The cytosolic isoform of glutaredoxin 2 promotes cell migration and invasion.

BACKROUND: Cytosolic glutaredoxin 2 (Grx2c) controls axonal outgrowth and is specifically induced in many cancer cell lines. We thus hypothesized that Grx2c promotes cell motility and invasiveness. METHODS: We characterized the impact of Grx2c expression in cell culture models. We combined stable isotope labeling, phosphopeptide enrichment, and high-accuracy mass spectrometry to characterize the underlying mechanisms. RESULTS: The most prominent associations were found with actin dynamics, cellular adhesion, and receptor-mediated signal transduction, processes that are crucial for cell motility. For instance, collapsin response mediator protein 2, a protein involved in the regulation of cytoskeletal dynamics, is regulated by Grx2c through a redox switch that controls the phosphorylation state of the protein as well. Cell lines expressing Grx2c showed dramatic alterations in morphology. These cells migrated two-fold faster and gained the ability to infiltrate a collagen matrix. CONCLUSIONS: The expression of Grx2c promotes cell migration, and may negatively correlate with cancer-specific survival. GENERAL SIGNIFICANCE: Our results imply critical roles of Grx2c in cytoskeletal dynamics, cell adhesion, and cancer cell invasiveness.

Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

Nucleoredoxin-Dependent Targets and Processes in Neuronal Cells.

Nucleoredoxin (Nrx) is an oxidoreductase of the thioredoxin family of proteins. It was shown to act as a signal transducer in some pathways; however, so far, no comprehensive analysis of its regulated substrates and functions was available. Here, we used a combination of two different strategies to fill this gap. First, we analyzed the thiol-redox state of the proteome of SH-SY5Y neuroblastoma cells depleted of Nrx compared to control cells using a differential thiol-labeling technique and quantitative mass spectrometry. 171 proteins were identified with an altered redox state; 161 of these were more reduced in the absence of Nrx. This suggests functions of Nrx in the oxidation of protein thiols. Second, we utilized the active site mutant Cys208Ser of Nrx, which stabilizes a mixed disulfide intermediate with its substrates and therefore trapped interacting proteins from the mouse brain (identifying 1710 proteins) and neuronal cell culture extracts (identifying 609 proteins). Profiling of the affected biological processes and molecular functions in cells of neuronal origin suggests numerous functions of Nrx in the redox regulation of metabolic pathways, cellular morphology, and signal transduction. These results characterize Nrx as a cellular oxidase that itself may be oxidized by the formation of disulfide relays with peroxiredoxins.

Correction to: Thiol-Redox Proteomics to Study Reversible Protein Thiol Oxidations in Bacteria.

This protocol was originally published © Springer Science+Business Media, LLC, part of Springer Nature 2018, but has now been made available © The Author(s) under a CC BY 4.0 license.

Thiol-Redox Proteomics to Study Reversible Protein Thiol Oxidations in Bacteria.

Thiol-redox proteomics methods are rapidly developing tools in redox biology. These are applied to identify and quantify proteins with reversible thiol oxidations that are formed under normal growth and oxidative stress conditions inside cells. The proteins with reversible thiol oxidations are usually prepared by alkylation of reduced thiols, subsequent reduction of disulfide bonds followed by a second differential alkylation of newly released thiols. Here, we describe two methods for detection of protein S-thiolations in Gram-positive bacteria using the direct shotgun approach and the fluorescent-label thiol-redox proteomics method that have been successfully applied in our previous work.

GvmR - A Novel LysR-Type Transcriptional Regulator Involved in Virulence and Primary and Secondary Metabolism of Burkholderia pseudomallei.

Burkholderia pseudomallei is a soil-dwelling bacterium able to survive not only under adverse environmental conditions, but also within various hosts which can lead to the disease melioidosis. The capability of B. pseudomallei to adapt to environmental changes is facilitated by the large number of regulatory proteins encoded by its genome. Among them are more than 60 uncharacterized LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, disruption of gvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 genes were upregulated and 141 genes were downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1, and T6SS2), which could explain the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assembly, and syrbactin metabolism (secondary metabolite) was decreased in the mutant. On the other hand, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin, and porin genes. Quantitative real-time PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes essential for the survival in the animal host and under nutrient limitation.

Comprehensive Redox Profiling of the Thiol Proteome of Clostridium difficile.

The strictly anaerobic bacterium C. difficile has become one of the most problematic hospital acquired pathogens and a major burden for health care systems. Although antibiotics work effectively in most C. difficile infections (CDIs), their detrimental effect on the intestinal microbiome paves the way for recurrent episodes of CDI. To develop alternative, non-antibiotics-based treatment strategies, deeper knowledge on the physiology of C. difficile, stress adaptation mechanisms and regulation of virulence factors is mandatory. The focus of this work was to tackle the thiol proteome of C. difficile and its stress-induced alterations, because recent research has reported that the amino acid cysteine plays a central role in the metabolism of this pathogen. We have developed a novel cysteine labeling approach to determine the redox state of protein thiols on a global scale. Applicability of this technique was demonstrated by inducing disulfide stress using the chemical diamide. The method can be transferred to any kind of redox challenge and was applied in this work to assess the effect of bile acids on the thiol proteome of C. difficile We present redox-quantification for more than 1,500 thiol peptides and discuss the general difficulty of redox analyses of peptides possessing more than a single cysteine residue. The presented method will be especially useful not only when determining redox status, but also for providing information on protein quantity. Additionally, our comprehensive data set reveals protein cysteine sites particularly susceptible to oxidation and builds a groundwork for redox proteomics studies in C. difficile.

Staphylococcus aureus α-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells.

Exposure of cultured human airway epithelial model cells (16HBE14o-, S9) to Staphylococcus aureus α-toxin (hemolysin A, Hla) induces changes in cell morphology and cell layer integrity that are due to the inability of the cells to maintain stable cell-cell or focal contacts and to properly organize their actin cytoskeletons. The aim of this study was to identify Hla-activated signaling pathways involved in regulating the phosphorylation level of the actin-depolymerizing factor cofilin. We used recombinant wild-type hemolysin A (rHla) and a variant of Hla (rHla-H35L) that is unable to form functional transmembrane pores to treat immortalized human airway epithelial cells (16HBE14o-, S9) as well as freshly isolated human nasal tissue. Our results indicate that rHla-mediated changes in cofilin phosphorylation require the formation of functional Hla pores in the host cell membrane. Formation of functional transmembrane pores induced hypophosphorylation of cofilin at Ser3, which was mediated by rHla-induced attenuation of p21-activated protein kinase and LIM kinase activities. Because dephosphorylation of pSer3-cofilin results in activation of this actin-depolymerizing factor, treatment of cells with rHla resulted in loss of actin stress fibers from the cells and destabilization of cell shape followed by the appearance of paracellular gaps in the cell layers. Activation of protein kinase A or activation of small GTPases (Rho, Rac, Cdc42) do not seem to be involved in this response.

Modulation of MHC class I surface expression in B16F10 melanoma cells by methylseleninic acid.

The essential trace element selenium (Se) might play a role in cancer prevention as well as for cancer therapy. Its metabolite methylselenol is able to kill cells through distinct mechanisms including induction of reactive oxygen species, DNA damage and apoptosis. Since methylselenol affects innate immune responses by modulating the expression of NKG2D ligands, the aim of this study was to determine whether the methylselenol generating compound methylseleninic acid (MSA) influences the expression of the MHC class I surface antigens and growth properties thereby reverting immune escape. Treatment of B16F10 melanoma cells expressing low basal MHC class I surface antigens with dimethyldiselenide (DMDSe) and MSA, but not with selenomethionine and selenite resulted in a dose-dependent upregulation of MHC class I cell surface antigens. This was due to a transcriptional upregulation of some major components of the antigen processing machinery (APM) and the interferon (IFN) signaling pathway and accompanied by a reduced migration of B16F10 melanoma cells in the presence of MSA. Comparative "ome"-based profilings of untreated and MSA-treated melanoma cells linked the anti-oxidative response system with MHC class I antigen processing. Since MSA treatment enhanced MHC class I surface expression also on different human tumors cell lines, MSA might affect the malignant phenotype of various tumor cells by restoring MHC class I APM component expression due to an altered redox status and by partially mimicking IFN-gamma signaling thereby providing a novel mechanism for the chemotherapeutic potential of methylselenol generating Se compounds.

Real-Time Imaging of the Bacillithiol Redox Potential in the Human Pathogen Staphylococcus aureus Using a Genetically Encoded Bacilliredoxin-Fused Redox Biosensor.

AIMS: Bacillithiol (BSH) is utilized as a major thiol-redox buffer in the human pathogen Staphylococcus aureus. Under oxidative stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolation, which can be reversed by bacilliredoxins (Brx). In eukaryotes, glutaredoxin-fused roGFP2 biosensors have been applied for dynamic live imaging of the glutathione redox potential. Here, we have constructed a genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus. RESULTS: The Brx-roGFP2 biosensor showed a specific and rapid response to low levels of bacillithiol disulfide (BSSB) in vitro that required the active-site Cys of Brx. Dynamic live imaging in two methicillin-resistant S. aureus (MRSA) USA300 and COL strains revealed fast and dynamic responses of the Brx-roGFP2 biosensor under hypochlorite and hydrogen peroxide (H2O2) stress and constitutive oxidation of the probe in different BSH-deficient mutants. Furthermore, we found that the Brx-roGFP2 expression level and the dynamic range are higher in S. aureus COL compared with the USA300 strain. In phagocytosis assays with THP-1 macrophages, the biosensor was 87% oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes, indicating that antibiotics do not cause oxidative stress in S. aureus. Conclusion and Innovation: This Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be applied for dynamic live imaging of redox changes in S. aureus and other BSH-producing Firmicutes. Antioxid. Redox Signal. 26, 835-848.

Redox regulation of mitochondrial proteins and proteomes by cysteine thiol switches.

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals.

Quantitative Proteomics Reveals the Dynamics of Protein Phosphorylation in Human Bronchial Epithelial Cells during Internalization, Phagosomal Escape, and Intracellular Replication of Staphylococcus aureus.

Internalization of Staphylococcus aureus by nonprofessional phagocytic cells is a major suspected cause of persistent and difficult-to-treat infections, including pneumonia. In this study, we established an infection model with 16HBE14o- human bronchial epithelial cells and demonstrated internalization, escape from phagosomal clearance, and intracellular replication of S. aureus HG001 within the first 4 h postinfection. We used quantitative phosphoproteomics to identify characteristic signaling networks in the host at different infection stages. Although we found only minor changes in protein abundance, the infection was accompanied by highly dynamic alterations in phosphorylation events primarily in proteins that are associated with pathways of cytoskeleton dynamics, cell-cell and cell-matrix contacts, vesicle trafficking, autophagy, and GTPase signaling. Analyses of host protein kinases by kinase-substrate mapping, active regulatory site immunoblotting, and prediction algorithms highlighted known and novel host kinases with putative critical roles in S. aureus infection-accompanied signaling including FAK, PKA, PKC, and CDK. Targeted pharmacological inhibition of these kinases resulted in a significant reduction of intracellular S. aureus cells. The current study constitutes a valuable resource for better understanding the infection-relevant molecular pathomechanisms of airway cells and for developing novel host-centric anti-infective strategies for treating S. aureus infections.

Staphylococcus aureus Infection Reduces Nutrition Uptake and Nucleotide Biosynthesis in a Human Airway Epithelial Cell Line.

The Gram positive opportunistic human pathogen Staphylococcus aureus induces a variety of diseases including pneumonia. S. aureus is the second most isolated pathogen in cystic fibrosis patients and accounts for a large proportion of nosocomial pneumonia. Inside the lung, the human airway epithelium is the first line in defence with regard to microbial recognition and clearance as well as regulation of the immune response. The metabolic host response is, however, yet unknown. To address the question of whether the infection alters the metabolome and metabolic activity of airway epithelial cells, we used a metabolomics approach. The nutrition uptake by the human airway epithelial cell line A549 was monitored over time by proton magnetic resonance spectroscopy (¹H-NMR) and the intracellular metabolic fingerprints were investigated by gas chromatography and high performance liquid chromatography (GC-MS) and (HPLC-MS). To test the metabolic activity of the host cells, glutamine analogues and labelled precursors were applied after the infection. We found that A549 cells restrict uptake of essential nutrients from the medium after S. aureus infection. Moreover, the infection led to a shutdown of the purine and pyrimidine synthesis in the A549 host cell, whereas other metabolic routes such as the hexosamine biosynthesis pathway remained active. In summary, our data show that the infection with S. aureus negatively affects growth, alters the metabolic composition and specifically impacts the de novo nucleotide biosynthesis in this human airway epithelial cell model.

Proteomic discovery of host kinase signaling in bacterial infections.

Protein phosphorylation catalyzed by protein kinases acts as a reversible molecular switch in signal transduction, providing a mechanism for the control of protein function in cellular processes. During microbial infection, cellular signaling essentially contributes to immune control to restrict the dissemination of invading pathogens within the host organism. However, pathogenic microbes compete for the control of host signaling to create a beneficial environment for successful invasion and infection. Although efforts to achieve a better understanding of the host-pathogen interaction and its molecular consequences have been made, there is urgent need for a comprehensive characterization of infection-related host signaling processes. System-wide and hypothesis-free analysis of phosphorylation-mediated host signaling during host-microbe interactions by mass spectrometry (MS)-based methods is not only promising in view of a greater understanding of the pathogenesis of the infection but also may result in the identification of novel host targets for preventive or therapeutic intervention. Here, we review state-of-the-art MS-based techniques for the system-wide identification and quantitation of protein phosphorylation and compare them to array-based phosphoprotein analyses. We also provide an overview of how phosphoproteomics and kinomics have contributed to our understanding of protein kinase-driven phosphorylation networks that operate during host-microbe interactions.

Resolution of Novel Pancreatic Ductal Adenocarcinoma Subtypes by Global Phosphotyrosine Profiling.

Comprehensive characterization of signaling in pancreatic ductal adenocarcinoma (PDAC) promises to enhance our understanding of the molecular aberrations driving this devastating disease, and may identify novel therapeutic targets as well as biomarkers that enable stratification of patients for optimal therapy. Here, we use immunoaffinity-coupled high-resolution mass spectrometry to characterize global tyrosine phosphorylation patterns across two large panels of human PDAC cell lines: the ATCC series (19 cell lines) and TKCC series (17 cell lines). This resulted in the identification and quantification of over 1800 class 1 tyrosine phosphorylation sites and the consistent segregation of both PDAC cell line series into three subtypes with distinct tyrosine phosphorylation profiles. Subtype-selective signaling networks were characterized by identification of subtype-enriched phosphosites together with pathway and network analyses. This revealed that the three subtypes characteristic of the ATCC series were associated with perturbations in signaling networks associated with cell-cell adhesion and epithelial-mesenchyme transition, mRNA metabolism, and receptor tyrosine kinase (RTK) signaling, respectively. Specifically, the third subtype exhibited enhanced tyrosine phosphorylation of multiple RTKs including the EGFR, ERBB3 and MET. Interestingly, a similar RTK-enriched subtype was identified in the TKCC series, and 'classifier' sites for each series identified using Random Forest models were able to predict the subtypes of the alternate series with high accuracy, highlighting the conservation of the three subtypes across the two series. Finally, RTK-enriched cell lines from both series exhibited enhanced sensitivity to the small molecule EGFR inhibitor erlotinib, indicating that their phosphosignature may provide a predictive biomarker for response to this targeted therapy. These studies highlight how resolution of subtype-selective signaling networks can provide a novel taxonomy for particular cancers, and provide insights into PDAC biology that can be exploited for improved patient management.

Induction of Macrophage Function in Human THP-1 Cells Is Associated with Rewiring of MAPK Signaling and Activation of MAP3K7 (TAK1) Protein Kinase.

Macrophages represent the primary human host response to pathogen infection and link the immediate defense to the adaptive immune system. Mature tissue macrophages convert from circulating monocyte precursor cells by terminal differentiation in a process that is not fully understood. Here, we analyzed the protein kinases of the human monocytic cell line THP-1 before and after induction of macrophage differentiation by using kinomics and phosphoproteomics. When comparing the macrophage-like state with the monocytic precursor, 50% of the kinome was altered in expression and even 71% of covered kinase phosphorylation sites were affected. Kinome rearrangements are for example characterized by a shift of overrepresented cyclin-dependent kinases associated with cell cycle control in monocytes to calmodulin-dependent kinases and kinases involved in proinflammatory signaling. Eventually, we show that monocyte-to-macrophage differentiation is associated with major rewiring of mitogen-activated protein kinase signaling networks and demonstrate that protein kinase MAP3K7 (TAK1) acts as the key signaling hub in bacterial killing, chemokine production and differentiation. Our study proves the fundamental role of protein kinases and cellular signaling as major drivers of macrophage differentiation and function. The finding that MAP3K7 is central to macrophage function suggests MAP3K7 and its networking partners as promising targets in host-directed therapy for macrophage-associated disease.

Quantitative phosphotyrosine profiling of patient-derived xenografts identifies therapeutic targets in pediatric leukemia.

Activating mutations in tyrosine kinases (TKs) drive pediatric high-risk acute lymphoblastic leukemia (ALL) and confer resistance to standard chemotherapy. Therefore, there is urgent need to characterize dysregulated TK signaling axes in patients with ALL and identify actionable kinase targets for the development of therapeutic strategies. Here, we present the first study to quantitatively profile TK activity in xenografted patient biopsies of high-risk pediatric ALL. We integrated a quantitative phosphotyrosine profiling method with 'spike-in' stable isotope labeling with amino acids in cell culture (SILAC) and quantified 1394 class I phosphorylation sites in 16 ALL xenografts. Moreover, hierarchical clustering of phosphotyrosine sites could accurately classify these leukemias into either B or T-cell lineages with the high-risk early T-cell precursor (ETP) and Ph-like ALL clustering as a distinct group. Furthermore, we validated this approach by using specific kinase pathway inhibitors to perturb ABL1, FLT3, and JAK TK signaling in four xenografted patient samples. By quantitatively assessing the tyrosine phosphorylation status of activated kinases in xenograft models of ALL, we were able to identify and validate clinically relevant targets. Therefore, this study highlights the application and potential of phosphotyrosine profiling for identifying clinically relevant kinase targets in leukemia.

A multi-omics approach identifies key hubs associated with cell type-specific responses of airway epithelial cells to staphylococcal alpha-toxin.

Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways.

INTRODUCTION: Although aberrant tyrosine kinase signalling characterises particular breast cancer subtypes, a global analysis of tyrosine phosphorylation in mouse models of breast cancer has not been undertaken to date. This may identify conserved oncogenic pathways and potential therapeutic targets. METHODS: We applied an immunoaffinity/mass spectrometry workflow to three mouse models: murine stem cell virus-Neu, expressing truncated Neu, the rat orthologue of human epidermal growth factor receptor 2, Her2 (HER2); mouse mammary tumour virus-polyoma virus middle T antigen (PyMT); and the p53-/- transplant model (p53). Pathways and protein-protein interaction networks were identified by bioinformatics analysis. Molecular mechanisms underpinning differences in tyrosine phosphorylation were characterised by Western blot analysis and array comparative genomic hybridisation. The functional role of mesenchymal-epithelial transition factor (Met) in a subset of p53-null tumours was interrogated using a selective tyrosine kinase inhibitor (TKI), small interfering RNA (siRNA)-mediated knockdown and cell proliferation assays. RESULTS: The three models could be distinguished on the basis of tyrosine phosphorylation signatures and signalling networks. HER2 tumours exhibited a protein-protein interaction network centred on avian erythroblastic leukaemia viral oncogene homologue 2 (Erbb2), epidermal growth factor receptor and platelet-derived growth factor receptor α, and they displayed enhanced tyrosine phosphorylation of ERBB receptor feedback inhibitor 1. In contrast, the PyMT network displayed significant enrichment for components of the phosphatidylinositol 3-kinase signalling pathway, whereas p53 tumours exhibited increased tyrosine phosphorylation of Met and components or regulators of the cytoskeleton and shared signalling network characteristics with basal and claudin-low breast cancer cells. A subset of p53 tumours displayed markedly elevated cellular tyrosine phosphorylation and Met expression, as well as Met gene amplification. Treatment of cultured p53-null cells exhibiting Met amplification with a selective Met TKI abrogated aberrant tyrosine phosphorylation and blocked cell proliferation. The effects on proliferation were recapitulated when Met was knocked down using siRNA. Additional subtypes of p53 tumours exhibited increased tyrosine phosphorylation of other oncogenes, including Peak1/SgK269 and Prex2. CONCLUSION: This study provides network-level insights into signalling in the breast cancer models utilised and demonstrates that comparative phosphoproteomics can identify conserved oncogenic signalling pathways. The Met-amplified, p53-null tumours provide a new preclinical model for a subset of triple-negative breast cancers.

Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o- under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o- cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.