RESUMEN
A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs -2, -9, and -13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M-1 s-1 and 4300 M-1 s-1, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (kcat/Km) of 5200 M-1 s-1. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 µM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Colorantes Fluorescentes/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ácidos Hidroxámicos/farmacología , Cinética , Péptidos/química , Péptidos/metabolismoRESUMEN
A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities.
Asunto(s)
Proteínas ADAM/análisis , Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Colorantes Fluorescentes/química , Proteínas ADAM/química , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia , Humanos , Hidrólisis , Análisis de Regresión , SolubilidadRESUMEN
We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.
Asunto(s)
Proteínas ADAM/metabolismo , Colorimetría/métodos , Pruebas de Enzimas/métodos , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Bencidinas/química , Dipéptidos/farmacología , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Peróxido de Hidrógeno/química , Cinética , Imanes/química , Metaloproteinasa 2 de la Matriz/orina , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Microesferas , Ácido Nitrilotriacético/análogos & derivados , Ácido Nitrilotriacético/química , Compuestos Organometálicos/química , Péptidos/química , Péptidos/metabolismo , Especificidad por SustratoRESUMEN
Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.23.4) are typically used to catalyze the enzymatic step. The most commonly used method to study chymosin activity is the relative milk-clotting activity test that measures the end point of the enzymatic and aggregation step. This method showed that camel chymosin has a 2-fold higher milk-clotting activity toward bovine milk than bovine chymosin. To enable a study of the enzymatic step independent of the aggregation step, a fluorescence resonance energy transfer assay has been developed using a peptide substrate derived from the 98-108 sequence of bovine κ-casein. This assay and Michaelis-Menten kinetics were employed to determine the enzymatic activity of camel and bovine chymosin under milk clotting-like conditions (pH 6.65, ionic strength 80 mM). The results obtained show that the catalytic efficiency of camel chymosin is 3-fold higher than bovine chymosin. The substrate affinity and catalytic activity of bovine and camel chymosin increase at lower pH (6.00 and 5.50). The glycosylation of bovine and camel chymosin did not affect binding of the fluorescence resonance energy transfer substrate, but doubly glycosylated camel chymosin seems to have slightly higher catalytic efficiency. In the characterization of the enzymes, the developed assay is easier and faster to use than the traditionally used relative milk-clotting activity test method.
Asunto(s)
Caseínas/metabolismo , Quimosina/metabolismo , Transferencia Resonante de Energía de Fluorescencia/veterinaria , Leche/enzimología , Animales , Camelus , Bovinos , Transferencia Resonante de Energía de Fluorescencia/métodos , Glicosilación , CinéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with <5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK1/2 and higher MMP activities. For biological function, ADAM8 requires multimerization and associates with ß1 integrin on the cell surface. A peptidomimetic ADAM8 inhibitor, BK-1361, designed by structural modelling of the disintegrin domain, prevents ADAM8 multimerization. In PDAC cells, BK-1361 affects ADAM8 function leading to reduced invasiveness, and less ERK1/2 and MMP activation. BK-1361 application in mice decreased tumour burden and metastasis of implanted pancreatic tumour cells and provides improved metrics of clinical symptoms and survival in a Kras(G12D)-driven mouse model of PDAC. Thus, our data integrate ADAM8 in pancreatic cancer signalling and validate ADAM8 as a target for PDAC therapy.
Asunto(s)
Proteínas ADAM/metabolismo , Proteínas de la Membrana/metabolismo , Terapia Molecular Dirigida , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas ADAM/antagonistas & inhibidores , Animales , Western Blotting , Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Espacio Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Integrina beta1/metabolismo , Estimación de Kaplan-Meier , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Invasividad Neoplásica , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/uso terapéutico , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Transducción de Señal/efectos de los fármacosRESUMEN
Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The donor/quencher pair used were 5-carboxy fluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)- NH2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)- NH2, and C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala-Lys(Fam)- NH2 cleavage sites were effectively processed by ADAM9 with turnover numbers of 100 +/- 20 x 10(-2) min(-1), 20 +/- 10 x 10(-2) min(-1), and 10 +/- 3 x 10(-2) min(-1). In addition, a peptide based on the 33 kDa cleavage site of the low affinity receptor for IgE, CD23, Dabcyl-Leu-Arg-Ala-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(Fam)- NH2 was processed as well but with less efficiency. A more selective substrate for ADAM9 was found based on the betacellulin cleavage site. However, the valine containing precursor TNF-alpha based substrate was used to measure IC50 values of metalloproteinase inhibitors against ADAM9 since it was processed the most efficiently. The tightest binding inhibitor was the Wyeth Aerst compound, TMI-1, with an IC50 of 2.1 +/- 0.3 nM. In addition, GI254023, previously identified as a selective inhibitor of ADAM10, also inhibited ADAM9 with an IC50 of 280 +/- 110 nM. These results demonstrate that sensitive substrates for ADAM9 can be developed that are useful in high-throughput screening assays for ADAM9.
Asunto(s)
Proteínas ADAM/metabolismo , Colorantes Fluorescentes/química , Proteínas de la Membrana/metabolismo , Proteínas ADAM/química , Líquidos Corporales/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Hidrólisis , Proteínas de la Membrana/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In this paper we describe novel fluorescent substrates for the human ADAM family members ADAM17, ADAM10, ADAM8, and ADAM12 that have good specificity constants and are useful for high-throughput screening of inhibitors. The fluorescence resonance energy transfer substrates contain a 4-(4-dimethylaminophenylazo)benzoyl and 5-carboxyfluorescein (Dabcyl/Fam) pair and are based on known cleavage sequences in precursor tumor necrosis factor-alpha (TNF-alpha) and CD23. The precursor TNF-alpha-based substrate, Dabcyl-Leu-Ala-Gln-Ala-Homophe-Arg-Ser-Lys(Fam)-NH2, is a good substrate for all the ADAMs tested, including ADAM12 for which there is no reported fluorescent substrate. The CD23-based substrate, Dabcyl-His-Gly-Asp-Gln-Met-Ala-Gln-Lys-Ser-Lys(Fam)-NH2, is more selective, being hydrolyzed efficiently only by ADAM8 and ADAM10. The substrates were used to obtain inhibition constants for four inhibitors that are commonly used in shedding assays: TMI-1, GM6001, GW9471, and TAPI-2. The Wyeth Aerst compound, TMI-1, is a potent inhibitor against all of the ADAMs tested and is slow binding against ADAM17.
Asunto(s)
Proteínas ADAM/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas de la Membrana/metabolismo , Inhibidores de Proteasas/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAM10 , Proteína ADAM12 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Dipéptidos/química , Dipéptidos/metabolismo , Dipéptidos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/metabolismo , Ácidos Hidroxámicos/farmacología , Cinética , Proteínas de la Membrana/antagonistas & inhibidores , Morfolinas/química , Morfolinas/metabolismo , Morfolinas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Unión ProteicaRESUMEN
Matrix metalloproteinases (MMPs) are a family of enzymes that are up-regulated in many diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). Here we report on a novel technique that can be used to simultaneously measure activity levels for a panel of enzymes, such as the MMPs. The technique, termed the multiple-enzyme/multiple-reagent assay system (MEMRAS), relies on the use of reagents such as substrates with varying selectivity profiles against a group of enzymes. When reaction rates are measured by following a change in fluorescence with time, for mixtures of enzymes, an equation with unknown concentrations for each activity is generated for each reagent used. Simultaneously solving the set of equations leads to a solution for the unknown concentrations. We have applied this mathematical technique to measure activity levels for mixtures of MMPs such as collagenase 3 and gelatinase A. In addition, because we were most interested in determining collagenase 3 levels as a potential biological marker for OA, we developed highly selective substrates for this enzyme by using results found in previous bacteriophage substrate-mapping experiments. Some of the best substrates tested have specific activities for collagenase 3 that are 37,000-, 17,000-, 90-, and 200-fold selective over stromelysin 1, collagenase 1, and gelatinases A and B, respectively.
Asunto(s)
Metaloproteinasas de la Matriz/química , Oligopéptidos/química , p-Dimetilaminoazobenceno/análogos & derivados , Compuestos Cromogénicos , Colagenasas/química , Dinitrobencenos/química , Activación Enzimática , Fluoresceína , Fluorometría , Gelatina , Humanos , Indicadores y Reactivos , Cinética , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 3 de la Matriz/química , Metaloproteinasa 9 de la Matriz/química , Relación Estructura-Actividad , Especificidad por Sustrato , Líquido Sinovial/químicaRESUMEN
Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.