Ralph Schlapbach

Community proteogenomics reveals insights into the physiology of phyllosphere bacteria

Aerial plant surfaces represent the largest biological interface on Earth and provide essential services as sites of carbon dioxide fixation, molecular oxygen release, and primary biomass production. Rather than existing as axenic organisms, plants are colonized by microorganisms that affect both their health and growth. To gain insight into the physiology of phyllosphere bacteria under in situ conditions, we performed a culture-independent analysis of the microbiota associated with leaves of soybean, clover, and Arabidopsis thaliana plants using a metaproteogenomic approach. We found a high consistency of the communities on the 3 different plant species, both with respect to the predominant community members (including the alphaproteobacterial genera Sphingomonas and Methylo bacterium) and with respect to their proteomes. Observed known proteins of Methylobacterium were to a large extent related to the ability of these bacteria to use methanol as a source of carbon and energy. A remarkably high expression of various TonB-dependent receptors was observed for Sphingomonas. Because these outer membrane proteins are involved in transport processes of various carbohydrates, a particularly large substrate utilization pattern for Sphingomonads can be assumed to occur in the phyllosphere. These adaptations at the genus level can be expected to contribute to the success and coexistence of these 2 taxa on plant leaves. We anticipate that our results will form the basis for the identification of unique traits of phyllosphere bacteria, and for uncovering previously unrecorded mechanisms of bacteria-plant and bacteria-bacteria relationships.

A high-quality catalog of the Drosophila melanogaster proteome.

Understanding how proteins and their complex interaction networks convert the genomic information into a dynamic living organism is a fundamental challenge in biological sciences. As an important step towards understanding the systems biology of a complex eukaryote, we cataloged 63% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis-driven experimentation feedback loops, whereby data collection is guided by statistical analysis of prior data. We show that high-quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models. We also present experimentally identified proteotypic peptides matching ∼50% of D. melanogaster gene models. This library of proteotypic peptides should enable fast, targeted and quantitative proteomic studies to elucidate the systems biology of this model organism.

Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

A system-wide analysis of protein phosphorylation in yeast reveals robustness in the network of kinases and phosphatases. Holistic Approach Protein kinases and phosphatases make attractive targets for therapies. Although various such enzymes have been characterized individually in vitro, an understanding of their roles in vivo, in the context of the entire network of kinases and phosphatases, is lacking. Indeed, inadequate knowledge of the downstream, indirect consequences of targeting a particular enzyme has led to the discontinuation of potential therapies. Bodenmiller et al. (listen to the accompanying Podcast) individually targeted most of the kinases and phosphatases in yeast, and they performed phosphoproteomic analysis of the effects of these deletions or mutations on the cellular phosphorylation network. They found that the network was surprisingly robust to perturbations in individual enzymes and that a large number of changes occurred in phosphoproteins that were not direct substrates of the targeted kinase or phosphatase. This approach should serve as a starting point toward understanding the complexity of phosphorylation regulation in yeast and other organisms. The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery—and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

Qualitative and Quantitative Analyses of Protein Phosphorylation in Naive and Stimulated Mouse Synaptosomal Preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

PhosphoPep--a phosphoproteome resource for systems biology research in Drosophila Kc167 cells.

The ability to analyze and understand the mechanisms by which cells process information is a key question of systems biology research. Such mechanisms critically depend on reversible phosphorylation of cellular proteins, a process that is catalyzed by protein kinases and phosphatases. Here, we present PhosphoPep, a database containing more than 10 000 unique high‐confidence phosphorylation sites mapping to nearly 3500 gene models and 4600 distinct phosphoproteins of the Drosophila melanogaster Kc167 cell line. This constitutes the most comprehensive phosphorylation map of any single source to date. To enhance the utility of PhosphoPep, we also provide an array of software tools that allow users to browse through phosphorylation sites on single proteins or pathways, to easily integrate the data with other, external data types such as protein–protein interactions and to search the database via spectral matching. Finally, all data can be readily exported, for example, for targeted proteomics approaches and the data thus generated can be again validated using PhosphoPep, supporting iterative cycles of experimentation and analysis that are typical for systems biology research.

TNF‐α and IFN‐γ render microglia sensitive to Fas ligand‐induced apoptosis by induction of Fas expression and down‐regulation of Bcl‐2 and Bcl‐xL

The immune response in the central nervous system (CNS) involves microglial cells which represent intraparenchymal antigen‐presenting cells (APC). To control immune effector mechanisms it may be required to induce apoptosis of APC and thereby limit reactivation of T cells that have invaded the CNS. In the present study we investigated the susceptibility of primary murine microglia and of the murine microglial cell line BV‐2 to undergo Fas‐mediated apoptosis. Whereas resting microglia are resistant to Fas ligand (FasL) treatment, induction of FasL‐mediated apoptosis was achieved by treatment with TNF‐α or IFN‐γ. The effect of these cytokines was paralleled by up‐regulation of Fas expression and down‐regulation of Bcl‐2 and Bcl‐xL but not Bax. Activation of microglia by TNF‐α and IFN‐γ was also accompanied by increased amounts of mRNA for the apoptosis inhibit FLIP, an effect which did not protect the cells from FasL‐induced apoptosis. The FasL‐induced cell death pathway in microglia involves reactive oxygen intermediates because the antioxidants N‐acetyl‐cysteine and glutathione interfere with induction of apoptosis. Surprisingly, microglia constitutively express FasL on the cell surface. However, blocking of endogenous Fas‐FasL inter action with Fas‐Fc fusion protein did not enhance the survival of microglia, excluding the possibility of suicide or fratricide mechanisms. By their expression of FasL and their TNF‐α / IFN‐γ‐dependent sensitivity to the pro‐apoptotic effect of exogenous FasL, microglial cells may influence the course of T cell‐mediated diseases of the CNS.

Myc-mediated repression of microRNA-34a promotes high-grade transformation of B-cell lymphoma by dysregulation of FoxP1

Gastric marginal zone B-cell lymphoma of MALT type (MALT lymphoma) arises in the context of chronic inflammation induced by the bacterial pathogen Helicobacter pylori. Although generally considered an indolent disease, MALT lymphoma may transform to gastric diffuse large B-cell lymphoma (gDLBCL) through mechanisms that remain poorly understood. By comparing microRNA expression profiles of gastric MALT lymphoma and gDLBCL, we have identified a signature of 27 deregulated microRNAs(miRNAs) that share the characteristic of being transcriptionally repressed by Myc. Myc overexpression was consequently detected in 80% of gDLBCL but only 20% of MALT lymphomas spotted on a tissue microarray. A highly similar signature of Myc-repressed miRNAs was further detected in nodal DLBCL. Small interfering RNA-mediated knock-down of Myc blocked proliferation of DLBCL cell lines. Of the Myc-repressed miRNAs down-regulated in malignant lymphoma, miR-34a showed the strongest antiproliferative properties when overexpressed in DLBCL cells. We could further attribute miR-34as tumor-suppressive effects to deregulation of its target FoxP1. FoxP1 overexpression was detected in gDLBCL but not in gastric MALT lymphoma; FoxP1 knock-down efficiently blocked DLBCL proliferation. In conclusion, our results elucidate a novel Myc- and FoxP1-dependent pathway of malignant transformation and suggest miR-34a replacement therapy as a promising strategy in lymphoma treatment.

Characterization of the Interactome of the Human MutL Homologues MLH1, PMS1, and PMS2

Postreplicative mismatch repair (MMR) involves the concerted action of at least 20 polypeptides. Although the minimal human MMR system has recently been reconstituted in vitro, genetic evidence from different eukaryotic organisms suggests that some steps of the MMR process may be carried out by more than one protein. Moreover, MMR proteins are involved also in other pathways of DNA metabolism, but their exact role in these processes is unknown. In an attempt to gain novel insights into the function of MMR proteins in human cells, we searched for interacting partners of the MutL homologues MLH1 and PMS2 by tandem affinity purification and of PMS1 by large scale immunoprecipitation. In addition to proteins known to interact with the MutL homologues during MMR, mass spectrometric analyses identified a number of other polypeptides, some of which bound to the above proteins with very high affinity. Whereas some of these interactors may represent novel members of the mismatch repairosome, others appear to implicate the MutL homologues in biological processes ranging from intracellular transport through cell signaling to cell morphology, recombination, and ubiquitylation.

Implementation and evaluation of relative and absolute quantification in shotgun proteomics with label-free methods.

Tandem mass spectrometry allows for fast protein identification in a complex sample. As mass spectrometers get faster, more sensitive and more accurate, methods were devised by many academic research groups and commercial suppliers that allow protein research also in quantitative respect. Since label-free methods are an attractive alternative to labeling approaches for proteomics researchers seeking for accurate quantitative results we evaluated several open-source analysis tools in terms of performance on two reference data sets, explicitly generated for this purpose. In this paper we present an implementation, T3PQ (Top 3 Protein Quantification), of the method suggested by Silva and colleagues for LC-MS(E) applications and we demonstrate its applicability to data generated on FT-ICR instruments acquiring in data dependent acquisition (DDA) mode. In order to validate this method and to show its usefulness also for absolute protein quantification, we generated a reference data set of a sample containing four different proteins with known concentrations. Furthermore, we compare three other label-free quantification methods using a complex biological sample spiked with a standard protein in defined concentrations. We evaluate the applicability of these methods and the quality of the results in terms of robustness and dynamic range of the spiked-in protein as well as other proteins also detected in the mixture. We discuss drawbacks of each method individually and consider crucial points for experimental designs. The source code of our implementation is available under the terms of the GNU GPLv3 and can be downloaded from sourceforge (http://fqms.svn.sourceforge.net/svnroot/fqms). A tarball containing the data used for the evaluation is available on the FGCZ web server (http://fgcz-data.uzh.ch/public/T3PQ.tgz).

TGFβ directs gene expression of activated microglia to an anti-inflammatory phenotype strongly focusing on chemokine genes and cell migratory genes

In experimental autoimmune encephalomyelitis, the acute phase of the disease is produced by T‐helper lymphocyte type 1 (TH1), which produces mainly TNFα and IFNγ. Recovery from the disease is mediated by T‐helper lymphocyte types 2 and 3 (TH2/TH3), which, among other cytokines, produce transforming growth factor β (TGFβ). To address the influence of TGFβ on TH1‐induced gene expression, microarray technology was used on murine primary microglial cells stimulated with IFNγ and TNFα in the absence or presence of TGFβ. The resulting data from an investigation of up to 5,500 genes provided the notion that TGFβ prevents the induction of a proinflammatory gene program within microglia exposed to a TH1 milieu. TH1 cytokines upregulated 175 genes comprising cytokine, chemokine, and genes involved in host response to infection and the TNFα/IFNγ intracellular signaling pathway. It is observed that TGFβ inhibits expression of 25% of the TNFα/IFNγ‐induced genes and a further 66 TNFα/IFNγ‐independent genes. The focus of TGFβ inhibition is observed to be directed in genes involved in chemotaxis (IL‐15, CXCL1, CXCL2, CCL3, CCL4, CCL5, CCL9), chemokine receptors (CCR5, CCR9), LIF receptor, and FPR2, and on genes mediating cell migration (MMP9, MMP13, MacMARCKS, endothelin receptor B, Ena/VASP, Gas7), apoptosis (FAS, TNF, TNF receptor, caspase‐1 and ‐11), and host response to infection (toll‐like receptor 6, Mx‐1, and MARCO). Taken collectively, the data strongly suggest that one of the main effects of TGFβ is to impair cell entry into the CNS and to hinder migration of microglia in the CNS parenchyma.