RESUMEN
Chronic inflammation is a major risk factor for colorectal cancer. The p38/MAPKAP Kinase 2 (MK2) kinase axis controls the synthesis of proinflammatory cytokines that mediate both chronic inflammation and tumor progression. Blockade of this pathway has been previously reported to suppress inflammation and to prevent colorectal tumorigenesis in a mouse model of inflammation-driven colorectal cancer, by mechanisms that are still unclear. Here, using whole-animal and tissue-specific MK2 KO mice, we show that MK2 activity in the myeloid compartment promotes tumor progression by supporting tumor neoangiogenesis in vivo. Mechanistically, we demonstrate that MK2 promotes polarization of tumor-associated macrophages into protumorigenic, proangiogenic M2-like macrophages. We further confirmed our results in human cell lines, where MK2 chemical inhibition in macrophages impairs M2 polarization and M2 macrophage-induced angiogenesis. Together, this study provides a molecular and cellular mechanism for the protumorigenic function of MK2.
Asunto(s)
Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/epidemiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/enzimología , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular , Citoplasma/enzimología , Daño del ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Transporte Activo de Núcleo Celular , Antibióticos Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Ciclo Celular/efectos de la radiación , Núcleo Celular/enzimología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Reparación del ADN , Doxorrubicina/farmacología , Exorribonucleasas/metabolismo , Retroalimentación Fisiológica , Células HeLa , Neoplasias de Cabeza y Cuello/enzimología , Neoplasias de Cabeza y Cuello/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Mitosis , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de la radiación , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/efectos de la radiación , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Rayos Ultravioleta , Fosfatasas cdc25/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Signaling networks regulate cellular responses to external stimuli through post-translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large-scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel-screening platform developed for the proteome-wide substrate-analysis of specific kinases. Here, we aimed to identify substrates of mitogen-activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen-activated protein kinase cascade. An ATP analog-sensitive mutant of Mek1 (Mek1-as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC-MS/MS of IMAC-enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/química , Alineación de Secuencia , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
Chronic inflammation increases the risk for colorectal cancer through a variety of mechanisms involving the tumor microenvironment. MAPK-activated protein kinase 2 (MK2), a major effector of the p38 MAPK stress and DNA damage response signaling pathway, and a critical regulator of pro-inflammatory cytokine production, has been identified as a key contributor to colon tumorigenesis under conditions of chronic inflammation. We have previously described how genetic inactivation of MK2 in an inflammatory model of colon cancer results in delayed tumor progression, decreased tumor angiogenesis, and impaired macrophage differentiation into a pro-tumorigenic M2-like state. The molecular mechanism responsible for the impaired angiogenesis and tumor progression, however, has remained contentious and poorly defined. Here, using RNA expression analysis, assays of angiogenesis factors, genetic models, in vivo macrophage depletion and reconstitution of macrophage MK2 function using adoptive cell transfer, we demonstrate that MK2 activity in macrophages is necessary and sufficient for tumor angiogenesis during inflammation-induced cancer progression. We identify a critical and previously unappreciated role for MK2-dependent regulation of the well-known pro-angiogenesis factor CXCL-12/SDF-1 secreted by tumor associated-macrophages, in addition to MK2-dependent regulation of Serpin-E1/PAI-1 by several cell types within the tumor microenvironment.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Neoplasias Asociadas a Colitis/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neovascularización Patológica , Proteínas Serina-Treonina Quinasas/metabolismo , Macrófagos Asociados a Tumores/enzimología , Traslado Adoptivo , Proteínas Angiogénicas/genética , Animales , Células Cultivadas , Neoplasias Asociadas a Colitis/genética , Neoplasias Asociadas a Colitis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Transcripción Genética , Microambiente Tumoral , Macrófagos Asociados a Tumores/trasplanteRESUMEN
In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Reparación del ADN/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Células HCT116 , Humanos , Immunoblotting , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Nanomedicina/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
The epidermal growth factor receptor (EGFR/ErbB1/Her1) belongs to the ErbB family of receptor tyrosine kinases (RTKs) and is a key player in the regulation of cell proliferation, differentiation, survival, and migration. Overexpression and mutational changes of EGFR have been identified in a variety of human cancers and the regulation of EGFR signaling plays a critical role in tumor development and progression. Due to its biological significance the EGFR signaling network is a widely used model system for the development of analytical techniques. Novel quantitative proteomics and phosphoproteomics approaches play an important role in the characterization of signaling pathways in a time and stimulus dependent manner. Recent studies discussed in this review provide new insights into different aspects of EGFR signal transduction, such as regulation and dynamics of its phosphorylation sites, association with interaction partners and identification of regulated phosphoproteins. Correlation of data from functional proteomics studies with results from other fields of signal transduction research by systems biology will be necessary to integrate and translate these findings into successful clinical applications.
Asunto(s)
Receptores ErbB/fisiología , Fosfoproteínas/metabolismo , Proteómica/métodos , Animales , Sitios de Unión , Bases de Datos Factuales , Humanos , Fosforilación , Mapeo de Interacción de Proteínas , Receptor ErbB-2/fisiología , Receptor ErbB-3/fisiología , Transducción de Señal , Biología de SistemasRESUMEN
The potential of an organic monolith with incorporated titanium dioxide (TiO(2)) and zirconium dioxide (ZrO(2)) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano- and microscale TiO(2) and ZrO(2) powders. Eleven phosphopeptides from alpha-casein digest could be recovered by an optimized mixture of microscale TiO(2)/ZrO(2) particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano-structured material. When compared to conventional immobilized metal-ion affinity chromatography and commercial phosphorylation-enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from alpha-casein and five from beta-casein digests by using TiO(2) and ZrO(2) based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI-MS/MS measurements and a following MASCOT database search.
Asunto(s)
Fosfopéptidos/análisis , Proteínas/metabolismo , Estirenos , Titanio , Circonio , Animales , Glutatión Transferasa/genética , Indicadores y Reactivos , Proteína Quinasa 1 Activada por Mitógenos/análisis , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/análisis , Proteína Quinasa 3 Activada por Mitógenos/genética , Nanopartículas , Fragmentos de Péptidos/análisis , Fosforilación , Porosidad , Polvos , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , TripsinaRESUMEN
In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Resistencia a Antineoplásicos/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Mutación , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Anciano , Animales , Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cisplatino/uso terapéutico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Pleiotropía Genética , Células HEK293 , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de SupervivenciaRESUMEN
The evolutionary history of the genus Omphalotus was inferred from DNA sequences of the ITS1-5.8S-ITS2 rDNA region. We analyzed 32 collections from different geographical areas: O. olearius (Europe), O. illudens (Europe, North America), O. subilludens (North America), O. olivascens var. olivascens (North America) and var. indigo (Mexico), O. mexicanus (Middle America), O. nidiformis (Australia), and O. japonicus (Japan). Phylogenetic analysis was performed declaring Nothopanus eugrammus as outgroup. Our analyses show that the genus Omphalotus is split into two major clades, the first consisting of O. illudens and O. mexicanus and the second comprising O. olearius, O. olivascens, O. japonicus, O. nidiformis and O. subilludens. Moreover, the often discussed synonymy of O. illudens and O. olearius is rejected. Omphalotus japonicus, a species formerly placed in the genus Lampteromyces Sing. for morphological reasons, clustered as the sister group of O. olearius.
RESUMEN
A fundamental limitation in devising new therapeutic strategies for killing cancer cells with DNA damaging agents is the need to identify synthetic lethal interactions between tumor-specific mutations and components of the DNA damage response (DDR) in vivo. The stress-activated p38 mitogen-activated protein kinase (MAPK)/MAPKAP kinase-2 (MK2) pathway is a critical component of the DDR network in p53-deficient tumor cells in vitro. To explore the relevance of this pathway for cancer therapy in vivo, we developed a specific gene targeting strategy in which Cre-mediated recombination simultaneously creates isogenic MK2-proficient and MK2-deficient tumors within a single animal. This allows direct identification of MK2 synthetic lethality with mutations that promote tumor development or control response to genotoxic treatment. In an autochthonous model of non-small-cell lung cancer (NSCLC), we demonstrate that MK2 is responsible for resistance of p53-deficient tumors to cisplatin, indicating synthetic lethality between p53 and MK2 can successfully be exploited for enhanced sensitization of tumors to DNA-damaging chemotherapeutics in vivo.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Reparación del ADN/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Antineoplásicos/farmacología , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular , Daño del ADN/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
DNA damage signaling and checkpoint control pathways are among the most commonly mutated networks in human tumors. Emerging data suggest that synthetic lethal interactions between mutated oncogenes or tumor suppressor genes with molecules involved in the DNA damage response and DNA repair pathways can be therapeutically exploited to preferentially kill cancer cells. In this review, we discuss the concept of synthetic lethality with a focus on p53, a commonly lost tumor suppressor gene, in the context of DNA damage signaling. We describe several recent examples in which this concept was successfully applied to target tumor cells in culture or in mouse models, as well as in human cancer patients.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Daño del ADN/genética , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/terapia , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , HumanosRESUMEN
In response to DNA damage, cells activate a complex, kinase-based signaling network that consist of two components--a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes to arrest the cell cycle along with recruitment of repair machinery to damaged DNA, followed by a delayed transcriptional response that promotes cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves post-transcriptional control of mRNA stability, splicing, and translation as a critical part of the DNA damage response. Here, we describe recent work implicating DNA damage-dependent modification of RNA-binding proteins that are responsible for some of these mRNA effects, highlighting recent work on post-transcriptional regulation of the cell cycle checkpoint protein/apoptosis inducer Gadd45a by the checkpoint kinase MAPKAP Kinase-2.
Asunto(s)
Daño del ADN/genética , Biosíntesis de Proteínas/genética , Procesamiento Postranscripcional del ARN/genética , Empalme del ARN/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Animales , Humanos , Transducción de Señal/genéticaRESUMEN
Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2-DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel-free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or 'chemical genetics' and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.
Asunto(s)
Fosfoproteínas/fisiología , Proteómica , Transducción de Señal/fisiología , Animales , Ratones , Fosfoproteínas/clasificación , Fosfoproteínas/aislamiento & purificaciónRESUMEN
In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.
Asunto(s)
Biomarcadores/sangre , Neoplasias de la Mama/sangre , Electroforesis en Gel Bidimensional/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adulto , Apolipoproteína A-I/análisis , Apolipoproteína C-III , Apolipoproteínas A/sangre , Apolipoproteínas C/sangre , Regulación hacia Abajo , Femenino , Haptoglobinas/análisis , Hemoglobinas/análisis , Humanos , Persona de Mediana Edad , Posmenopausia , Premenopausia , Precursores de Proteínas/sangre , Transferrina/análisis , Regulación hacia ArribaRESUMEN
Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Colorantes Fluorescentes/química , Fosfoproteínas/análisis , Proteoma/análisis , Proteómica/métodos , Animales , Ratones , Fosfoproteínas/química , Fosfoproteínas/aislamiento & purificación , Proteoma/química , Proteoma/aislamiento & purificaciónRESUMEN
We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.
Asunto(s)
Albúminas/aislamiento & purificación , Proteínas Sanguíneas/análisis , Cromatografía de Afinidad/métodos , Inmunoglobulina G/aislamiento & purificación , Proteoma/análisis , Acetonitrilos/química , Albúminas/química , Electroforesis en Gel Bidimensional , Humanos , Inmunoglobulina G/química , Desnaturalización ProteicaRESUMEN
The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound Fe(III) as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-Fe(III) for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe(III) being 25.4 micromol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe(III) and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of beta-casein deriving phosphopeptides, the established system was extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).
Asunto(s)
Hierro/análisis , Fosfoproteínas/química , Proteómica/métodos , Secuencia de Aminoácidos , Caseínas/química , Cromatografía de Afinidad , Compuestos Epoxi/química , Glutatión Transferasa/metabolismo , Iminoácidos/química , Hierro/química , Espectrometría de Masas , Metacrilatos/química , Microscopía Electrónica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Mioglobina/química , Péptidos/química , Fosfopéptidos/química , Fosforilación , Polímeros/química , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , Tripsina/farmacología , Compuestos de Vinilo/químicaRESUMEN
Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.