Matthias Selbach

Publications (31)297.63 Total impact

• Article: Argonaute2 regulates the pancreatic β-cell secretome.

  Sudhir G Tattikota, Matthias D Sury, Thomas Rathjen, Hans-Hermann Wessels, Amit K Pandey, Xintian You, Clinton Becker, Wei Chen, Matthias Selbach, Matthew N Poy
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  ABSTRACT: Argonaute2 (Ago2) is an established component of the microRNA-induced silencing complex. Similar to miR-375 loss-of-function studies, inhibition of Ago2 in the pancreatic β-cell resulted in enhanced insulin release underlining the relationship between these two genes. Moreover, as the most abundant microRNA in pancreatic endocrine cells, miR-375 was also observed to be enriched in Ago2-associated complexes. Both Ago2 and miR-375 regulate the pancreatic β-cell secretome and we identified using quantitative mass spectrometry the enhanced release of a set of proteins or secretion signature in response to a glucose stimulus using the murine β-cell line, MIN6. In addition, loss of Ago2 resulted in the increased expression of miR-375 target genes, including gephyrin and ywhaz. These targets positively contribute to exocytosis indicating they may mediate the functional role of both miR-375 and Ago proteins in the pancreatic β-cell by influencing the secretory pathway. This study specifically addresses the role of Ago2 in the systemic release of proteins from β-cells and highlights the contribution of the microRNA pathway to the function of this cell type.
  Molecular &amp Cellular Proteomics 01/2013; · 7.40 Impact Factor
• Article: Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease.

  André Möller, Sheila Q Xie, Fabian Hosp, Benjamin Lang, Hemali P Phatnani, Sonya James, Francisco Ramirez, Gayle B Collin, Jürgen K Naggert, M Madan Babu, Arno L Greenleaf, Matthias Selbach, Ana Pombo
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  ABSTRACT: RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.
  Molecular &amp Cellular Proteomics 12/2011; 11(6):M111.011767. · 7.40 Impact Factor
• Article: Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR.

  Svetlana Lebedeva, Marvin Jens, Kathrin Theil, Björn Schwanhäusser, Matthias Selbach, Markus Landthaler, Nikolaus Rajewsky
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  ABSTRACT: Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ∼26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.
  Molecular cell 06/2011; 43(3):340-52. · 14.61 Impact Factor
• Source

  Available from: umd.edu

  Article: Global quantification of mammalian gene expression control.

  Björn Schwanhäusser, Dorothea Busse, Na Li, Gunnar Dittmar, Johannes Schuchhardt, Jana Wolf, Wei Chen, Matthias Selbach
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  ABSTRACT: Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.
  Nature 05/2011; 473(7347):337-42. · 36.28 Impact Factor
• Article: Analyzing protein-protein interactions by quantitative mass spectrometry.

  Florian E Paul, Fabian Hosp, Matthias Selbach
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  ABSTRACT: Since most cellular processes depend on interactions between proteins, information about protein-protein interactions (PPIs) provide valuable insights into protein function. Over the last years, quantitative affinity purification followed by mass spectrometry (q-AP-MS) has become a powerful approach to investigate PPIs in an unbiased manner. In q-AP-MS the protein of interest is biochemically enriched together with its interaction partners. In parallel, a control experiment is performed to control for non-specific binding. Quantitative mass spectrometry is then employed to compare protein levels in both samples and to exclude non-specific contaminants. Here, we provide two detailed q-AP-MS protocols for pull-downs with immobilized bait proteins or transient transfection of tagged expression constructs. We discuss benefits and limitations of q-AP-MS and highlight critical parameters that need to be considered. The protocols and background information presented here allow the reader to adapt the generic q-AP-MS strategy for a wide range of biological questions.
  Methods 03/2011; 54(4):387-95. · 4.01 Impact Factor
• Source

  Available from: edoc.mdc-berlin.de

  Article: Whole cell proteome regulation by microRNAs captured in a pulsed SILAC mass spectrometry approach.

  Olivia A Ebner, Matthias Selbach
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  ABSTRACT: Since gene expression is controlled on many different levels in a cell, capturing a comprehensive snapshot of all regulatory processes is a difficult task. One possibility to monitor effective changes within a cell is to directly quantify changes in protein synthesis, which reflects the accumulative impact of regulatory mechanisms on gene expression. Pulsed stable isotope labeling by amino acids in cell culture (pSILAC) has been shown to be a viable method to investigate de novo protein synthesis on a proteome-wide scale (Schwanhausser et al., Proteomics 9:205-209, 2009; Selbach et al., Nature 455:58-63, 2008). One application of pSILAC is to study the regulation of protein expression by microRNAs. Here, we describe how pSILAC in conjunction with shotgun mass spectrometry can assess differences in the protein profile between cells transfected with a microRNA and non-transfected cells.
  Methods in molecular biology (Clifton, N.J.) 01/2011; 725:315-31.
• Article: The SILAC fly allows for accurate protein quantification in vivo.

  Matthias D Sury, Jia-Xuan Chen, Matthias Selbach
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  ABSTRACT: Stable isotope labeling by amino acids in cell culture (SILAC) is widely used to quantify protein abundance in tissue culture cells. Until now, the only multicellular organism completely labeled at the amino acid level was the laboratory mouse. The fruit fly Drosophila melanogaster is one of the most widely used small animal models in biology. Here, we show that feeding flies with SILAC-labeled yeast leads to almost complete labeling in the first filial generation. We used these "SILAC flies" to investigate sexual dimorphism of protein abundance in D. melanogaster. Quantitative proteome comparison of adult male and female flies revealed distinct biological processes specific for each sex. Using a tudor mutant that is defective for germ cell generation allowed us to differentiate between sex-specific protein expression in the germ line and somatic tissue. We identified many proteins with known sex-specific expression bias. In addition, several new proteins with a potential role in sexual dimorphism were identified. Collectively, our data show that the SILAC fly can be used to accurately quantify protein abundance in vivo. The approach is simple, fast, and cost-effective, making SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics.
  Molecular &amp Cellular Proteomics 10/2010; 9(10):2173-83. · 7.40 Impact Factor
• Article: The versatility of Helicobacter pylori CagA effector protein functions: The master key hypothesis.

  Steffen Backert, Nicole Tegtmeyer, Matthias Selbach
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  ABSTRACT: Several bacterial pathogens inject virulence proteins into host target cells that are substrates of eukaryotic tyrosine kinases. One of the key examples is the Helicobacter pylori CagA effector protein which is translocated by a type-IV secretion system. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinases. CagA then binds to and activates/inactivates multiple signaling proteins in a phosphorylation-dependent and phosphorylation-independent manner. A recent proteomic screen systematically identified eukaryotic binding partners of the EPIYA phosphorylation sites of CagA and similar sites in other bacterial effectors by high-resolution mass spectrometry. Individual phosphorylation sites recruited a surprisingly high number of interaction partners suggesting that each phosphorylation site can interfere with many downstream pathways. We now count 20 reported cellular binding partners of CagA, which represents the highest quantitiy among all yet known virulence-associated effector proteins in the microbial world. This complexity generates a highly remarkable and puzzling scenario. In addition, the first crystal structure of CagA provided us with new information on the function of this important virulence determinant. Here we review the recent advances in characterizing the multiple binding signaling activities of CagA. Injected CagA can act as a 'master key' that evolved the ability to highjack multiple host cell signalling cascades, which include the induction of membrane dynamics, actin-cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and anti-apoptotic nuclear responses. The discovery that different pathogens use this common strategy to subvert host cell functions suggests that more examples will emerge soon.
  Helicobacter 06/2010; 15(3):163-76. · 3.15 Impact Factor
• Source

  Available from: Henning Lauterbach

  Article: Quantitative proteomics reveals subset-specific viral recognition in dendritic cells.

  Christian A Luber, Jürgen Cox, Henning Lauterbach, Ben Fancke, Matthias Selbach, Jurg Tschopp, Shizuo Akira, Marian Wiegand, Hubertus Hochrein, Meredith O'Keeffe, Matthias Mann
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  ABSTRACT: Dendritic cell (DC) populations consist of multiple subsets that are essential orchestrators of the immune system. Technological limitations have so far prevented systems-wide accurate proteome comparison of rare cell populations in vivo. Here, we used high-resolution mass spectrometry-based proteomics, combined with label-free quantitation algorithms, to determine the proteome of mouse splenic conventional and plasmacytoid DC subsets to a depth of 5,780 and 6,664 proteins, respectively. We found mutually exclusive expression of pattern recognition pathways not previously known to be different among conventional DC subsets. Our experiments assigned key viral recognition functions to be exclusively expressed in CD4(+) and double-negative DCs. The CD8alpha(+) DCs largely lack the receptors required to sense certain viruses in the cytoplasm. By avoiding activation via cytoplasmic receptors, including retinoic acid-inducible gene I, CD8alpha(+) DCs likely gain a window of opportunity to process and present viral antigens before activation-induced shutdown of antigen presentation pathways occurs.
  Immunity 02/2010; 32(2):279-89. · 21.64 Impact Factor
• Source

  Available from: Daniel Besser

  Article: The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development.

  Katja S Grossmann, Hagen Wende, Florian E Paul, Cyril Cheret, Alistair N Garratt, Sandra Zurborg, Konstantin Feinberg, Daniel Besser, Herbert Schulz, Elior Peles, Matthias Selbach, Walter Birchmeier, Carmen Birchmeier
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  ABSTRACT: The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.
  Proceedings of the National Academy of Sciences 09/2009; 106(39):16704-9. · 9.68 Impact Factor
• Source

  Available from: PubMed Central

  Article: Novel insights into proteomic technologies and their clinical perspective.

  Gunnar Dittmar, Matthias Selbach
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  ABSTRACT: A report on the Proteomic Forum 2009 conference, Berlin, Germany, 29 March to 2 April, 2009.
  Genome Medicine 06/2009; 1(5):53.
• Source

  Available from: Sabine Brandt

  Article: Host cell interactome of tyrosine-phosphorylated bacterial proteins.

  Matthias Selbach, Florian Ernst Paul, Sabine Brandt, Patrick Guye, Oliver Daumke, Steffen Backert, Christoph Dehio, Matthias Mann
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  ABSTRACT: Selective interactions between tyrosine-phosphorylated proteins and their cognate, SH2-domain containing ligands play key roles in mammalian signal transduction. Several bacterial pathogens use secretion systems to inject tyrosine kinase substrates into host cells. Upon phosphorylation, these effector proteins recruit cellular binding partners to manipulate host cell functions. So far, only a few interaction partners have been identified. Here we report the results of a proteomic screen to systematically identify binding partners of all known tyrosine-phosphorylated bacterial effectors by high-resolution mass spectrometry. We identified 39 host interactions, all mediated by SH2 domains, including four of the five already known interaction partners. Individual phosphorylation sites recruited a surprisingly high number of cellular interaction partners suggesting that individual phosphorylation sites can interfere with multiple cellular signaling pathways. Collectively, our results indicate that tyrosine-phosphorylation sites of bacterial effector proteins have evolved as versatile interaction modules that can recruit a rich repertoire of cellular SH2 domains.
  Cell host & microbe 05/2009; 5(4):397-403. · 13.02 Impact Factor
• Source

  Available from: Juergen Cox

  Article: A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.

  Jürgen Cox, Ivan Matic, Maximiliane Hilger, Nagarjuna Nagaraj, Matthias Selbach, Jesper V Olsen, Matthias Mann
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  ABSTRACT: MaxQuant is a quantitative proteomics software package designed for analyzing large mass spectrometric data sets. It is specifically aimed at high-resolution mass spectrometry (MS) data. Currently, Thermo LTQ-Orbitrap and LTQ-FT-ICR instruments are supported and Mascot is used as a search engine. This protocol explains step by step how to use MaxQuant on stable isotope labeling by amino acids in cell culture (SILAC) data obtained with double or triple labeling. Complex experimental designs, such as time series and drug-response data, are supported. A standard desktop computer is sufficient to fulfill the computational requirements. The workflow has been stress tested with more than 1,000 liquid chromatography/mass spectrometry runs in a single project. In a typical SILAC proteome experiment, hundreds of thousands of peptides and thousands of proteins are automatically and reliably quantified. Additional information for identified proteins, such as Gene Ontology, domain composition and pathway membership, is provided in the output tables ready for further bioinformatics analysis. The software is freely available at the MaxQuant home page.
  Nature Protocol 02/2009; 4(5):698-705. · 8.36 Impact Factor
• Article: Global analysis of cellular protein translation by pulsed SILAC.

  Björn Schwanhäusser, Manfred Gossen, Gunnar Dittmar, Matthias Selbach
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  ABSTRACT: Current methods for system-wide gene expression analysis detect changes in mRNA abundance, but neglect regulation at the level of translation. Pulse labeling with stable isotopes has been used to measure protein turnover rates, but this does not directly provide information about translation rates. Here, we developed pulsed stable isotope labeling by amino acids in cell culture (pSILAC) with two heavy isotope labels to directly quantify protein translation on a proteome-wide scale. We applied the method to cellular iron homeostasis as a model system and demonstrate that it can confidently identify proteins that are translationally regulated by iron availability.
  Proteomics 01/2009; 9(1):205-9. · 4.43 Impact Factor
• Article: Complex kinase requirements for Chlamydia trachomatis Tarp phosphorylation.

  Adrian Mehlitz, Sebastian Banhart, Simone Hess, Matthias Selbach, Thomas F Meyer
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  ABSTRACT: Chlamydia trachomatis translocates the effector protein Tarp (translocated actin-recruiting phosphoprotein) into the host cell cytoplasm where it is quickly tyrosine phosphorylated. Abl and Src kinases have been implicated in Tarp phosphorylation; however, we observed that the situation is more complex. Chemical inhibition of Src family kinases confirmed a role for these kinases in Tarp phosphorylation. Infection of Src, Yes, Fyn (SYF)-deficient cells showed a dampened, but incompletely blocked, Tarp phosphorylation. Inhibition of Abl in an SYF background still did not completely block Tarp phosphorylation. Consequently, we tested additional kinases and found that Syk, but not Btk or Jak2, is a potent kinase of Tarp in vitro. Inhibition of Syk in an SYF background further blocked Tarp phosphorylation. Under these conditions, inclusion formation still proceeded normally. These data reveal a highly promiscuous substrate property of Tarp and set the stage for further functional characterization of Tarp phosphorylation during host cell infection.
  FEMS Microbiology Letters 11/2008; 289(2):233-40. · 2.04 Impact Factor
• Source

  Available from: ncku.edu.tw

  Article: Widespread changes in protein synthesis induced by microRNAs.

  Matthias Selbach, Björn Schwanhäusser, Nadine Thierfelder, Zhuo Fang, Raya Khanin, Nikolaus Rajewsky
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  ABSTRACT: Animal microRNAs (miRNAs) regulate gene expression by inhibiting translation and/or by inducing degradation of target messenger RNAs. It is unknown how much translational control is exerted by miRNAs on a genome-wide scale. We used a new proteomic approach to measure changes in synthesis of several thousand proteins in response to miRNA transfection or endogenous miRNA knockdown. In parallel, we quantified mRNA levels using microarrays. Here we show that a single miRNA can repress the production of hundreds of proteins, but that this repression is typically relatively mild. A number of known features of the miRNA-binding site such as the seed sequence also govern repression of human protein synthesis, and we report additional target sequence characteristics. We demonstrate that, in addition to downregulating mRNA levels, miRNAs also directly repress translation of hundreds of genes. Finally, our data suggest that a miRNA can, by direct or indirect effects, tune protein synthesis from thousands of genes.
  Nature 10/2008; 455(7209):58-63. · 36.28 Impact Factor
• Article: Role of type IV secretion in Helicobacter pylori pathogenesis.

  Steffen Backert, Matthias Selbach
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  ABSTRACT: Helicobacter pylori is a highly successful human-specific gastric pathogen that colonizes more than half the world's population. Infection with this bacterium can induce gastric pathologies ranging from chronic gastritis to peptic ulcers and even cancer. Virulent H. pylori isolates harbour the cag (cytotoxin-associated genes) pathogenicity island, a 40 kb stretch of DNA that encodes components of a type IV secretion system (T4SS). This T4SS forms a pilus for the injection of virulence factors into host target cells such as the CagA oncoprotein. This is accomplished by a specialized adhesin of the pilus surface, the CagL protein, which binds to and activates host cell integrins for subsequent delivery of CagA across the host cell membrane. Injected CagA becomes tyrosine-phosphorylated by Src and Abl family kinases and mimics a host cell protein in binding and activation of multiple signalling factors. Here we review the recent advances in the characterization of phosphorylation-dependent and phosphorylation-independent signalling activities of CagA and the T4SS which include the induction of membrane dynamics, actin cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and anti-apoptotic nuclear responses. The contribution of these signalling cascades to H. pylori pathogenesis is discussed.
  Cellular Microbiology 05/2008; 10(8):1573-81. · 5.46 Impact Factor
• Article: The Helicobacter pylori CagA protein disrupts matrix adhesion of gastric epithelial cells by dephosphorylation of vinculin.

  Stefan Moese, Matthias Selbach, Volker Brinkmann, Alexander Karlas, Beatrice Haimovich, Steffen Backert, Thomas F Meyer
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  ABSTRACT: Helicobacter pylori colonizes the human stomach, contributing to or causing several diseases. Translocation of the CagA bacterial protein into gastric epithelial cells has been linked to an increased risk of peptic ulcer disease and gastric carcinoma. Upon translocation, CagA is tyrosine phosphorylated by Src family kinases (SFKs), which themselves become inactivated via a negative feedback loop. Here, we show that tyrosine-phosphorylated CagA disrupts adhesion of AGS cells to the extracellular matrix. Owing to the inactivation of c-Src via CagA interaction, vinculin is dephosphorylated at tyrosine residues, 100 and 1065, by corresponding phosphatases. Vinculin dephosphorylation disturbs the interaction and recruitment of the actin-related protein 2/3 (Arp2/3) complex by p34Arc, resulting in a reduction of focal adhesion complexes. These defects can be mimicked by downregulating vinculin using RNA interference in non-infected cells. Tyrosine dephosphorylation of vinculin results in severe cellular deficiencies in cell-matrix adhesion, cell spreading and wound repair. We hypothesize that CagA-mediated inactivation of vinculin is a key step in the mechanism by which H. pylori induces damage to the gastric epithelium and represents an important step in disease development.
  Cellular Microbiology 06/2007; 9(5):1148-61. · 5.46 Impact Factor
• Source

  Available from: uhkt.cz

  Article: Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK).

  Matthias Selbach, Matthias Mann
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  ABSTRACT: Present screening methods for protein-protein interactions (PPIs) rely on the overexpression of artificial fusion proteins, making it difficult to assess in vivo relevance. Here we combine stable isotope labeling with amino acids in cell culture (SILAC), RNA interference (RNAi), coimmunoprecipitation and quantitative mass-spectrometry analysis to detect cellular interaction partners of endogenous proteins in mammalian cells with very high confidence. We used this screen to identify interaction partners of beta-catenin and Cbl.
  Nature Methods 01/2007; 3(12):981-3. · 19.28 Impact Factor
• Source

  Available from: ncu.edu.tw

  Article: Robust Salmonella metabolism limits possibilities for new antimicrobials.

  Daniel Becker, Matthias Selbach, Claudia Rollenhagen, Matthias Ballmaier, Thomas F Meyer, Matthias Mann, Dirk Bumann
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  ABSTRACT: New antibiotics are urgently needed to control infectious diseases. Metabolic enzymes could represent attractive targets for such antibiotics, but in vivo target validation is largely lacking. Here we have obtained in vivo information about over 700 Salmonella enterica enzymes from network analysis of mutant phenotypes, genome comparisons and Salmonella proteomes from infected mice. Over 400 of these enzymes are non-essential for Salmonella virulence, reflecting extensive metabolic redundancies and access to surprisingly diverse host nutrients. The essential enzymes identified were almost exclusively associated with a small subgroup of pathways, enabling us to perform a nearly exhaustive screen. Sixty-four enzymes identified as essential in Salmonella are conserved in other important human pathogens, but almost all belong to metabolic pathways that are inhibited by current antibiotics or that have previously been considered for antimicrobial development. Our comprehensive in vivo analysis thus suggests a shortage of new metabolic targets for broad-spectrum antibiotics, and draws attention to some previously known but unexploited targets.
  Nature 04/2006; 440(7082):303-7. · 36.28 Impact Factor