RESUMEN
Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.
Asunto(s)
Arabidopsis/enzimología , Ácidos Hidroxámicos/química , Inhibidores de la Metaloproteinasa de la Matriz , Proteínas de Plantas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Hojas de la Planta/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem , Rayos UltravioletaRESUMEN
The central role of kinases in cell signaling has set them in the focus of biomedical research. In functional proteomics analyses, large- scale profiling of kinases has become feasible through the use of affinity pulldown beads that carry immobilized kinase inhibitors. As an alternative approach to solid phase beads, Capture Compound Mass Spectrometry (CCMS) enables the functional isolation of protein-classes on the basis of small molecule-protein interactions in solution. Capture Compounds are trifunctional probes: a selectivity function interacts with the native target proteins in equilibrium, upon irradiation a photoactivatable reactivity function forms an irreversible covalent bond to the target proteins, and a sorting function allows the captured proteins to be isolated from a complex protein mixture. We report the design and application of a novel, fully water-soluble Capture Compound that carries the broadband kinase inhibitor staurosporine as selectivity function. We used this Capture Compound to profile the kinome of the human liver-derived cell line HepG2 and identified one hundred kinases. HepG2 cells are a widely used model system for hepatocarcinoma, hepatitis, and for investigation of drug toxicity effects. CCMS experiments in membrane fractions of human placenta are given as example for the applicability to human tissue.
Asunto(s)
Hepatocitos/efectos de los fármacos , Espectrometría de Masas/métodos , Fosfotransferasas/metabolismo , Estaurosporina/farmacología , Línea Celular , Electroforesis en Gel de Poliacrilamida , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Modelos Moleculares , Estaurosporina/metabolismoRESUMEN
Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.
Asunto(s)
Metiltransferasas/aislamiento & purificación , S-Adenosilhomocisteína/análogos & derivados , Reactivos de Enlaces Cruzados/química , Cinética , Magnetismo , Procesos Fotoquímicos , S-Adenosilhomocisteína/síntesis química , S-Adenosilhomocisteína/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estreptavidina/química , Estreptavidina/metabolismo , Rayos UltravioletaRESUMEN
Capture Compound Mass Spectrometry (CCMS) is a platform technology for the functional isolation of subproteomes. Here we report the synthesis of two new kinase Capture Compounds (CCs) based on the tyrosine-kinase specific inhibitors dasatinib and imatinib and compare their interaction profiles to that of our previously reported staurosporine-CCs. CCs are tri-functional molecules: they comprise a sorting function (e.g. the small molecule or drug of interest) which interacts with target proteins, a photo-activatable reactivity function to covalently trap the interacting proteins, and a sorting function to isolate the CC-protein conjugates from complex biological samples for protein identification by liquid chromatography/mass spectrometry (LC-MS/MS). We present data of CCMS experiments from human HepG2 cells and compare the profiles of the kinases isolated with dasatinib, imatinib and staurosporine CC, respectively. Dasatinib and imatinib have a more selective kinase binding profile than staurosporine. Moreover, the new CCs allow isolation and identification of additional kinases, complementing the staurosporine CC. The family of kinase CCs will be a valuable tool for the proteomic profiling of this important protein class. Besides sets of expected kinases we identified additional specific interactors; these off-targets may be of relevance in the view of the pharmacological profile of dasatinib and imatinib.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteómica/métodos , Pirimidinas/farmacología , Estaurosporina/farmacología , Tiazoles/farmacología , Benzamidas , Cromatografía Liquida/métodos , Dasatinib , Células Hep G2 , Humanos , Mesilato de Imatinib , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Espectrometría de Masas/métodos , Piperazinas/química , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/genética , Pirimidinas/química , Estaurosporina/química , Tiazoles/químicaRESUMEN
S-Adenosyl-L-methionine (AdoMet) is the major methyl donor for biological methylation reactions catalyzed by methyltransferases. We report the first chemical synthesis of AdoMet analogs with extended carbon chains replacing the methyl group and their evaluation as cofactors for all three classes of DNA methyltransferases. Extended groups containing a double or triple bond in the beta position to the sulfonium center were transferred onto DNA in a catalytic and sequence-specific manner, demonstrating a high utility of such synthetic cofactors for targeted functionalization of biopolymers.
Asunto(s)
Carbono/química , Coenzimas/síntesis química , Metilasas de Modificación del ADN/metabolismo , S-Adenosilmetionina/síntesis química , Sitios de Unión , Catálisis , Metilación de ADN , Metilasas de Modificación del ADN/clasificación , Mitosis , Modelos Químicos , S-Adenosilmetionina/análogos & derivados , Estereoisomerismo , Especificidad por Sustrato , Compuestos de Sulfonio/química , Compuestos de Sulfonio/metabolismoRESUMEN
Here we describe a one-step synthetic procedure for the preparation of S-adenosyl-L-methionine (AdoMet) analogs with extended carbon chains replacing the methyl group. These AdoMet analogs function as efficient cofactors for DNA methyltransferases (MTases), and we provide a protocol for sequence-specific transfer of extended side chains from these AdoMet analogs to DNA by DNA MTases. Direct chemoselective allylation or propargylation of S-adenosyl-L-homocysteine (AdoHcy) at sulfur is achieved under the acidic conditions needed to protect other nucleophilic positions in AdoHcy. The unsaturated bonds in beta position to the sulfonium center of the resulting AdoMet analogs are designed to stabilize the transition state formed upon DNA MTase-catalyzed nucleophilic attack at the carbon next to the sulfonium center and lead to efficient transfer of the extended side chains to DNA. Using these protocols, sequence-specific functionalized DNA can be obtained within one to two weeks.
Asunto(s)
Metilasas de Modificación del ADN/metabolismo , ADN/metabolismo , S-Adenosilmetionina/análogos & derivados , Alquilación , S-Adenosilmetionina/químicaRESUMEN
Incorporation of sulfoximines as backbone modifying element results in two new pseudopeptide bonds which display enhanced (bond A) and strongly reduced reactivity (bond B) towards hydrolysis by Proteinase K.