RESUMEN
Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).
Asunto(s)
Anticuerpos/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/normas , Proteínas de Neoplasias/inmunología , Neoplasias/metabolismo , Proteómica/métodos , Humanos , Inmunoprecipitación , Espectrometría de Masas , Proteínas de Neoplasias/metabolismo , Neoplasias/inmunología , Células Tumorales CultivadasRESUMEN
We report a tool for the analysis of subcellular proteomics data, called MetaMass, based on the use of standardized lists of subcellular markers. We analyzed data from 11 studies using MetaMass, mapping the subcellular location of 5,970 proteins. Our analysis revealed large variations in the performance of subcellular fractionation protocols as well as systematic biases in protein annotation databases. The Excel and R versions of MetaMass should enhance transparency and reproducibility in subcellular proteomics.
Asunto(s)
Metaanálisis como Asunto , Proteínas/metabolismo , Proteómica/métodos , Fracciones Subcelulares/metabolismo , Algoritmos , Animales , Biomarcadores/metabolismo , Células Cultivadas , Células Madre Embrionarias/metabolismo , Ratones , Proteómica/estadística & datos numéricosRESUMEN
Atherosclerotic plaque development involves multiple extra- and intra-cellular signals engaging cells from the immune system and from the vasculature. Pro-inflammatory pathways activated by interferon gamma (IFNγ) and toll-like receptor 4 (TLR4) ligands are profoundly involved in plaque formation and have been shown to involve cross-talk in all atheroma-interacting cell types leading to increased activation of signal transducer and activator of transcription-1 (STAT1) and elevated expression of pro-inflammatory mediators. Here we demonstrate that in Gene Expression Omnibus repository (GEO) deposited microarray datasets, obtained from human coronary and carotid atherosclerotic plaques, a significant increase in expression of pro-inflammatory and immunomodulatory genes can be detected. Moreover, increased expression of multiple chemokines, adhesion molecules and matrix-remodeling molecules was commonly detected in both plaque types and correlated with the presence of putative STAT1 binding sites in their promoters, suggesting strong involvement of STAT1 in plaque development. We also provide evidence to suggest that STAT1-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) or STAT1-interferon-regulated factor (IRF) regulatory modules are over-represented in the promoters of these inflammatory genes, which points to a possible contribution of IFNγ and TLR4 cross-talk in the process of atherogenesis. Finally, a subset of these genes encodes for secreted proteins that could serve as a basis of a non-invasive diagnostic assay. The results of our in silico analysis in vitro provide potential evidence that STAT1-dependent IFNγ-TLR4 cross-talk plays a crucial role in coronary and carotid artery plaque development and identifies a STAT1-dependent gene signature that could represent a novel diagnostic tool to monitor and diagnose plaque progression in human atherosclerosis.
Asunto(s)
Minería de Datos , Placa Aterosclerótica/genética , Factor de Transcripción STAT1/metabolismo , Transcriptoma/genética , Enfermedades Vasculares/metabolismo , Humanos , Factores Reguladores del Interferón/metabolismo , Interferón gamma/metabolismo , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Receptor Toll-Like 4/metabolismo , Enfermedades Vasculares/genéticaRESUMEN
Inflammation plays an important role in host defenses against infectious agents and injury, but it also contributes to the pathophysiology of atherosclerosis. Signal transducer and activated transcription 1 (STAT1) has been identified as a point of convergence for the cross talk between the pro-inflammatory cytokine interferon γ (IFNγ) and the Toll-like receptor-4 (TLR4) ligand LPS in immune cells. However, there is no information available on the role of STAT1 in TLR4-mediated progression of atherosclerosis and on potential synergism between lipopolysaccharides (LPS) and IFNγ signaling in cells from the vasculature. Cultured human microvascular endothelial cells (HMECs) exposed to LPS activated STAT1 in a delayed manner that was inhibited by cycloheximide treatment. Pretreatment of HMECs as well as primary vascular smooth muscle cells (VSMCs) with IFNγ followed by LPS resulted in a significant increase in STAT1 phosphorylation compared with both factors alone. Increased STAT1 protein levels, strictly mediated by IFNγ, correlated with the augmented STAT1 phosphorylation that was absent in TLR4(-/-) cells. As assessed by PCR, Western analysis, and ELISA, this coincided with increased expression of the chemokine interferon gamma-induced protein 10 kDa (IP-10) and the adhesion molecule ICAM-1 in a TLR4-dependent manner.The STAT1-inhibitor fludarabine markedly reduced these effects as well as IFNγ and LPS-dependent adhesion of U937 cells to endothelial cells, emphasizing the potential importance of STAT1 in the integration of both signals. With the established roles of IFNγ and TLRs in atherosclerotic pathology, the STAT1-dependent signal integration between IFNγ and TLR in ECs and VSMCs in response to exogenous and endogenous atherogenic ligands could result in amplification of pro-inflammatory responses in the damaged vessel and be a novel mechanism involved in the initiation and progression of atherosclerosis.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Células Endoteliales , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Monocitos , Miocitos del Músculo Liso , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/fisiología , Antineoplásicos/farmacología , Aterosclerosis/fisiopatología , Adhesión Celular/fisiología , Línea Celular , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Expresión Génica , Humanos , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/fisiología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Fosforilación , Factor de Transcripción STAT1/genética , Receptor Toll-Like 4/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologíaRESUMEN
Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io .
Asunto(s)
Proteoma/metabolismo , Proteómica , Transducción de Señal , Animales , Fenómenos Biológicos , Fraccionamiento Celular , Células HeLa , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Presión Osmótica , Fosforilación , Fracciones Subcelulares/metabolismo , Flujo de TrabajoRESUMEN
Antibody array analysis of labeled proteomes has high throughput and is simple to perform, but validation remains challenging. Here, we used differential detergent fractionation and size exclusion chromatography in sequence for high-resolution separation of biotinylated proteins from human primary keratinocytes and leukocytes. Ninety-six sample fractions from each cell type were analyzed with microsphere-based antibody arrays and flow cytometry (microsphere affinity proteomics; MAP). Monomeric proteins and multi-molecular complexes in the cytosol, cytoplasmic organelles, membranes and nuclei were resolved as discrete peaks of antibody reactivity across the fractions. The fractionation also provided a two-dimensional matrix for assessment of specificity. Thus, antibody reactivity peaks were considered to represent specific binding if the position in the matrix was in agreement with published information about i) subcellular location, ii) size of the intended target, and iii) cell type-dependent variation in protein expression. Similarities in the reactivity patterns of either different antibodies to the same protein or antibodies to similar proteins were used as additional supporting evidence. This approach provided validation of several hundred proteins and identification of monomeric proteins and protein complexes. High-resolution MAP solves many of the problems associated with obtaining specificity with immobilized antibodies and a protein label. Thus, laboratories with access to chromatography and flow cytometry can perform large-scale protein analysis on a daily basis. This opens new possibilities for cell biology research in dermatology and validation of antibodies.
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Queratinocitos/metabolismo , Leucocitos/metabolismo , Análisis por Matrices de Proteínas/métodos , Proteínas/metabolismo , Adulto , Anticuerpos , Biotinilación , Cromatografía en Gel , Citometría de Flujo , Expresión Génica , Humanos , Queratinocitos/citología , Leucocitos/citología , Microesferas , Persona de Mediana Edad , Cultivo Primario de Células , Unión Proteica , Proteínas/inmunología , Proteómica , Piel/citología , Piel/metabolismoRESUMEN
It is known that the micromotion between implant and bone inhibits direct bone growth either on or into implant surfaces in vivo. Nevertheless, biocompatibility tests in vitro of biomaterials for bone/implant interfaces are mainly performed under static conditions. This work describes a dynamic, in vitro experimental simulation of the effect of mutual, small-scale implant surface-tissue displacement on adhered cells. Disks of simulated tissue (PVP hydrogel) were subjected to cyclic micromotion ranging from 0 at the center to 1000 microm at the periphery at approximately 13 Hz, relative to biomaterial surfaces or tissue culture polystyrene controls populated with human osteoblasts in standard tissue culture plate wells. The effect of the interfacial micromotion on the number of cells remaining attached was quantitated by XTT assay. The activity level of the remaining cells was determined by an alkaline phosphatase assay, and cell stress was evaluated by nitrogen assay. Significantly more cells (ANOVA) became detached from similarly prepared surfaces of titanium, hydroxyapatite, and alumina compared to the polystyrene control, and detachment from alumina was greater than for the other two materials. The activity of the remaining attached cells was lower as compared to the static (no micromotion) control but not significantly different among the biomaterials. All nitrogen assays were negative, suggesting minimal cell stress occurred. The method is proposed as a useful and discriminating in vitro tool for biocompatibility studies focused on cell adhesion to biomaterials under conditions related to those which exist at the implant/bone interface in vivo, and it allows subsequent studies of the still-viable cells by other methods.
Asunto(s)
Ensayo de Materiales/métodos , Movimiento (Física) , Osteoblastos/citología , Prótesis e Implantes/efectos adversos , Materiales Biocompatibles/química , Materiales Biocompatibles/normas , Huesos/citología , Adhesión Celular , Recuento de Células , Técnicas de Cultivo de Célula , Supervivencia Celular , Células Cultivadas , Humanos , Mecánica , Modelos Biológicos , Prótesis e Implantes/normas , Estrés FisiológicoRESUMEN
Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.
Asunto(s)
Aterosclerosis/genética , Factor de Transcripción STAT1/genética , Receptor Toll-Like 4/genética , Aterosclerosis/patología , Células Sanguíneas , Quimiocina CXCL9/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/biosíntesis , Interferón gamma/biosíntesis , Interferón gamma/genética , FN-kappa B/biosíntesis , FN-kappa B/genética , Fosforilación , Factor de Transcripción STAT1/biosíntesis , Transducción de Señal/genética , Receptor Toll-Like 4/biosíntesisRESUMEN
Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors.
Asunto(s)
Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Células Cultivadas , Simulación por Computador , Óxidos S-Cíclicos/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Interferón-alfa/farmacología , Interferón gamma/farmacología , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Unión Proteica , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT2/antagonistas & inhibidores , Factor de Transcripción STAT2/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Alineación de Secuencia , Vidarabina/análogos & derivados , Vidarabina/farmacología , Dominios Homologos srcRESUMEN
Atherosclerosis is characterized by early endothelial dysfunction and altered vascular smooth muscle cells (VSMCs) contractility. The forming atheroma is a site of excessive production of cytokines and inflammatory ligands by various cell types that mediate inflammation and immune responses. Key factors contributing to early stages of plaque development are IFNγ and TLR4. This review provides insight in the differential STAT1-dependent signal integration between IFNγ and TLR4 signals in vascular cells and atheroma interacting immune cells. This results in increased leukocyte attraction and adhesion and VSMC proliferation and migration, which are important characteristics of EC dysfunction and early triggers of atherosclerosis.
RESUMEN
Inflammation participates importantly in host defenses against infectious agents and injury, but it also contributes to the pathophysiology of atherosclerosis. Recruitment of blood leukocytes to the injured vascular endothelium characterizes the initiation and progression of atherosclerosis and involves many inflammatory mediators, modulated by cells of both innate and adaptive immunity. The pro-inflammatory cytokine, interferon (IFN)-γ derived from T cells, is vital for both innate and adaptive immunity and is also expressed at high levels in atherosclerotic lesions. As such IFN-γ plays a crucial role in the pathology of atherosclerosis through activation of signal transducer and activator of transcription (STAT) 1. Toll-like receptors (TLRs) are innate immune pattern recognition receptors (PRRs) expressed on a variety of cells, and thus initiate and sustain the inflammatory response in atherosclerosis. More recent studies have revealed that STAT1 is involved in the signaling events mediated by TLR4, leading to increased expression of several pro-inflammatory and pro-atherogenic mediators. By upregulating members of the Suppressors Of Cytokine Signaling (SOCS) family that regulate cellular responsiveness to immune signals, IFNγ and TLR4-activated pathways have also shown to inhibit IL-6 STAT3-dependent anti-inflammatory signaling and potentially shift IL-6 to a STAT1 activating pro-inflammatory cytokine. Consequently, STAT1 has been identified as a point of convergence for the cross-talk between the pro-atherogenic IFN-γ, TLR4 and IL-6 activated pathways in immune as well as vascular cells, as such amplifying pro-inflammatory signals. This results in augmented smooth muscle cell (SMC) and leukocyte migration, leukocyte to endothelial cell (EC) adhesion and foam cell formation, and could encompass a novel mechanism involved in the initiation and progression of atherosclerosis. Therefore, application of small inhibitory compounds that specifically interact with the SH2-phosphotyrosine pocket of STAT1, proposed here as a novel working mechanism for the known STAT1 inhibitor fludarabine, could be a promising tool in the development of a therapeutical strategy for atherosclerosis.
Asunto(s)
Aterosclerosis/terapia , Interferón gamma/inmunología , Interleucina-6/inmunología , Factor de Transcripción STAT1/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Animales , Aterosclerosis/inmunología , Humanos , Inmunidad Innata , Inflamación/inmunología , Inflamación/terapia , Músculo Liso Vascular/inmunología , Miocitos del Músculo Liso/inmunología , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT3/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunologíaRESUMEN
The prevalence of cardiovascular disease in patients with renal failure is extremely high and accounts for a large part of the morbidity and mortality. Inflammation participates importantly in host defense against infectious agents and injury, but also contributes to the pathophysiology of many diseases, including cardiovascular atherosclerosis, which is a main problem in patients with renal failure. Recruitment of blood leukocytes to the injured vascular endothelium characterizes the initiation and progression of atherosclerosis and involves many inflammatory mediators, modulated by cells of both innate and adaptive immunity. Excessive inflammatory and immune responses, communicated by these different cell types, are driven by inflammatory cytokines that promote associated tissue damage if cytokine signaling pathways remain unregulated. Thus, pathways capable of suppressing proinflammatory cytokine signaling hold the potential to limit life-threatening cardiovascular events caused by atherogenesis. Suppressor of cytokine signaling (SOCS) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. Accumulating evidence supports the idea that dysregulation of cytokine signaling by differential SOCS expression is involved in the pathogenesis of various inflammatory, and immunological diseases, including atherosclerosis. Based on recent observations, in which SOCS expression levels are profoundly altered in kidney disease, we discuss the possibilities of SOCS as new intracellular markers of inflammation as well as their potential atherogenic properties in renal failure related cardiovascular disease.