RESUMEN
Compounds containg catechol or bisphosphonate were tested as inhibitors of the zinc metalloproteases, thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) which are bacterial virulence factors, and the human matrix metalloproteases MMP-9 and -14. Inhibition of virulence is a putative strategy in the development of antibacterial drugs, but the inhibitors should not interfere with human enzymes. Docking indicated that the inhibitors bound MMP-9 and MMP-14 with the phenyl, biphenyl, chlorophenyl, nitrophenyl or methoxyphenyl ringsystem in the S1'-subpocket, while these ringsystems entered the S2'- or S1 -subpockets or a region involving amino acids in the S1'- and S2'-subpockets of the bacterial enzymes. An arginine conserved among the bacterial enzymes seemed to hinder entrance deeply into the S1'-subpocket. Only the bisphosphonate containing compound RC2 bound stronger to PLN and TLN than to MMP-9 and MMP-14. Docking indicated that the reason was that the conserved arginine (R203 in TLN and R198 in PLN) interacts with phosphate groups of RC2.
Asunto(s)
Antibacterianos/farmacología , Catecoles/farmacología , Difosfonatos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Bacterias/enzimología , Catecoles/síntesis química , Catecoles/química , Difosfonatos/síntesis química , Difosfonatos/química , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/síntesis química , Inhibidores de la Metaloproteinasa de la Matriz/química , Metaloendopeptidasas/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Células THP-1RESUMEN
Inhibition of bacterial virulence is believed to be a new treatment option for bacterial infections. In the present study, we tested dipicolylamine (DPA), tripicolylamine (TPA), tris pyridine ethylene diamine (TPED), pyridine and thiophene derivatives as putative inhibitors of the bacterial virulence factors thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) and the human zinc metalloproteases, matrix metalloprotease-9 (MMP-9) and matrix metalloprotease-14 (MMP-14). These compounds have nitrogen or sulfur as putative donor atoms for zinc chelation. In general, the compounds showed stronger inhibition of MMP-14 and PLN than of the other enzymes, with Ki values in the lower µM range. Except for DPA, none of the compounds showed significantly stronger inhibition of the virulence factors than of the human zinc metalloproteases. TPA and Zn230 were the only compounds that inhibited all five zinc metalloproteinases with a Ki value in the lower µM range. The thiophene compounds gave weak or no inhibition. Docking indicated that some of the compounds coordinated zinc by one oxygen atom from a hydroxyl or carbonyl group, or by oxygen atoms both from a hydroxyl group and a carbonyl group, and not by pyridine nitrogen as in DPA and TPA.
Asunto(s)
Quelantes/química , Quelantes/farmacología , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Compuestos de Zinc/química , Compuestos de Zinc/farmacología , Aminoácidos , Bacterias/efectos de los fármacos , Bacterias/enzimología , Dominio Catalítico , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Previous studies have shown that THP-1 cells produced an SDS-stable and reduction-sensitive complex between proMMP-9 and a chondroitin sulfate proteoglycan (CSPG) core protein. The complex could be reconstituted in vitro using purified serglycin (SG) and proMMP-9 and contained no inter-disulfide bridges. It was suggested that the complex involved both the FnII module and HPX domain of proMMP-9. The aims of the present study were to resolve the interacting regions of the molecules that form the complex and the types of interactions involved. In order to study this, we expressed and purified full-length and deletion variants of proMMP-9, purified CSPG and SG, and performed in vitro reconstitution assays, peptide arrays, protein modelling, docking, and molecular dynamics (MD) simulations. ProMMP-9 variants lacking both the FnII module and the HPX domain did not form the proMMP-9âCSPG/SG complex. Deletion variants containing at least the FnII module or the HPX domain formed the proMMP-9âCSPG/SG complex, as did the SG core protein without CS chains. The interacting parts covered large surface areas of both molecules and implicated dynamic and complementary ionic, hydrophobic, and hydrogen bond interactions. Hence, no short single interacting linear motifs in the two macromolecules could explain the strong SDS-stable and reduction-sensitive binding.
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Metaloproteinasa 9 de la Matriz/química , Metaloproteinasa 9 de la Matriz/metabolismo , Proteoglicanos/química , Proteoglicanos/metabolismo , Eliminación de Secuencia , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Animales , Sitios de Unión , Línea Celular , Humanos , Enlace de Hidrógeno , Metaloproteinasa 9 de la Matriz/genética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Dominios Proteicos , Células Sf9 , Células THP-1RESUMEN
BACKGROUND: Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. METHODS: Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - ß1 (TGF-ß1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. RESULTS: We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-ß1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. CONCLUSIONS: These results show that soluble factors in the tumour microenvironment, such as TGF-ß1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.
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Carcinoma de Células Escamosas/genética , Neoplasias de la Boca/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Factor de Crecimiento Transformador beta1/genética , Animales , Carcinoma de Células Escamosas/patología , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias de la Boca/patología , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Transducción de Señal , Microambiente Tumoral/genéticaRESUMEN
BACKGROUND: Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho-hydroxy N-acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs. METHODS: Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching. RESULTS: Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC50 values over 100µM were obtained after the enzyme activities were corrected for quenching. These IC50 values are far above the concentrations needed to activate or inhibit the caspases. CONCLUSION: The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs. GENERAL SIGNIFICANCE: Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders.
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Hidrazonas/química , Isatina/química , Metaloproteinasa 14 de la Matriz/química , Metaloproteinasa 9 de la Matriz/química , Simulación del Acoplamiento Molecular , Piperazinas/química , Inhibidores de Proteasas/química , Dominio Catalítico , Línea Celular Tumoral , HumanosRESUMEN
Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme implicated in motility, differentiation, and regeneration of skeletal muscle fibers through processing of extracellular substrates. Although MMP-2 has been found to be localized intracellularly in cardiomyocytes where the enzyme is thought to contribute to post-ischemic loss of contractility, little is known about intracellular MMP-2 activity in skeletal muscle fibers. In the present study we demonstrate intracellular MMP-2 in normal skeletal muscle by immunohistochemical staining. Immunogold electron microscopic analyses indicated that the enzyme was concentrated in Z-lines of the sarcomers, in the nuclear membrane, and in mitochondria. By use of in situ zymography, we found that gelatinolytic activity in muscle fibers was co-localized with immunofluorecent staining for MMP-2. Staining for MMP-9, the other member of the gelatinase group of the MMPs, was negative. The broad-spectrum metalloprotease inhibitor EDTA and the selective gelatinase inhibitor CTT2, but not the cysteine inhibitor E64, strongly reduced the gelatinolytic activity. The intracellular gelatinolytic activity was much more prominent in fast twitch type II fibers than in slow twitch type I fibers, and there was a decrease in intracellular gelatinolytic activity and MMP-2 expression in muscles from mice exposed to high intensity interval training. Together our results indicate that MMP-2 is part of the intracellular proteolytic network in normal skeletal muscle, especially in fast twitch type II fibers. Further, the results suggest that intracellular MMP-2 in skeletal muscle fibers is active during normal homeostasis, and affected by the level of physical activity.
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Metaloproteinasa 2 de la Matriz/metabolismo , Fibras Musculares de Contracción Rápida/enzimología , Fibras Musculares de Contracción Lenta/enzimología , Animales , Diferenciación Celular , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Ácido Edético/farmacología , Gelatinasas/antagonistas & inhibidores , Leucina/análogos & derivados , Leucina/farmacología , Masculino , Metaloproteinasa 2 de la Matriz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Células Musculares/citología , Mioblastos/citología , Péptidos Cíclicos/farmacología , Condicionamiento Físico Animal , Sarcómeros/metabolismoRESUMEN
Biomarkers are used as tools in cancer diagnostics and in treatment stratification. In most cancers, there are increased levels of one or several members of the matrix metalloproteinases (MMPs). This is a family of proteolytic enzymes that are involved in many phases of cancer progression, including angiogenesis, invasiveness, and metastasis. It has therefore been expected that MMPs could serve as both diagnostic and prognostic markers in cancer patients, but despite a huge number of studies, it has been difficult to establish MMPs as cancer biomarkers. In the present paper, we assess some of the challenges associated with MMP research as well as putative reasons for the conflicting data on the value of these enzymes as diagnostic and prognostic markers in cancer patients. We also review the prognostic value of a number of MMPs in patients with lung, colorectal, breast, and prostate cancers. The review also discusses MMPs as potential target molecules for therapeutic agents and new strategies for development of such drugs.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Neoplasias/enzimología , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neovascularización Patológica , PronósticoRESUMEN
Increased expression of the invasion- and metastasis-associated protein S100A4 is found in many types of cancer, but the regulation of S100A4 expression is poorly understood. The microenvironment surrounding tumors has a significant effect on tumor progression, and in the present study, we investigated the role of the microenvironment in the expression of S100A4. Tumors of three different human carcinoma cell lines were established in the tongue or skin of mice, and S100A4 expression was assessed by quantitative RT-PCR, Western blotting, and immunohistochemical analysis in tumors and stromal tissue and in cancer cells grown in vitro. Tongue tumors of the oral squamous cell carcinoma cell line HSC-4 showed a pronounced increase in S100A4 expression during tumor growth, whereas only a minor increase was detected in skin tumors of the same cell line. The S100A4 expression correlated with the methylation status of cytosine-guanine sites in the first intron of the gene. For all cell lines, S100A4 expression in the tumor stroma was related to the presence of inflammatory cells rather than to the level of S100A4 in the tumor cells.
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Carcinoma/metabolismo , Carcinoma/patología , Proteínas S100/biosíntesis , Microambiente Tumoral , Animales , Western Blotting , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión al Calcio S100A4 , Trasplante HeterólogoRESUMEN
BACKGROUND: Nephropathy is serious complication of diabetes. We have previously shown that level of the proteoglycan syndecan-1 in blood is associated with ultrastructural kidney changes in young persons with type 1 diabetes. Dysregulation of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) may contribute to the development of nephropathy. The aim of this study was to investigate if the levels of MMPs in blood samples are potential markers of early nephropathy in type 1 diabetes. METHODS: Blood samples were collected from type 1 diabetes patients after 11 years of diabetes (n = 15) and healthy volunteers (n = 12) and stored at /80 degrees C until measurement. Levels and activities of serum MMP-2, MMP-9, TIMP-1 and TIMP- 2 were analyzed and compared to those of control individuals using ELISA, SDS-PAGE gelatin zymography, and Western blot analysis. RESULTS: The serum levels of both MMP-9 and MMP-2 were significantly higher in subjects with type 1 diabetes, compared to controls (p = 0.016 and p = 0.008 respectively). Western blotting revealed no differences between the two groups in the levels of TIMP-1 or TIMP-2, respectively. CONCLUSION: Our MMP analysis of serum from a limited number of patients with type 1 diabetes suggest that such analysis is potentially useful as markers in studies of people at risk of progression to chronic kidney disease.
RESUMEN
To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present an in situ gelatin zymography method that allows for a precise localization of active gelatin-degrading enzymes in a tissue section. In this method, dye-quenched gelatin is put on top of a tissue section. During an incubation period, active gelatinolytic enzymes will degrade the substrate and fluorescent signals are emitted from the locations of these enzymes.
Asunto(s)
Pruebas de Enzimas/métodos , Gelatinasas/metabolismo , Microscopía Fluorescente/métodos , Microtomía/métodos , Animales , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Gelatina/análisis , Gelatina/metabolismo , Gelatinasas/análisis , Humanos , Glándulas Mamarias Humanas/química , Glándulas Mamarias Humanas/enzimología , Glándulas Mamarias Humanas/ultraestructura , Ratones , Especificidad por Sustrato , Adhesión del Tejido/métodos , Fijación del Tejido/métodosRESUMEN
To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF).
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Pruebas de Enzimas/métodos , Gelatinasas/metabolismo , Animales , Línea Celular , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Furanos/análisis , Furanos/metabolismo , Gelatina/análisis , Gelatina/metabolismo , Gelatinasas/análisis , Humanos , Riñón/química , Riñón/enzimología , Hígado/química , Hígado/enzimología , Ratones , Ratones Endogámicos BALB C , Piel/química , Piel/enzimologíaRESUMEN
Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD(+) complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.
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Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/ultraestructura , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/ultraestructura , Modelos Químicos , Modelos Moleculares , Sitios de Unión , Catálisis , Simulación por Computador , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The two matrix metalloproteinases, MMP-2 and MMP-9, are known to form various dimer complexes. In the present review, some of these complexes are described along with their biological and pathobiological functions.
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Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteoglicanos/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Animales , Dimerización , Haptoglobinas/metabolismo , Humanos , Lipocalinas/metabolismo , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 9 de la Matriz/química , alfa-Fetoproteínas/metabolismoRESUMEN
The S100A4 protein as well as the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are associated with diseases such as arthritis and cancer. This mini review focuses on in vitro and in vivo studies indicating S100A4 involvement in regulation of MMPs and TIMPs, and the biological and pathobiological consequences of this regulation.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Neoplasias/metabolismo , Proteínas S100/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Animales , Regulación hacia Abajo , Humanos , Proteína de Unión al Calcio S100A4 , Regulación hacia ArribaRESUMEN
Cell adhesion and migration is largely dependent on integrin binding to extracellular matrix, and several signalling pathways involved in these processes have been shown to be modified by reactive oxygen species (ROS). In fact, integrin activation is linked to increased ROS production by NADPH-oxidases, 5-lipoxygenase, and release from mitochondria. Cell migration is intimately linked to degradation of the extracellular matrix, and activated matrix metalloproteinases (MMPs) are a prerequisite for cancer cell invasion and metastasis. In this minireview, we focus on the interplay between integrin-mediated ROS production and MMP expression as well as its biological and pathobiological significance.
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Matriz Extracelular/metabolismo , Integrinas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Humanos , NADPH Oxidasas/metabolismo , Neoplasias/metabolismo , Transducción de SeñalRESUMEN
Proteases are probably underestimated exposure agents in bioaerosols. Their roles as barrier disrupters in allergic sensitization and activators of innate inflammation call for more attention in exposure-response studies. The main objectives of this study was (i) to establish a suitable method for detection of small quantities of proteases in filtered air samples and (ii) to utilize the method to characterize exposure to proteases in a salmon industry work environment. Analysis of proteases in filtered air samples was based on zymography, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 0.1% gelatin as substrate added in the polyacrylamide gel. Gelatinase activity was evident as cleared (unstained) regions. The area of these regions was quantified using image analysis (UVP Vision Works®). Standard curves with known amounts of active porcine trypsin were added to each gel. Validation of 11 non-linear standard curves showed R2 (range) = 0.8989-0.9882, limit of detection = 0.056 nM, lower limit of quantification = 0.161 nM, and coefficients of variations (range) = 20-28%. Sampling of bioaerosols in salmon industry was performed using polytetrafluoretylene filters with an airflow of 3 l min-1. All samples contained visible bands close to the size of porcine trypsin (23.3 kDa). The bands did not disappear in the presence of EDTA but abolished by Pefabloc, demonstrating that the enzyme is a serine protease, most likely salmon trypsin. Airborne levels of active protease were below the statistical detection limit in the filleting department but quantifiable in extract samples from the slaughter department. Three filtered air samples from the slaughter department showed air concentrations of 6.2, 16.5, and 27.0 ng m-3 air. We conclude that zymography is a sensitive and reliable method for exposure assessment of active proteases in indoor environmental samples. We recommend this assay for use in occupational studies to characterize and quantify exposure to active proteases in bioaerosols.
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Aerosoles/análisis , Contaminantes Ocupacionales del Aire/análisis , Bioensayo/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Explotaciones Pesqueras , Exposición Profesional/análisis , Péptido Hidrolasas/análisis , Animales , Endopeptidasas/análisis , Humanos , SalmónRESUMEN
Inhibitors targeting bacterial enzymes should not interfere with enzymes of the host, and knowledge about structural determinants for selectivity is important for designing inhibitors with a therapeutic potential. We have determined the binding strengths of two hydroxamate compounds, galardin and compound 1b for the bacterial zinc metalloproteases, thermolysin, pseudolysin and auerolysin, known to be bacterial virulence factors, and the two human zinc metalloproteases MMP-9 and MMP-14. The active sites of the bacterial and human enzymes have huge similarities. In addition, we also studied the enzyme-inhibitor interactions by molecular modelling. The obtained Ki values of galardin for MMP-9 and MMP-14 and compound 1b for MMP-9 are approximately ten times lower than previously reported. Compound 1b binds stronger than galardin to both MMP-9 and MMP-14, and docking studies indicated that the diphenyl ether moiety of compound 1b obtains more favourable interactions within the S´1-subpocket than the 4-methylpentanoyl moiety of galardin. Both compounds bind stronger to MMP-9 than to MMP-14, which appears to be due to a larger S´1-subpocket in the former enzyme. Galardin, but not 1b, inhibits the bacterial enzymes, but the galardin Ki values were much larger than for the MMPs. The docking indicates that the S´1-subpockets of the bacterial proteases are too small to accommodate the diphenyl ether moiety of 1b, while the 4-methylpentanoyl moiety of galardin enters the pocket. The present study indicates that the size and shape of the ligand structural moiety entering the S´1-subpocket is an important determinant for selectivity between the studied MMPs and bacterial MPs.
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Antibacterianos/farmacología , Dipéptidos/farmacología , Ácidos Hidroxámicos/farmacología , Metaloproteinasas de la Matriz/metabolismo , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Inhibidores de Proteasas/farmacología , Animales , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Dipéptidos/química , Dipéptidos/metabolismo , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Metaloproteinasas de la Matriz/química , Metaloproteasas/química , Metaloproteasas/genética , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Células Sf9 , Células THP-1RESUMEN
Pro-inflammatory cells, cytokines, and chemokines are essential in promoting a tumor supporting microenvironment. Chemerin is a chemotactic protein and a natural ligand for the receptors CMKLR1, GPR1, and CCRL2. The chemerin/CMKLR1 axis is involved in immunity and inflammation, and it has also been implicated in obesity and cancer. In neuroblastoma, a childhood tumor of the peripheral nervous system we identified correlations between high CMKLR1 and GPR1 expression and reduced overall survival probability. CMKLR1, GPR1, and chemerin RNA and protein were detected in neuroblastoma cell lines and neuroblastoma primary tumor tissue. Chemerin induced calcium mobilization, increased MMP-2 synthesis as well as MAP-kinase- and Akt-mediated signaling in neuroblastoma cells. Stimulation of neuroblastoma cells with serum, TNFα or IL-1ß increased chemerin secretion. The small molecule CMKLR1 antagonist α-NETA reduced the clonogenicity and viability of neuroblastoma cell lines indicating the chemerin/CMKLR1 axis as a promoting factor in neuroblastoma tumorigenesis. Furthermore, nude mice carrying neuroblastoma SK-N-AS cells as xenografts showed impaired tumor growth when treated daily with α-NETA from day 1 after tumor cell injection. This study demonstrates the potential of the chemerin/CMKLR1 axis as a prognostic factor and possible therapeutic target in neuroblastoma.
RESUMEN
Madin-Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules.
Asunto(s)
Riñón/citología , Riñón/metabolismo , Péptido Hidrolasas/metabolismo , Proteoglicanos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Western Blotting , Catálisis , Células Cultivadas , Clonación Molecular , Perros , Activación Enzimática/efectos de los fármacos , Gelatina/química , Gelatina/metabolismo , Humanos , Oligopéptidos/farmacología , Péptido Hidrolasas/efectos de los fármacos , Proteoglicanos/genética , Sulfatos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Proteínas de Transporte Vesicular/genéticaRESUMEN
In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.