RESUMEN
Cellular contractility is governed by a control system of proteins that integrates internal and external cues to drive diverse shape change processes. This contractility controller includes myosin II motors, actin crosslinkers and protein scaffolds, which exhibit robust and cooperative mechanoaccumulation. However, the biochemical interactions and feedback mechanisms that drive the controller remain unknown. Here, we use a proteomics approach to identify direct interactors of two key nodes of the contractility controller in the social amoeba Dictyostelium discoideum: the actin crosslinker cortexillin I and the scaffolding protein IQGAP2. We highlight several unexpected proteins that suggest feedback from metabolic and RNA-binding proteins on the contractility controller. Quantitative in vivo biochemical measurements reveal direct interactions between myosin II and cortexillin I, which form the core mechanosensor. Furthermore, IQGAP1 negatively regulates mechanoresponsiveness by competing with IQGAP2 for binding the myosin II-cortexillin I complex. These myosin II-cortexillin I-IQGAP2 complexes are pre-assembled into higher-order mechanoresponsive contractility kits (MCKs) that are poised to integrate into the cortex upon diffusional encounter coincident with mechanical inputs.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Dictyostelium/metabolismo , Proteínas de Microfilamentos/metabolismo , Miosina Tipo II/metabolismo , Proteínas Protozoarias/metabolismo , Actinas/genética , Citoesqueleto/genética , Dictyostelium/genética , Proteínas de Microfilamentos/genética , Miosina Tipo II/genética , Proteínas Protozoarias/genéticaRESUMEN
Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes encoding proteins that enable cells to adapt to reduced O2 availability. Proteins encoded by HIF-1 target genes play a central role in mediating physiological processes that are dysregulated in cancer and heart disease. These diseases are also characterized by increased production of cyclic adenosine monophosphate (cAMP), the allosteric activator of cAMP-dependent protein kinase A (PKA). Using glutathione S-transferase pull-down, coimmunoprecipitation, and mass spectrometry analyses, we demonstrated that PKA interacts with HIF-1α in HeLa cervical carcinoma cells and rat cardiomyocytes. PKA phosphorylated Thr(63) and Ser(692) on HIF-1α in vitro and enhanced HIF transcriptional activity and target gene expression in HeLa cells and rat cardiomyocytes. PKA inhibited the proteasomal degradation of HIF-1α in an O2-independent manner that required the phosphorylation of Thr(63) and Ser(692) and was not affected by prolyl hydroxylation. PKA also stimulated the binding of the coactivator p300 to HIF-1α to enhance its transcriptional activity and counteracted the inhibitory effect of asparaginyl hydroxylation on the association of p300 with HIF-1α. Furthermore, increased cAMP concentrations enhanced the expression of HIF target genes encoding CD39 and CD73, which are enzymes that convert extracellular adenosine 5'-triphosphate to adenosine, a molecule that enhances tumor immunosuppression and reduces heart rate and contractility. These data link stimuli that promote cAMP signaling, HIF-1α-dependent changes in gene expression, and increased adenosine, all of which contribute to the pathophysiology of cancer and heart disease.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Transcripción Genética , 5'-Nucleotidasa/metabolismo , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , AMP Cíclico/metabolismo , Progresión de la Enfermedad , Proteínas Ligadas a GPI/metabolismo , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Terapia de Inmunosupresión , Ratones , Miocitos Cardíacos/metabolismo , Fosforilación , Unión ProteicaRESUMEN
Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.
Asunto(s)
Separación Celular/métodos , Glicoproteínas/análisis , Inmunofenotipificación/métodos , Proteínas de la Membrana/análisis , Células Madre Pluripotentes/metabolismo , Proteómica/métodos , Animales , Células Cultivadas , Receptor gp130 de Citocinas/análisis , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Espectrometría de Masas , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Microscopía Confocal , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Teratoma/metabolismo , Teratoma/patologíaRESUMEN
The current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem cell line, R1. Proteins from whole-cell lysates were subjected either to 2-D-LC, or 1-DE, or were unfractionated prior to enzymatic digestion and subsequent analysis by MS. The results yielded 1895 identified non-redundant proteins and, for 128 of these, the specific isoform could be determined based on detection of an isoform-specific peptide. When compared with two previously published proteomic studies that used the same cell line, the current study reveals 612 new proteins.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Animales , Línea Celular , Bases de Datos de Proteínas , Ratones , Células Madre Pluripotentes/metabolismoRESUMEN
Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and beta-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mioblastos/metabolismo , Proteoma/química , Proteómica/métodos , Animales , Acuaporina 1/química , Sitios de Unión , Diferenciación Celular , Membrana Celular/metabolismo , Glicoproteínas/química , Glicosilación , Ratones , Modelos Biológicos , Sarcoglicanos/químicaRESUMEN
High abundance proteins in serum and plasma (e.g., albumin) are routinely removed during proteomic sample processing as they can mask lower abundance proteins and peptides of biological/clinical interest. A common method of albumin depletion is based on immunoaffinity capture, and many immunoaffinity devices are designed for multiple uses. In this case, it is critical that the albumin captured on the affinity matrix is stripped from the column prior to regeneration of the matrix and processing of subsequent samples, to ensure no carryover and that maximal binding sites are available for subsequent samples. The current study examines the ability of a manufacturer's protocol to remove the proteins and peptides captured by an immunoaffinity spin column. The data presented in the current work illustrate the difficulty in completely removing albumin from the immunoaffinity device, and consequently, may explain the variability and decreased efficiency shown for this device in previous studies. In summary, the current data present important considerations for the implementation of multiple-use immunoaffinity devices for processing subsequent clinical samples in a proteomic workflow.
Asunto(s)
Cromatografía de Afinidad/instrumentación , Inmunoquímica/métodos , Proteómica , Albúmina Sérica , Cromatografía Líquida de Alta Presión , Equipo Reutilizado , Humanos , Albúmina Sérica/química , Albúmina Sérica/aislamiento & purificaciónRESUMEN
Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity of most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine, which is highly transported into proliferating cells, is a major source of energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here we report that the c-Myc (hereafter referred to as Myc) oncogenic transcription factor, which is known to regulate microRNAs and stimulate cell proliferation, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase, in human P-493 B lymphoma cells and PC3 prostate cancer cells. This leads to upregulation of glutamine catabolism. Glutaminase converts glutamine to glutamate, which is further catabolized through the tricarboxylic acid cycle for the production of ATP or serves as substrate for glutathione synthesis. The unique means by which Myc regulates glutaminase uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species homeostasis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glutaminasa/metabolismo , Glutamina/metabolismo , MicroARNs/metabolismo , Mitocondrias/enzimología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Regiones no Traducidas 3'/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
BACKGROUND: The pyruvic acid analog 3-bromopyruvate (3BrPA) is an alkylating agent known to induce cancer cell death by blocking glycolysis. The anti-glycolytic effect of 3BrPA is considered to be the inactivation of glycolytic enzymes. Yet, there is a lack of experimental documentation on the direct interaction of 3BrPA with any of the suggested targets during its anticancer effect. METHODS AND RESULTS: In the current study, using radiolabeled ((14)C) 3BrPA in multiple cancer cell lines, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the primary intracellular target of 3BrPA, based on two-dimensional (2D) gel electrophoretic autoradiography, mass spectrometry and immunoprecipitation. Furthermore, in vitro enzyme kinetic studies established that 3BrPA has marked affinity to GAPDH. Finally, Annexin V staining and active caspase-3 immunoblotting demonstrated that apoptosis was induced by 3BrPA. CONCLUSION: GAPDH pyruvylation by 3BrPA affects its enzymatic function and is the primary intracellular target in 3BrPA mediated cancer cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/patología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Neoplasias Hepáticas/patología , Piruvatos/farmacología , Animales , Apoptosis/fisiología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/enzimología , Electroforesis en Gel Bidimensional , Humanos , Immunoblotting , Inmunoprecipitación , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/enzimología , Conejos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
This study assesses the ability of a novel family of machine learning algorithms to identify changes in relative protein expression levels, measured using 2-D DIGE data, which support accurate class prediction. The analysis was done using a training set of 36 total cellular lysates comprised of six normal and three cancer biological replicates (the remaining are technical replicates) and a validation set of four normal and two cancer samples. Protein samples were separated by 2-D DIGE and expression was quantified using DeCyder-2D Differential Analysis Software. The relative expression reversal (RER) classifier correctly classified 9/9 training biological samples (p<0.022) as estimated using a modified version of leave one out cross validation and 6/6 validation samples. The classification rule involved comparison of expression levels for a single pair of protein spots, tropomyosin isoforms and alpha-enolase, both of which have prior association as potential biomarkers in cancer. The data was also analyzed using algorithms similar to those found in the extended data analysis package of DeCyder software. We propose that by accounting for sources of within- and between-gel variation, RER classifiers applied to 2-D DIGE data provide a useful approach for identifying biomarkers that discriminate among protein samples of interest.
Asunto(s)
Biomarcadores de Tumor/análisis , Electroforesis en Gel Bidimensional/métodos , Proteínas/análisis , Algoritmos , Línea Celular , Proteínas de Unión al ADN/análisis , Humanos , Datos de Secuencia Molecular , Reconocimiento de Normas Patrones Automatizadas , Fosfopiruvato Hidratasa/análisis , Isoformas de Proteínas/análisis , Reproducibilidad de los Resultados , Programas Informáticos , Tropomiosina/análisis , Proteínas Supresoras de Tumor/análisisRESUMEN
Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.