RESUMO
Increased triglyceride accumulation in adipocytes caused by a misbalance between energy intake and energy consumption, results in increased adipocyte size, excess adipose tissue, increased body weight and ultimately, obesity. It is well established that enlarged adipocytes exhibit malfunctions that contribute to whole body insulin resistance, a key factor for the development of type 2 diabetes. However, the underlying molecular cause for dysfunctional adipocyte behavior and signaling is poorly understood. Since the adipocyte cell surface proteome, or surfaceome, represents the cellular signaling gateway to the microenvironment, we studied the contribution of this subproteome to adipocyte malfunctions in obesity. By using the chemoproteomic Cell Surface Capture (CSC) technology, we established surfaceome maps of primary adipocytes derived from different mouse models for metabolic disorders. Relative quantitative comparison between these surfaceome maps revealed a set of cell surface glycoproteins with modulated location-specific abundance levels. RNAi mediated targeting of a subset of the detected obesity modulated cell surface glycoproteins in an in vitro model system provided functional evidence for their role in adiponectin secretion and the lipolytic activity of adipocytes. Thus, we conclude that the identified cell surface glycoproteins which exhibit obesity induced abundance changes and impact adipocyte function at the same time contribute to adipocyte malfunction in obesity. The regulation of their concerted activities could improve adipocyte function in obesity.
Assuntos
Adipócitos/patologia , Glicoproteínas de Membrana/metabolismo , Obesidade/patologia , Adipócitos/metabolismo , Adiponectina/metabolismo , Animais , Células Cultivadas , Lipólise , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismoRESUMO
Glioblastoma is the most common primary brain tumor in adults with low average survival time after diagnosis. In order to improve glioblastoma treatment, new drug-accessible targets need to be identified. Cell surface glycoproteins are prime drug targets due to their accessibility at the surface of cancer cells. To overcome the limited availability of suitable antibodies for cell surface protein detection, we performed a comprehensive mass spectrometric investigation of the glioblastoma surfaceome. Our combined cell surface capturing analysis of primary ex vivo glioblastoma cell lines in combination with established glioblastoma cell lines revealed 633 N-glycoproteins, which vastly extends the known data of surfaceome drug targets at subcellular resolution. We provide direct evidence of common glioblastoma cell surface glycoproteins and an approximate estimate of their abundances, information that could not be derived from genomic and/or transcriptomic glioblastoma studies. Apart from our pharmaceutically valuable repertoire of already and potentially drug-accessible cell surface glycoproteins, we built a mass-spectrometry-based toolbox enabling directed, sensitive, and repetitive glycoprotein measurements for clinical follow-up studies. The included Skyline Glioblastoma SRM assay library provides an elevated starting point for parallel testing of the abundance level of the detected glioblastoma surfaceome members in future drug perturbation experiments.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoma/metabolismo , Idoso , Sequência de Aminoácidos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Glicosilação , Humanos , Masculino , Glicoproteínas de Membrana/química , Pessoa de Meia-Idade , Dados de Sequência Molecular , Terapia de Alvo Molecular , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteômica , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
During maturation, erythropoietic cells extrude their nuclei but retain their ability to respond to oxidant stress by tightly regulating protein translation. Several studies have reported microRNA-mediated regulation of translation during terminal stages of erythropoiesis, even after enucleation. In the present study, we performed a detailed examination of the endogenous microRNA machinery in human red blood cells using a combination of deep sequencing analysis of microRNAs and proteomic analysis of the microRNA-induced silencing complex. Among the 197 different microRNAs detected, miR-451a was the most abundant, representing more than 60% of all read sequences. In addition, miR-451a and its known target, 14-3-3ζ mRNA, were bound to the microRNA-induced silencing complex, implying their direct interaction in red blood cells. The proteomic characterization of endogenous Argonaute 2-associated microRNA-induced silencing complex revealed 26 cofactor candidates. Among these cofactors, we identified several RNA-binding proteins, as well as motor proteins and vesicular trafficking proteins. Our results demonstrate that red blood cells contain complex microRNA machinery, which might enable immature red blood cells to control protein translation independent of de novo nuclei information.
Assuntos
Eritrócitos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Proteômica/métodos , Complexo de Inativação Induzido por RNA/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Massas , MicroRNAs/metabolismo , Ligação Proteica , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Reticulócitos/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.
Assuntos
Espectrometria de Massas/métodos , Proteínas de Membrana/química , Proteômica/métodos , Animais , Linhagem Celular , Bases de Dados de Proteínas , Humanos , CamundongosRESUMO
Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS-one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a biotin tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., insulin) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.
Assuntos
Biotina/análogos & derivados , Hidrazinas/química , Ligantes , Biologia Molecular/métodos , Receptores de Superfície Celular/análise , Succinimidas/química , Biotina/química , Receptores ErbB/metabolismo , Glicina/química , Glicosilação , Humanos , Espectrometria de Massas/métodos , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Receptor ErbB-2/análise , Receptor ErbB-2/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties--one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.
Assuntos
Especificidade de Órgãos , Receptores de Superfície Celular/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Biotina/análogos & derivados , Biotina/química , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Hidrazinas/química , Insulina/farmacologia , Ligantes , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteômica , Succinimidas/químicaRESUMO
Fetal hemoglobin, HbF (α(2)γ(2)), is the main hemoglobin synthesized up to birth, but it subsequently declines and adult hemoglobin, HbA (α(2)ß(2)), becomes predominant. Several studies have indicated that expression of the HbF subunit γ-globin might be regulated post-transcriptionally. This could be confered by â¼22-nucleotide long microRNAs that associate with argonaute proteins to specifically target γ-globin mRNAs and inhibit protein expression. Indeed, applying immunopurifications, we found that γ-globin mRNA was associated with argonaute 2 isolated from reticulocytes that contain low levels of HbF (<1%), whereas association was significantly lower in reticulocytes with high levels of HbF (90%). Comparing microRNA expression in reticulocytes from cord blood and adult blood, we identified several miRNAs that were preferentially expressed in adults, among them miRNA-96. The overexpression of microRNA-96 in human ex vivo erythropoiesis decreased γ-globin expression by 50%, whereas the knock-down of endogenous microRNA-96 increased γ-globin expression by 20%. Moreover, luciferase reporter assays showed that microRNA-96 negatively regulates expression of γ-globin in HEK293 cells, which depends on a seedless but highly complementary target site located within the coding sequence of γ-globin. Based on these results we conclude that microRNA-96 directly suppresses γ-globin expression and thus contributes to HbF regulation.