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Martinsried, Germany

Schaab C.,KINAXO Biotechnologies
Methods in molecular biology (Clifton, N.J.) | Year: 2011

Regulation of protein phosphorylation plays an important role in many cellular processes, particularly in signal transduction. Diseases such as cancer and inflammation are often linked to aberrant signaling pathways. Mass spectrometry-based methods allow monitoring the phosphorylation status in an unbiased and quantitative manner. The analysis of this data requires the application of advanced statistical methods, some of which can be borrowed from the gene expression analysis field. Nevertheless, these methods have to be enhanced or complemented by new methods. After reviewing the key concepts of phosphoproteomics and some major data analysis methods, these tools are applied to a real-world data set. Source


Schmid A.B.,TU Munich | Lagleder S.,TU Munich | Lagleder S.,King Abdulaziz University | Grawert M.A.,TU Munich | And 13 more authors.
EMBO Journal | Year: 2012

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo. © 2012 European Molecular Biology Organization. Source


Klammer M.,KINAXO Biotechnologies | Godl K.,KINAXO Biotechnologies | Tebbe A.,KINAXO Biotechnologies | Schaab C.,KINAXO Biotechnologies | Schaab C.,Max Planck Institute of Biochemistry
BMC Bioinformatics | Year: 2010

Background: Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation). Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action.Results: Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor.Conclusions: SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data. © 2010 Klammer et al; licensee BioMed Central Ltd. Source


Mausbacher N.,Max Planck Institute of Biochemistry | Mausbacher N.,TU Munich | Schreiber T.B.,Max Planck Institute of Biochemistry | Daub H.,Max Planck Institute of Biochemistry | Daub H.,KINAXO Biotechnologies
Molecular and Cellular Proteomics | Year: 2010

The lipid mediator lysophosphatidic acid (LPA) is a serum component that regulates cellular functions such as proliferation, migration, and survival via specific G protein-coupled receptors. The underlying signaling mechanisms are still incompletely understood, including those that operate at the plasma membrane to modulate cell-cell and cell-matrix interactions in LPA-promoted cell migration. To explore LPA-evoked phosphoregulation with a focus on cell surface proteins, we combined glycoproteome enrichment by immobilized lectins with SILAC-based quantitative phosphoproteomics. We performed biological replicate analyses in SCC-9 squamous cell carcinoma cells and repeatedly quantified the effect of 1.5- and 5-min LPA treatment on more than 700 distinct phosphorylations in lectin-purified proteins. We detected many regulated phosphorylation events on various types of plasma membrane proteins such as cell adhesion molecules constituting adherens junctions, desmosomes, and hemidesmosomes. Several of these LPA-regulated phosphorylation sites have been characterized in a biological context other than G protein-coupled receptor signaling, and the transfer of this functional information suggests coordinated and multifactorial cell adhesion control in LPA-induced cell migration. Additionally, we identified LPA-mediated activation loop phosphorylation of the serine/threonine kinase Wnk1 and verified a role of Wnk1 for LPA-induced cell migration in knock-down experiments. In conclusion, the glycoproteome phosphoproteomics strategy described here sheds light on incompletely understood mechanisms in LPA-induced cell migratory behavior. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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