RESUMEN
Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-GsαKO mice) have abnormal myelopoiesis, osteopenia, and reduced adipose tissue. We previously reported that the severe osteopenia and the changes in adipose tissue present in these mice were mediated by increased sclerostin, which suppress osteoblast functions and promote browning of white adipocytes. Inversely, the myeloproliferation was driven by granulocyte colony-stimulating factor (G-CSF) and administration of neutralizing antibodies against G-CSF only partially restored the myeloproliferation, suggesting that additional osteocyte-derived factors might be involved. We hypothesized that osteocytes secrete Gsα-dependent factor(s) which regulate the myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsαcont and Ocy454-GsαKO ) to delineate the osteocyte "secretome" and its regulation by Gsα. Here we reported that factors secreted by osteocytes increased the number of myeloid colonies and promoted macrophage proliferation. The proliferation of myeloid cells was further promoted by osteocytes lacking Gsα expression. Myeloid cells can differentiate into bone-resorbing osteoclasts, therefore, we hypothesized that osteocyte-secreted factors might also regulate osteoclastogenesis in a Gsα-dependent manner. Conditioned medium (CM) from Ocy454 (both Gsαcont and GsαKO ) significanlty increased the proliferation of bone marrow mononuclear cells (BMNC) and, at the same time, inhibited their differentiation into mature osteoclasts via a Gsα-dependent mechanism. Proteomics analysis of CM from Ocy454 Gsαcont and GsαKO cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-GsαKO cells compared to Ocy454-Gsαcont , whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsαcont cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-GsαKO cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signaling.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células Mieloides/metabolismo , Células Mieloides/fisiología , Osteocitos/metabolismo , Osteocitos/fisiología , Animales , Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/fisiopatología , Médula Ósea/metabolismo , Médula Ósea/fisiología , Resorción Ósea/metabolismo , Resorción Ósea/fisiopatología , Línea Celular , Medios de Cultivo Condicionados/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Mielopoyesis/fisiología , Osteoclastos/metabolismo , Osteoclastos/fisiología , Osteogénesis/fisiología , Transducción de Señal/fisiologíaRESUMEN
Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.-Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.
Asunto(s)
Células Endoteliales/metabolismo , Glutarredoxinas/metabolismo , Melanoma/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Animales , Femenino , Arteria Femoral/cirugía , Glutarredoxinas/genética , Miembro Posterior/irrigación sanguínea , Miembro Posterior/cirugía , Isquemia , Ligadura , Masculino , Ratones , Ratones Transgénicos , Neoplasias ExperimentalesRESUMEN
Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. Although current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-associated viruses (AAV) are vectors of choice in gene therapy because of their well-characterized safety and efficacy profiles; however, although gene therapy has been successful in preclinical models of the disease, clinical trials in humans have failed to demonstrate efficacy. Significantly, all primary AAV receptors of the virus are glycans. We thus hypothesize that age related changes in glycan receptors of heparan sulfate (HS) proteoglycans (receptor for rAAV2), and/or N-glycans with terminal galactose (receptor for rAAV9) results in poor adeno-associated virus binding in either the striatum or substantia nigra, or both, affecting transduction and gene delivery. To test our hypothesis we analyzed the striatum and substantia nigra for changes in HS, N-glycans and proteomic signatures in young versus aged rat brain striatum and substantia nigra. We observed different brain region-specific HS disaccharide profiles in aged compared with young adult rats for brain region-specific profiles in striatum versus substantia nigra. We observed brain region- and age-specific N-glycan compositional profiles with respect to the terminal galactose units that serve as receptors for AAV9. We also observed brain region-specific changes in protein expression in the aging nigrostriatal pathway. These studies provide insight into age- and brain region-specific changes in glycan receptors and proteome that will inform design of improved viral vectors for Parkinson Disease (PD) gene therapy.
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Envejecimiento/metabolismo , Cuerpo Estriado/metabolismo , Glicómica , Proteoma/metabolismo , Proteómica , Sustancia Negra/metabolismo , Animales , Disacáridos/metabolismo , Galactosa/metabolismo , Heparitina Sulfato/metabolismo , Masculino , Especificidad de Órganos , Polisacáridos/metabolismo , Ratas Endogámicas F344RESUMEN
Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.
Asunto(s)
Factores Reguladores Miogénicos/metabolismo , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Trióxido de Arsénico , Arsenicales/farmacología , Ciclo Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina , Factores Reguladores Miogénicos/genética , Oligopéptidos/genética , Oligopéptidos/metabolismo , Óxidos/farmacología , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SUMO-1/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación , Transcripción GenéticaRESUMEN
One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2ß repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in ß-globin locus yeast artificial chromosome (ß-YAC) bone marrow cells. When WT ß-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2ß at the (A)γ-globin promoter is increased. In addition, OGT and Mi2ß recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human ß-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2ß is modified with O-GlcNAc, and both OGT and OGA interact with Mi2ß, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2ß repressor complex at the -566 GATA motif within the promoter.
Asunto(s)
Silenciador del Gen/fisiología , N-Acetilglucosaminiltransferasas/metabolismo , Elementos de Respuesta , beta-N-Acetilhexosaminidasas/metabolismo , gamma-Globinas/biosíntesis , Animales , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Humanos , Células K562 , Ratones , Ratones Transgénicos , N-Acetilglucosaminiltransferasas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta-N-Acetilhexosaminidasas/genética , gamma-Globinas/genéticaRESUMEN
Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes â¼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.
Asunto(s)
Transición Epitelial-Mesenquimal , Procesamiento Proteico-Postraduccional , Adenosina Trifosfato/metabolismo , Vías Biosintéticas , Línea Celular Tumoral , Inducción Enzimática , Glucosa/metabolismo , Glucógeno/metabolismo , Glicosilación , Hexosaminas/biosíntesis , Humanos , Ácido Láctico/metabolismo , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Ácido Pirúvico/metabolismo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrometry (LC-MS). The adoption of capillary electrophoresis-mass spectrometry methods (CE-MS) for glycomics and glycoproteomics is limited by the lack of convenient interfaces for coupling the CE devices to mass spectrometers. Here, we describe the application of a microfluidics-based CE-MS system for analysis of released glycans, glycopeptides and monosaccharides. We demonstrate a single CE method for three different modalities, thus contributing to comprehensive glycoproteomics analyses. In addition, we explored compatible sample derivatization methods. We used glycan TMT-labeling to improve electrophoretic migration and enable multiplexed quantitation by tandem MS. We used sialic acid linkage-specific derivatization methods to improve separation and the level of information obtained from a single analytical step. Capillary electrophoresis greatly improved glycoform separation for both released glycans and glycopeptides over that reported for chromatography modes more frequently employed for such analyses. Overall, the CE-MS method described here enables rapid setup and analysis of glycans and glycopeptides using mass spectrometry.
Asunto(s)
Glicopéptidos/análisis , Técnicas Analíticas Microfluídicas , Monosacáridos/análisis , Oligosacáridos/análisis , Electroforesis Capilar , Espectrometría de Masas , Modelos MolecularesRESUMEN
O-linked ß-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications including cardiomyopathy. However, mitochondrial O-GlcNAcylation, especially in response to chronic hyperglycemia in diabetes, has been poorly explored. We performed a comparative O-GlcNAc profiling of mitochondria from control and streptozotocin (STZ)-induced diabetic rat hearts by using an improved ß-elimination/Michael addition with isotopic DTT reagents (BEMAD) followed by tandem mass spectrometric analysis. In total, 86 mitochondrial proteins, involved in diverse pathways, were O-GlcNAcylated. Among them, many proteins have site-specific alterations in O-GlcNAcylation in response to diabetes, which suggests that protein O-GlcNAcylation is a novel layer of regulation mediating adaptive changes in mitochondrial metabolism during the progression of diabetic cardiomyopathy.
Asunto(s)
Acetilglucosamina/metabolismo , Cardiomiopatías Diabéticas/metabolismo , Proteínas Mitocondriales/metabolismo , Proteómica/métodos , Acilación , Animales , Diabetes Mellitus Experimental , Mitocondrias/metabolismo , Proteínas Mitocondriales/análisis , Miocardio/metabolismo , Ratas , Espectrometría de Masas en TándemRESUMEN
The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.
Asunto(s)
Diferenciación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Apoptosis/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Carbazoles/farmacología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1B1 , Células Eritroides/citología , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Células Nutrientes/citología , Células Nutrientes/efectos de los fármacos , Células Nutrientes/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genoma Humano/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Ratones , Receptores de Hidrocarburo de Aril/agonistasRESUMEN
Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified ≥1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, ß-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.
Asunto(s)
Cisteína/genética , Dieta/efectos adversos , Cardiopatías/etiología , Oxígeno/química , Animales , Cromatografía Liquida , Ciclo del Ácido Cítrico , Cisteína/química , Ácidos Grasos/química , Glucólisis , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica , Miocardio/metabolismo , Obesidad/metabolismo , Fosforilación Oxidativa , Fenotipo , Procesamiento Proteico-Postraduccional , Proteómica , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/química , Espectrometría de Masas en TándemRESUMEN
Pulmonary arterial hypertension (PAH) is a disease characterized by increased pulmonary vascular resistance and remodeling. Increase in the population of vascular smooth muscle cells is among the key events contributing to the remodeling. Endothelin-1 (ET-1), a potent vasoconstrictor, is linked to the etiology and progression of PAH. Here we analyze changes in protein expressions in response to ET-1 in pulmonary arterial smooth muscle cells (PASMC) from a healthy Control (non-PAH) and a PAH subject presenting a bone morphogenetic protein type II receptor (BMPR2) mutation with exon 1-8 deletion. Protein expressions were analyzed by proteomic mass spectrometry using label-free quantitation and the correlations were subjected to Ingenuity™ Pathway Analysis. The results point to eIF2/mTOR/p70S6K, RhoA/actin cytoskeleton/integrin and protein unbiquitination as canonical pathways whose protein expressions increase with the development of PAH. These pathways have an intimal function in the PAH-related physiology of smooth muscle proliferation, apoptosis, contraction and cellular stress. Exposure of the cells to ET-1 further increases protein expression within these pathways. Thus our results show changes in signaling pathways as a consequence of PAH and the effect of ET-1 interference on Control and PAH-affected cells.
RESUMEN
Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.
Asunto(s)
Mapas de Interacción de Proteínas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Ciclo Celular/fisiología , Humanos , Inmunoprecipitación , Espectrometría de Masas , Quinasas Relacionadas con NIMA , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteómica , Técnicas del Sistema de Dos HíbridosRESUMEN
Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFR Y1173 compared with the level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase Cγ, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y2 receptor mRNA expression increased, and after knockdown, migration and Ca(2+) mobilization were impaired. To examine phosphorylation mediated by P2Y2, cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry-based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFR Y974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in Src Y421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y2 receptor in mediating migration.
Asunto(s)
Señalización del Calcio , Movimiento Celular , Receptores ErbB/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patología , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Células Cultivadas , Receptores ErbB/genética , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Fosforilación/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Receptores Purinérgicos P2Y2/genética , Heridas y Lesiones/genética , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61±24% decrease for cardiac tissue and 38±7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane-bound HRas decreased growth factor-stimulated ERK phosphorylation (84±8.6% decrease) and increased apoptotic signaling (24±6.5-fold increase) after HPHG treatment that was prevented by overexpressing wild-type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17-HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Apoptosis/fisiología , Bovinos , Células Cultivadas , Cisteína/química , Dieta Alta en Grasa/efectos adversos , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/efectos adversos , Glucosa/administración & dosificación , Glucosa/efectos adversos , Lipoilación , Ratones , Ratones Endogámicos C57BL , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Estrés Fisiológico , Sacarosa/administración & dosificación , Sacarosa/efectos adversos , Superóxido Dismutasa/metabolismoRESUMEN
The biological functions of glycoconjugate glycans arise in the context of structural heterogeneity resulting from non-template driven biosynthetic reactions. Such heterogeneity is particularly apparent for the glycosaminoglycan (GAG) classes, of which heparan sulfate (HS) is of particular interest for its properties in binding to many classes of growth factors and growth factor receptors. The structures of HS chains vary according to spatial and temporal factors in biological systems as a mechanism where by the functions of the relatively limited number of associated proteoglycan core proteins is elaborated. Thus, there is a strong driver for the development of methods to discover functionally relevant structures in HS preparations for different sources. In the present work, a set of targeted tandem mass spectra were acquired in automated mode on HS oligosaccharides deriving from two different tissue sources. Statistical methods were used to determine the precursor and product ions, the abundances of which differentiate between the tissue sources. The results demonstrate considerable potential for using this approach to constrain the number of positional glycoform isomers present in different biological preparations toward the end of discovery of functionally relevant structures.
RESUMEN
Nitrite has recently emerged as an important bioactive molecule, capable of conferring cardioprotection and a variety of other benefits in the cardiovascular system and elsewhere. The mechanisms by which it accomplishes these functions remain largely unclear. To characterize the dose response and corresponding cardiac sequelae of transient systemic elevations of nitrite, we assessed the time course of oxidation/nitros(yl)ation, as well as the metabolomic, proteomic, and associated functional changes in rat hearts following acute exposure to nitrite in vivo. Transient systemic nitrite elevations resulted in: (1) rapid formation of nitroso and nitrosyl species; (2) moderate short-term changes in cardiac redox status; (3) a pronounced increase in selective manifestations of long-term oxidative stress as evidenced by cardiac ascorbate oxidation, persisting long after changes in nitrite-related metabolites had normalized; (4) lasting reductions in glutathione oxidation (GSSG/GSH) and remarkably concordant nitrite-induced cardioprotection, which both followed a complex dose-response profile; and (5) significant nitrite-induced protein modifications (including phosphorylation) revealed by mass spectrometry-based proteomic studies. Altered proteins included those involved in metabolism (eg, aldehyde dehydrogenase 2, ubiquinone biosynthesis protein CoQ9, lactate dehydrogenase B), redox regulation (eg, protein disulfide isomerase A3), contractile function (eg, filamin-C), and serine/threonine kinase signaling (eg, protein kinase A R1alpha, protein phosphatase 2A A R1-alpha). Thus, brief elevations in plasma nitrite trigger a concerted cardioprotective response characterized by persistent changes in cardiac metabolism, redox stress, and alterations in myocardial signaling. These findings help elucidate possible mechanisms of nitrite-induced cardioprotection and have implications for nitrite dosing in therapeutic regimens.
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Cardiotónicos/farmacología , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Nitritos/farmacología , Estrés Oxidativo/efectos de los fármacos , Proteoma/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Masculino , Oxidación-Reducción/efectos de los fármacos , Proteómica/métodos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a Top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-Orbitrap(™)) with automated sample introduction; automated data analysis is performed by our own software algorithm (BUPID topdown).The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle-skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. The fragment ion containing the amino acid substitution or modification can be identified by searching for a peak exhibiting the mass shift observed in the protein mass profile. This fragment ion can then be selected for MS/MS analysis in the ion trap to yield sequence information permitting the identification of the variant. Substantial sequence coverage has been obtained in this manner. This strategy allows for a stepwise MS/MS analysis of the protein structure. The sequence information obtained can be supplemented with whole protein NSD fragmentation and MS/MS analysis of specific protein charge states. The analyses of variant forms of TTR and hemoglobin are presented to illustrate the potential of the method.
RESUMEN
AIMS: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. RESULTS: Exposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. CONCLUSIONS: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.
Asunto(s)
Proteínas Nucleares , Sirtuina 1 , Animales , Apoptosis , Células Endoteliales/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno , Ratones , Oxidación-Reducción , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
The extreme pathological diversity of non-Hodgkin's lymphomas has made their accurate histological assessment difficult. New diagnostics and treatment modalities are urgently needed for these lymphomas, particularly in drug development for cancer-specific targets. Previously, we showed that a subset of B cell lymphoma, diffuse large B cell lymphoma, may be characterized by two major, orthogonal axes of gene expression: one set of transcripts that is differentially expressed between resting and proliferating, nonmalignant cells (ie, a "proliferative signature") and another set that is expressed only in proliferating malignant cells (ie, a "cancer signature"). A differential proteomic analysis of B cell proliferative states, similar to previous transcriptional profiling analyses, holds great promise either to reveal novel factors that participate in lymphomagenesis or to define biomarkers of onset or progression. Here, we use a murine model of diffuse large B cell lymphoma to conduct unbiased two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic analyses of malignant proliferating B cells and tissue-matched, normal resting, or normal proliferating cells. We show that the expression patterns of particular proteins or isoforms across these states fall into eight specific trends that provide a framework to identify malignancy-associated biomarkers and potential drug targets, a signature proteome. Our results support the central hypothesis that clusters of proteins of known function represent a panel of expression markers uniquely associated with malignancy and not normal proliferation.
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Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica/métodos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Proteómica/métodos , Animales , Dermatoglifia del ADN/métodos , Electroforesis en Gel Bidimensional , Femenino , Procesamiento de Imagen Asistido por Computador , Espectrometría de Masas , RatonesRESUMEN
In systemic amyloidoses, widespread deposition of protein as amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional polyacrylamide gel electrophoresis followed by matrix-assisted laser desorption/ionization mass spectrometry and peptide mass fingerprinting to directly characterize amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having amyloidoses typed as immunoglobulin light chain or transthyretin. Striking differences in the two-dimensional gel proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for transthyretin mutations, wild-type peptides and peptides containing amyloidogenic transthyretin variants were isolated in roughly equal amounts from the same protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the amyloidogenic proteins appeared in patient samples; some of these were identified as isoforms of serum amyloid P and apolipoprotein E, proteins that have been described previously to be associated with amyloid deposits. Finally changes in the normal expression pattern of resident adipose proteins, such as down-regulation of alphaB-crystallin, peroxiredoxin 6, and aldo-keto reductase I, were observed in apparent association with the presence of amyloid, although their levels did not strictly correlate with the grade of amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel proteins or protein post-translational modifications associated with amyloid infiltration.