RESUMEN
BACKGROUND: Skin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides. METHODS: Cathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry. RESULTS: Elastin samples derived from patients between 10 and 86â¯years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies. CONCLUSION: Cathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.
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Catepsina K/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Elastina/metabolismo , Envejecimiento de la Piel , Piel/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Catepsina K/análisis , Catepsinas/análisis , Niño , Cisteína Endopeptidasas/análisis , Elastina/análisis , Elastina/ultraestructura , Femenino , Humanos , Persona de Mediana Edad , Proteolisis , Adulto JovenRESUMEN
AIMS: Cathepsin V (CTSV/CTSL2) is a lysosomal cysteine proteinase and plays a role in extracellular matrix degradation. It is associated with poor prognosis in invasive breast cancer (IBC), but its role in breast ductal carcinoma in situ (DCIS) remains unclear. In this study, we aimed to evaluate the prognostic significance of CTSV in DCIS. METHODS: CTSV protein expression was immunohistochemically assessed in a well-characterised and annotated cohort of DCIS comprising pure DCIS (n=776) and DCIS coexisting with IBC (n=239). CTSV expression was analysed in tumour cells and surrounding stroma, including its association with clinicopathological parameters and outcome. RESULTS: In pure DCIS, high CTSV expression was observed in 29% of epithelial tumour cells and 20% of surrounding stroma. High expression in both components was associated with features of poor prognosis including higher nuclear grade, hormone receptor negativity and HER2 positivity. In addition, stromal CTSV expression was associated with larger DCIS size, comedo-type necrosis and high proliferation index. DCIS associated with IBC showed higher CTSV expression than pure DCIS either within the epithelial tumour cells or surrounding stroma (p<0.0001 and p=0.001, respectively). In DCIS/IBC, CTSV expression was higher in the invasive component than DCIS component either in tumour cells or surrounding stroma (both p<0.0001). CTSV stromal expression was associated with invasive recurrence independent of other prognostic factors in patients treated with breast conserving surgery (HR=3.0, p=0.005). CONCLUSION: High expression of CTSV is associated with poor outcome in DCIS and is a potential marker to predict DCIS progression to invasive disease.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/enzimología , Carcinoma Intraductal no Infiltrante/enzimología , Catepsinas/análisis , Cisteína Endopeptidasas/análisis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carcinoma Intraductal no Infiltrante/patología , Catepsinas/genética , Cisteína Endopeptidasas/genética , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Pronóstico , Regulación hacia ArribaRESUMEN
The USFDA-approved immunosuppressive drug rapamycin (Rapa), despite its potency, is limited by poor bioavailability and a narrow therapeutic index. In this study, we sought to improve bioavailability of Rapa with subcutaneous (SC) administration and to test its therapeutic feasibility and practicality in a murine model of Sjögren's syndrome (SS), a systemic autoimmune disease with no approved therapies. To improve its therapeutic index, we formulated Rapa with a carrier termed FAF, a fusion of the human cytosolic FK506-binding protein 12 (FKBP12) and an elastin-like polypeptide (ELP). The resulting 97 kDa FAF (i) has minimal burst release, (ii) is "humanized", (iii) is biodegradable, (iv) solubilizes two Rapa per FAF, and (v) avoids organic solvents or amphiphilic carriers. Demonstrating high stability, FAF remained soluble and monodisperse with a hydrodynamic radius of 8 nm at physiological temperature. A complete pharmacokinetic (PK) analysis of FAF revealed that the bioavailability of SC FAF was 60%, with significantly higher blood concentration during the elimination phase compared to IV FAF. The plasma concentration of Rapa delivered by FAF was 8-fold higher with a significantly increased plasma-to-whole blood ratio relative to free Rapa, 24 h after injection. To evaluate therapeutic effects, FAF-Rapa was administered SC every other day for 2 weeks to male non-obese diabetic (NOD) mice, which develop an SS-like autoimmune-mediated lacrimal gland (LG) inflammation and other characteristic features of SS. Both FAF-Rapa and free Rapa exhibited immunomodulatory effects by significantly suppressing lymphocytic infiltration, gene expression of IFN-γ, MHC II, type I collagen and IL-12a, and cathepsin S (CTSS) activity in LG compared to controls. Serum chemistry and histopathological analyses in major organs revealed no apparent toxicity of FAF-Rapa. Given its improved PK and equipotent therapeutic efficacy compared to free Rapa, FAF-Rapa is of further interest for systemic treatments for autoimmune diseases like SS.
Asunto(s)
Portadores de Fármacos/química , Composición de Medicamentos/métodos , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Péptidos/química , Sirolimus/administración & dosificación , Sirolimus/uso terapéutico , Síndrome de Sjögren/tratamiento farmacológico , Animales , Catepsinas/análisis , Modelos Animales de Enfermedad , Portadores de Fármacos/farmacocinética , Liberación de Fármacos , Estabilidad de Medicamentos , Elastina/química , Inmunosupresores/sangre , Inmunosupresores/química , Inyecciones Subcutáneas , Masculino , Ratones , Ratones Endogámicos NOD , Sirolimus/sangre , Sirolimus/química , Síndrome de Sjögren/sangre , Proteína 1A de Unión a Tacrolimus/químicaRESUMEN
Muscle wasting or cachexia is commonly associated with aging and many diseases such as cancer, infection, autoimmune disorders, and trauma. Decrease in muscle mass, or muscle atrophy, is often caused by dysfunction of protein proteolytic systems, such as lysosomes, which regulate protein turnover and homeostasis. Lysosomes contain many hydrolases and proteases and, thus, represent the major organelle that control protein turnover. Recently, lysosomes have emerged as a signaling hub to integrate cellular functions of nutrient sensing and metabolism, autophagy, phagocytosis, and endocytosis, which are all related to tissue homeostasis. In this chapter, we describe the protocol used to measure lysosomal proteinase (cathepsins) activity in the skeletal muscle. A better understanding of lysosomal function in muscle homeostasis is critical in developing new therapeutic approaches to prevent muscle wasting.
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Catepsinas/análisis , Lisosomas/enzimología , Músculo Esquelético/citología , Animales , Autofagia , Colorantes Fluorescentes/química , Homeostasis , Ratones , Microscopía Fluorescente , Músculo Esquelético/enzimología , Transducción de SeñalRESUMEN
Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.
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Catepsinas/metabolismo , Glioblastoma/patología , Nicho de Células Madre , Adulto , Anciano , Anciano de 80 o más Años , Arteriolas , Catepsina B/análisis , Catepsina B/metabolismo , Catepsina K , Catepsina Z/análisis , Catepsina Z/metabolismo , Catepsinas/análisis , Glioblastoma/enzimología , Glioblastoma/metabolismo , Humanos , Inmunohistoquímica , Persona de Mediana Edad , ProteolisisRESUMEN
BACKGROUND: Periodontitis is a prevalent oral disease with bone loss being it's hallmark. Clinical parameters used to measure periodontitis are retrospective and do not indicate active inflammation nor prognosis. GCF can be easily collected chairside and bone turnover biomarkers found in GCF can be evaluated to check for active inflammation and disease progression. This systematic review aims to evaluate the literature for association and predictive value of bone turnover biomarkers in GCF during periodontal disease. MATERIALS AND METHODS: This review was conducted and reported according to the PRISMA guidelines. The online databases Google Scholar and PubMed were used for data search. MeSH terms were used for PubMed search. All original studies from 1990 to 2017 conducted on human subjects in the English language were included in the review. Studies on non-human subjects, reviews and studies conducted in languages other than English were not considered. Reference lists of qualified articles were also searched. RESULTS: The search generated 2300 results whose titles were screened and 1571 articles were retreived. 23 articles were accepted in the review and full texts were accessed. These included 1 randomized controlled trial, 12 cross-sectional studies, five pre-post interventional studies, 4 longitudinal and 1 in-vitro in-vivo experimental study. The studies were conducted on patients of both genders ranging from 10 to 81 years in age. A total of 37 biomarkers were evalueted in the studies included in this review. Majority of the studies reported interleukin-1ß (IL-1ß) while receptor activated nuclear factor-kappa B ligand (RANKL) and matrix metalloproteinase-8 (MMP-8) were the other frequently reported biomarkers. Most of the studies evaluated more than two biomarkers. ELISA was the most commonly used biochemical test used for detection. CONCLUSION: A wide range of biomarkers have been established as indicators of alveolar bone resorption. Few of the biomarkers have also shown positive correlation with disease progression and outcome of periodontal therapies thus underscoring their predictive value in periodontal diagnosis and prognosis. Not one single biomarker has been reported to have a predictive advantage over another and a combination of two or more biomarkers along with clinical evaluation is recommended.
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Pérdida de Hueso Alveolar , Biomarcadores/análisis , Líquido del Surco Gingival/química , Periodontitis , Catepsinas/análisis , Humanos , Interleucina-1beta/análisis , Metaloproteinasa 8 de la Matriz/análisis , Ligando RANK/análisisRESUMEN
Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.
Asunto(s)
Proteasas de Cisteína/análisis , Electroforesis en Gel de Poliacrilamida/métodos , Pruebas de Enzimas/métodos , Animales , Catepsinas/análisis , Catepsinas/metabolismo , Proteasas de Cisteína/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Leucina/análogos & derivados , Leucina/metabolismo , Desnaturalización Proteica , Sustancias Reductoras/químicaRESUMEN
Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost. This chapter will demonstrate the utility and interpretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S, and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with densitometry.
Asunto(s)
Catepsinas/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Pruebas de Enzimas/métodos , Animales , Catepsina K/análisis , Catepsina K/metabolismo , Catepsina L/análisis , Catepsina L/metabolismo , Catepsinas/análisis , Células Cultivadas , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/metabolismo , Densitometría/métodos , Electroforesis en Gel de Poliacrilamida/instrumentación , Pruebas de Enzimas/instrumentación , Diseño de Equipo , Humanos , Coloración y Etiquetado/métodosRESUMEN
BACKGROUND: This study analyzes the effect of salt and acetic acid concentration, time, temperature and fish freezing on the activity and losses of cathepsins during the marinating of Atlantic and Baltic herrings. RESULTS: The highest contribution to meat general proteolytic activity was found for cathepsin D-like activity. This contribution decreased during the marinating process as a result of, among other things, cathepsin losses to brine. The methods of marinating had a significant impact on cathepsin activity losses. The average ratio of cathepsin D-like activity to L and B in brine accounted for 15:3.5:1.5, respectively. Depending on the method of calculation, cathepsin activity in brine was similar (per gram of tissue/milliliter of brine) or multiply higher (per gram protein in tissue/brine) than in the marinated herring meat. Statistical analysis demonstrated that the extent and structure of cathepsin losses were significantly correlated with the quantitative and qualitative composition of protein hydrolysis products in marinades. CONCLUSION: The presented results depict new phenomena of cathepsin losses and explain their impact on the process of fish marinating. Results allow better optimization of the process of meat ripening. The high activity of aspartyl and cysteine cathepsins in brine indicates the real feasibility of their application in the food industry for novel food design. © 2016 Society of Chemical Industry.
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Catepsinas/análisis , Peces , Manipulación de Alimentos/métodos , Alimentos Marinos/análisis , Ácido Acético/química , Animales , Congelación , Hidrólisis , Sales (Química)/química , Cloruro de Sodio/químicaRESUMEN
Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1ß and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1ß and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.
Asunto(s)
Catepsinas/fisiología , Periodontitis/etiología , Adolescente , Adulto , Animales , Autofagia/fisiología , Catepsinas/análisis , Células Cultivadas , Niño , Femenino , Encía/metabolismo , Humanos , Masculino , Periodontitis/enzimología , Ratas , Adulto JovenRESUMEN
BACKGROUND: Diabetes is a risk factor for atherosclerotic disease but negatively associated with the development and progression of abdominal aortic aneurysm (AAA). Advanced glycation end products (AGEs) are increased in diabetes and renders the vascular matrix more resistant to proteolysis. We assessed the concentration of AGEs in AAA biopsies obtained from diabetic and nondiabetic patients and hypothesized that (nonenzymatic) glycation of AAA tissue protects against proteolytic breakdown of collagen. METHODS: AAA biopsies were collected from 30 diabetic and 30 matched nondiabetic AAA patients at the time of open repair. Aortic control samples from 10 nondiabetic and 16 diabetic patients were collected, and concentrations of the AGE cross-link pentosidine was measured. Furthermore, noncross-linking AGEs (adducts), as well as proteolytic enzymes known to play a role in aneurysm development (matrix metalloproteinase [MMP]-2, MMP-9, cathepsin B and S) were quantified. Ex vivo, nondiabetic AAA biopsies were glycated and measured subsequently for collagen type I release. RESULTS: Pentosidine concentrations in AAA wall biopsies were increased in patients with diabetes compared with nondiabetics 9.4 (5.0-13.5) vs 6.0 (2.5-9.6) pmol/µmol lysine (P = .02). Increased pentosidine concentrations were also observed in nonaneurysmatic aortic wall biopsies from diabetic patients. In diabetic AAA vascular wall tissue, pentosidine concentration was negatively correlated with aortic diameter (r = -0.43; P = .02). Ex vivo glycated AAA biopsies were resistant against MMP-induced collagen type I degradation as compared with controls (7.0 vs 10.4 µg/L; P = .02). No differences were observed for AGEs that are not forming cross-links. CONCLUSIONS: These findings suggest that cross-linking AGEs like pentosidine play a protective role in AAA progression in diabetic patients.
Asunto(s)
Aorta Abdominal/química , Aneurisma de la Aorta Abdominal/metabolismo , Colágeno Tipo I/análisis , Diabetes Mellitus/metabolismo , Productos Finales de Glicación Avanzada/análisis , Anciano , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/patología , Arginina/análogos & derivados , Arginina/análisis , Estudios de Casos y Controles , Catepsinas/análisis , Citocinas/análisis , Femenino , Glicosilación , Humanos , Lisina/análogos & derivados , Lisina/análisis , Masculino , Metaloproteinasas de la Matriz/análisis , Estabilidad Proteica , ProteolisisRESUMEN
Cathepsins are a group of proteolytic enzymes of the endosomal/lysosomal pathway involved in the thymic development of T cells restricted by major histocompatibility complex class II molecules. In the normal thymus, cathepsin V (CTV) and cathepsin S (CTS) are expressed in cortical and medullary epithelial cells, respectively. To investigate whether cathepsins could serve as a diagnostic marker, we performed immunohistochemical analysis for CTV and CTS in 77 cases of thymic epithelial tumors. Almost all cases (59/60) of thymoma expressed CTV, whereas 28 of 60 cases of thymoma expressed CTS. Notably, CTS was expressed in most cases of type A and type AB thymomas, but not in type B thymoma. The expression of cathepsins in type AB thymoma showed a clear correlation with histologic features; CTV was found predominantly in the type B component, and CTS was frequently expressed in the type A component. In thymic carcinoma, CTV was expressed in less than half cases (7/17), and the ratio of CTS-positive cases was equivalent to that of thymoma (8/17). Cases of CTV-negative thymic carcinoma tended to have a higher incidence of recurrence than did CTV-positive cases. Although further studies with a larger number of cases are required to confirm the utility of cathepsin immunostaining, CTV and CTS appear to serve as auxiliary diagnostic and/or prognostic markers in thymic epithelial tumors.
Asunto(s)
Biomarcadores de Tumor/análisis , Catepsinas/análisis , Cisteína Endopeptidasas/análisis , Neoplasias Glandulares y Epiteliales/enzimología , Timoma/enzimología , Neoplasias del Timo/enzimología , Adolescente , Adulto , Anciano , Biopsia , Diagnóstico Diferencial , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Glandulares y Epiteliales/cirugía , Valor Predictivo de las Pruebas , Timoma/patología , Timoma/cirugía , Neoplasias del Timo/patología , Neoplasias del Timo/cirugía , Adulto JovenRESUMEN
BACKGROUND: Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) is a treatment option for peritoneal surface malignancies. The ability to detect microscopic foci of peritoneal metastasis intraoperatively may ensure the completeness of cytoreduction. In this study, we evaluated the suitability of a hand-held cathepsin-based fluorescent imaging system for intraoperative detection of appendiceal and colorectal peritoneal metastasis. METHODS: Peritoneal tumors and normal peritoneal tissues were collected from patients with appendiceal and colorectal peritoneal metastasis. Expression of different cathepsins (CTS-B, -D, -F, -G, -K, -L, -O, and -S) was determined by quantitative RT-PCR and immunohistochemistry. The hand-held cathepsin-based fluorescent imaging system was used to detect peritoneal xenografts derived from human colon cancer cells (HT29, LoVo and HCT116) in nu/nu mice. RESULTS: While the expression levels of CTS-B, -D, -L, and -S could be higher in peritoneal tumors than normal peritoneum with a median (range) of 6.1 (2.9-25.8), 2.0 (1.0-15.8), 1.4 (0.8-7.0), and 2.1 (1.6-13.9) folds by quantitative RT-PCR, respectively, CTS-B was consistently the major contributor of the overall cathepsin expression in appendiceal and colonic peritoneal tumors, including adenocarcinomas and low-grade appendiceal mucinous neoplasms. Using peritoneal xenograft mouse models, small barely visible colonic peritoneal tumors (<2.5 mm in maximum diameter) could be detected by the hand-held cathepsin-based fluorescent imaging system. CONCLUSIONS: Because cathepsin expression is higher in peritoneal tumors than underlying peritoneum, the hand-held cathepsin-based fluorescent imaging system could be useful for intraoperative detection of microscopic peritoneal metastasis during CRS-HIPEC and clinical trial is warranted.
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Neoplasias del Apéndice/patología , Catepsinas/análisis , Neoplasias Colorrectales/patología , Procedimientos Quirúrgicos de Citorreducción , Hipertermia Inducida , Imagen Óptica/instrumentación , Neoplasias Peritoneales/diagnóstico por imagen , Neoplasias Peritoneales/terapia , Adulto , Anciano , Animales , Catepsina B/análisis , Catepsinas/genética , Femenino , Fluorescencia , Células HCT116 , Células HT29 , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Imagen Óptica/métodos , Neoplasias Peritoneales/química , Neoplasias Peritoneales/secundario , Periodo Preoperatorio , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed.
Asunto(s)
Catepsinas/análisis , Técnicas Citológicas/métodos , Membranas Intracelulares/fisiología , Lisosomas/enzimología , Permeabilidad , MembranasRESUMEN
Proteomics at single-cell resolution can help to identify the heterogeneity among cell populations, shows more and more significance in current chemistry and biology. In this work, we demonstrated a new single cell chemical proteomic (SCCP) strategy with a membrane-permeable activity-based probe (ABP) to characterize the functional proteins in lysosome located in the cytosol. The ABP targeted to the cysteine cathepsin family protein, CpFABP-G, was designed for cysteine cathepsins labeling. The labeled HeLa cell of a cancer cell line was injected into a capillary and was lysed by SDS solution with heating. The lysate was then online readout by capillary electrophoresis-laser-induced fluorescence method. Due to the employment of highly specified ABP kicking out the uncorrelated proteins, the expression of cysteine cathepsins in individual HeLa cells was easily detected, and heterogeneity among those HeLa cells was readily discriminated. Further work was concentrated on SCCP analysis of the mouse leukemia cell of monocyte macrophage (RAW264.7). It was for the first time identifying two expression modes of cysteine cathepsins in RAW264.7, which could be undermined by the analysis of cell populations. We believed that SCCP would be one of the powerful alternatives for proteomics at single-cell resolution.
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Membranas Intracelulares/metabolismo , Lisosomas/química , Sondas Moleculares/análisis , Proteínas de Neoplasias/análisis , Neoplasias/química , Neoplasias/patología , Proteómica , Análisis de la Célula Individual , Animales , Catepsinas/análisis , Catepsinas/metabolismo , Línea Celular Tumoral , Electroforesis Capilar , Fluorescencia , Células HeLa , Humanos , Rayos Láser , Ratones , Sondas Moleculares/metabolismo , Proteínas de Neoplasias/metabolismo , Permeabilidad , Espectrometría de FluorescenciaRESUMEN
Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS using in-gel fluorescent scanning, obviating the necessity for downstream biotin-streptavidin conjugation and detection procedures.
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Catepsinas/análisis , Permeabilidad de la Membrana Celular , Química Clic/métodos , Cisteína/análisis , Técnicas de Sonda Molecular , Sondas Moleculares/química , Activación Enzimática , Células HEK293 , HumanosRESUMEN
Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or ß-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).
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Acetilglucosaminidasa/análisis , Catepsinas/análisis , Lisosomas/efectos de los fármacos , Membranas/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Línea Celular , Detergentes/metabolismo , Digitonina/metabolismo , Humanos , Lisosomas/enzimología , Membranas/fisiologíaRESUMEN
Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human diseases. Graphical Abstract á .
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Espectrometría de Masas/métodos , Neuropéptidos/metabolismo , Péptido Hidrolasas/metabolismo , Proteómica/métodos , Transducción de Señal , Secuencia de Aminoácidos , Catepsinas/análisis , Catepsinas/metabolismo , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/metabolismo , Encefalinas/análisis , Encefalinas/metabolismo , Humanos , Redes y Vías Metabólicas , Datos de Secuencia Molecular , Neuropéptidos/análisis , Péptido Hidrolasas/análisis , Vesículas Secretoras/química , Vesículas Secretoras/metabolismoRESUMEN
BACKGROUND: In cystic fibrosis (CF) the upper (UAW) and lower airways (LAW) are reservoirs for pathogens like Pseudomonas aeruginosa. The consecutive hosts' release of proteolytic enzymes contributes to inflammation and progressive pulmonary destruction. Objectives were to assess dynamics of protease : antiprotease ratios and pathogens in CF-UAW and LAW sampled by nasal lavage (NL) and sputum before and after intravenous- (IV-) antibiotic therapy. METHODS: From 19 IV-antibiotic courses of 17 CF patients NL (10 mL/nostril) and sputum were collected before and after treatment. Microbiological colonization and concentrations of NE/SLPI/CTSS (ELISA) and MMP-9/TIMP-1 (multiplex bead array) were determined. Additionally, changes of sinonasal symptoms were assessed (SNOT-20). RESULTS: IV-antibiotic treatment had more pronounced effects on inflammatory markers in LAW, whereas trends to decrease were also found in UAW. Ratios of MMP-9/TIMP-1 were higher in sputum, and ratios of NE/SLPI were higher in NL. Remarkably, NE/SLPI ratio was 10-fold higher in NL compared to healthy controls. SNOT-20 scores decreased significantly during therapy (P = 0.001). CONCLUSION: For the first time, changes in microbiological patterns in UAW and LAW after IV-antibiotic treatments were assessed, together with changes of protease/antiprotease imbalances. Delayed responses of proteases and antiproteases to IV-antibiotic therapy were found in UAW compared to LAW.
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Antibacterianos/administración & dosificación , Fibrosis Quística/tratamiento farmacológico , Pseudomonas aeruginosa/aislamiento & purificación , Inhibidor Tisular de Metaloproteinasa-1/análisis , Adolescente , Adulto , Estudios de Casos y Controles , Catepsinas/análisis , Niño , Fibrosis Quística/enzimología , Fibrosis Quística/microbiología , Femenino , Humanos , Inyecciones Intravenosas , Elastasa de Leucocito/análisis , Masculino , Metaloproteinasa 9 de la Matriz/análisis , Persona de Mediana Edad , Estudios Prospectivos , Inhibidor Secretorio de Peptidasas Leucocitarias/análisis , Esputo/microbiologíaRESUMEN
OBJECTIVES: Cathepsin S and cathepsin L are endosomal proteolytic enzymes involved in the degradation of extracellular matrixes, angiogenesis and antigen presentation. Cathepsins could thus play several roles in the disease process of RA. The aim of this study was to examine differences in cathepsin S and cathepsin L levels in serum and SF of RA patients with and without ACPA and RF. METHODS: In this study 121 patients with RA and clinical signs of knee synovitis were recruited. Patient characteristics were collected and matched samples of serum and SF were analysed for cathepsin S, cathepsin L, ACPA, IgA and IgM RF, CRP and MMP3. RESULTS: SF levels of cathepsin L, cathepsin S and MMP3 were significantly higher than in serum. Serum levels of both cathepsins were significantly higher in patients with ACPA, IgM-RF and IgA-RF compared with patients without these antibodies. SF levels of both cathepsins correlated with DAS28 and CRP in ACPA- and RF-positive but not in seronegative patients. CONCLUSION: The differences in cathepsin S and cathepsin L between RA patients with and without autoantibodies indicate that these cathepsins have a specific role in the disease process of seropositive RA. In this phenotype, cathepsin serum levels may reflect the autoimmune activity, whereas the levels in SF may reflect the local inflammatory and matrix degrading process in the joint.