA reduced CSTA expression. Furthermore, siRNA knockdown of CSTA resulted in cytoplasmic localization of Dsg2, perturbed cytokeratin 14 staining and reduced levels of desmoplakin in response to mechanical stretching. Both knockdown of either Dsg2 or CSTA induced loss of cell adhesion in a dispase-based assay and the effect was synergistic. Our findings here offer a novel pathway of CSTA regulation involving Dsg2 and a potential crosstalk between Dsg2 and CSTA that modulates cell adhesion. These results further support the recent human genetic findings that loss of function mutations in the CSTA gene result in skin fragility due to impaired cell-cell adhesion: autosomal-recessive exfoliative ichthyosis or acral peeling skin syndrome.
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Ciclo Celular , Cistatina A/metabolismo , Desmogleína 2/metabolismo , Redes Reguladoras de Genes , Animales , Adhesión Celular , Línea Celular Tumoral , Cistatina A/genética , Desmogleína 2/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , TranscriptomaRESUMEN
Autosomal recessive exfoliative ichthyosis (AREI) results from mutations in CSTA, encoding cysteine protease inhibitor A (cystatin A). We present a 25-year-old man from Iran with consanguineous parents, who presented with congenital erythroderma, hyperhidrosis and diffuse hyperkeratosis with coarse palmoplantar peeling of the skin, aggravated by exposure to water and by occlusion. Candidate gene analysis revealed a previously unknown homozygous loss-of-function mutation c.172C>T (p.Arg58Ter) in CSTA, and immunostaining showed absence of epidermal cystatin A, confirming the diagnosis of AREI. Ultrastructural analysis by transmission electron microscopy showed normal degradation of corneodesmosomes, mild intercellular oedema in the spinous layer but not in the basal layer, normal-appearing desmosomes, and prominent keratin filaments within basal keratinocytes. Thickness of cornified envelopes was reduced, lamellar lipid bilayers were disturbed, lamellar body secretion occurred prematurely and processing of secreted lamellar body contents was delayed. These barrier abnormalities were reminiscent of (albeit less severe than in) Netherton syndrome, which results from a deficiency of the serine protease inhibitor LEKTI. This work describes ultrastructural findings with evidence of epidermal barrier abnormalities in AREI.
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Cistatina A/genética , Mutación/genética , Enfermedades Cutáneas Genéticas/genética , Adulto , Diagnóstico Diferencial , Epidermis/patología , Dermatosis del Pie/genética , Dermatosis del Pie/patología , Dermatosis de la Mano/genética , Dermatosis de la Mano/patología , Homocigoto , Humanos , Masculino , Microscopía Electrónica de Transmisión , Síndrome de Netherton/patología , Enfermedades Cutáneas Genéticas/patologíaRESUMEN
Complementary DNAs encoding human stefins A (HSA) and B (HSB) were synthesized using Pichia-preferred codons by overlap extension PCR. The full-length genes were ligated downstream of the glyceraldehyde-3-phosphate dehydrogenase promoter in the Pichia expression vector pGAPZαC and successfully expressed in Pichia pastoris strain X-33. Functional recombinant HSA and HSB proteins were purified from culture medium at yields of 121.3 ± 13.5 (n = 3) and 95.4 ± 4.1 (n = 3) mg/L, respectively. Using this expression strategy, we demonstrated that high levels of bioactive recombinant HSA and HSB can be produced by fermentation in P. pastoris.