69 results on '"Gerrits B"'
Search Results
2. Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity
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Gerrits B, Stefano Ferrari, Daniel Hess, Karl-Heinz Klempnauer, Schubert S, Elisabetta Pani, Menigatti M, University of Zurich, and Ferrari, S
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Transcriptional Activation ,animal structures ,Blotting, Western ,Response element ,Electrophoretic Mobility Shift Assay ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,Biology ,Kidney ,Proto-Oncogene Mas ,Quail ,1307 Cell Biology ,Mice ,Proto-Oncogene Proteins c-myb ,Transactivation ,Pin1 ,Phosphorylation site ,1312 Molecular Biology ,Animals ,Humans ,Immunoprecipitation ,Point Mutation ,MYB ,RNA, Messenger ,Phosphorylation ,NIMA-Interacting Peptidylprolyl Isomerase ,Molecular Biology ,Cells, Cultured ,Reverse Transcriptase Polymerase Chain Reaction ,10061 Institute of Molecular Cancer Research ,fungi ,Kidney metabolism ,DNA ,Cell Biology ,Fibroblasts ,Peptidylprolyl Isomerase ,Molecular biology ,c-Myb ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,PIN1 ,570 Life sciences ,biology ,Ectopic expression ,U7 Systems Biology / Functional Genomics ,Transcription - Abstract
Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.
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- 2008
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3. Proteomic cell surface phenotyping of differentiating acute myeloid leukemia cells
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Hofmann, A, Gerrits, B, Schmidt, A, Bock, T, Bausch-Fluck, D, Aebersold, R, Wollscheid, B, University of Zurich, and Wollscheid, B
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1307 Cell Biology ,2403 Immunology ,1303 Biochemistry ,SX00 SystemsX.ch ,SX20 Research, Technology and Development Projects ,2720 Hematology ,570 Life sciences ,biology ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,SX06 InfectX - Published
- 2010
4. PhosphoPep--a database of protein phosphorylation sites in model organisms
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Bodenmiller B, Campbell D, Gerrits B, Lam H, Jovanovic M, Picotti P, Ralph Schlapbach, Aebersold R, University of Zurich, and Bodenmiller, B
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1502 Bioengineering ,1313 Molecular Medicine ,1305 Biotechnology ,570 Life sciences ,biology ,2402 Applied Microbiology and Biotechnology ,2204 Biomedical Engineering ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,U7 Systems Biology / Functional Genomics ,10124 Institute of Molecular Life Sciences - Published
- 2008
- Full Text
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5. Characterization of the interactome of the human MutL homologues MLH1, PMS1, and PMS
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Cannavo, E, Gerrits, B, Marra, Giancarlo, Schlapbach, R, Jiricny, J, University of Zurich, and Jiricny, J
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1307 Cell Biology ,1303 Biochemistry ,10061 Institute of Molecular Cancer Research ,1312 Molecular Biology ,570 Life sciences ,biology ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,U7 Systems Biology / Functional Genomics - Published
- 2007
6. Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast
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Bodenmiller, B., Wanka, S., Kraft, C., Urban, J., Campbell, D., Pedrioli, P.G., Gerrits, B., Picotti, P., Lam, Henry H N, Vitek, O., Brusniak, M.Y., Roschitzki, B., Zhang, C., Shokat, K.M., Schlapbach, R., Colman-Lerner, A., Nolan, G.P., Nesvizhskii, A.I., Peter, M., Loewith, R., Mering, C.V., Aebersold, R., Bodenmiller, B., Wanka, S., Kraft, C., Urban, J., Campbell, D., Pedrioli, P.G., Gerrits, B., Picotti, P., Lam, Henry H N, Vitek, O., Brusniak, M.Y., Roschitzki, B., Zhang, C., Shokat, K.M., Schlapbach, R., Colman-Lerner, A., Nolan, G.P., Nesvizhskii, A.I., Peter, M., Loewith, R., Mering, C.V., and Aebersold, R.
- Abstract
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 consequencesand 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. © 2008 American Association for the Advancement of Science.
- Published
- 2010
7. Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells - interplay of ErbB and IGF1 signalling through IGFBP3 regulation
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Worthington, J, Bertani, M, Chan, H L, Gerrits, B, Timms, J F, Worthington, J, Bertani, M, Chan, H L, Gerrits, B, and Timms, J F
- Abstract
BACKGROUND: Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression. METHODS: Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression. RESULTS: 775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signa
- Published
- 2010
8. Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster
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Goetze, S, Qeli, E, Mosimann, C, Staes, A, Gerrits, B, Roschitzki, B, Mohanty, S, Niederer, E M, Laczko, E, Timmerman, E, Lange, V, Hafen, E, Aebersold, R, Vandekerckhove, J, Basler, K; https://orcid.org/0000-0003-3534-1529, Ahrens, C H, Gevaert, K, Brunner, E, Goetze, S, Qeli, E, Mosimann, C, Staes, A, Gerrits, B, Roschitzki, B, Mohanty, S, Niederer, E M, Laczko, E, Timmerman, E, Lange, V, Hafen, E, Aebersold, R, Vandekerckhove, J, Basler, K; https://orcid.org/0000-0003-3534-1529, Ahrens, C H, Gevaert, K, and Brunner, E
- Abstract
Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.
- Published
- 2009
9. Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks
- Author
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Reiland, S, Messerli, G, Baerenfaller, K, Gerrits, B, Endler, A, Grossmann, J, Gruissem, W, Baginsky, S, Reiland, S, Messerli, G, Baerenfaller, K, Gerrits, B, Endler, A, Grossmann, J, Gruissem, W, and Baginsky, S
- Abstract
We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as well as two new motifs at the carboxyl terminus of ribosomal proteins. Using the phosphorylation motifs as a footprint for the activity of a specific kinase class, we connected the phosphoproteins with their putative kinases and constructed a chloroplast CKII phosphorylation network. The network topology suggests that CKII is a central regulator of different chloroplast functions. To provide insights into the dynamic regulation of protein phosphorylation, we analyzed the phosphoproteome at the end of day and end of night. The results revealed only minor changes in chloroplast kinase activities and phosphorylation site utilization. A notable exception was ATP synthase beta-subunit, which is found phosphorylated at CKII phosphorylation sites preferentially in the dark. We propose that ATP synthase is regulated in cooperation with 14-3-3 proteins by CKII-mediated phosphorylation of ATP synthase beta-subunit in the dark.
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- 2009
10. Neuronal Nogo-A modulates growth cone motility via Rho-GTP/LIMK1/cofilin in the unlesioned adult nervous system
- Author
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Montani, L, Gerrits, B, Gehrig, P, Kempf, A, Dimou, L, Wollscheid, B, Schwab, M E, Montani, L, Gerrits, B, Gehrig, P, Kempf, A, Dimou, L, Wollscheid, B, and Schwab, M E
- Abstract
Nogo-A has been extensively studied as a myelin-associated neurite outgrowth inhibitor in the lesioned adult central nervous system. However, its role in the intact central nervous system has not yet been clarified. Analysis of the intact adult nervous system of C57BL/6 Nogo-A knock-out (KO) versus wild-type (WT) mice by a combined two-dimensional gel electrophoresis and isotope-coded affinity tagging approach revealed regulation of cytoskeleton-, transport-, and signaling growth-related proteins, pointing to regulation of the actin cytoskeleton, the neuronal growth machinery, and in particular the Rho-GTPase/LIMK1/cofilin pathway. Nogo-A KO adult neurons showed enlarged, more motile growth cones compared with WT neurons. The phenotype was reproduced by acute in vitro neutralization of neuronal Nogo-A. LIMK1 phosphorylation was increased in Nogo-A KO growth cones, and its reduction caused the decrease of KO growth cone motility to WT levels. Our study suggests that in the unlesioned adult nervous system, neuronal Nogo-A can restrict neuronal growth through negative modulation of growth cone motility.
- Published
- 2009
11. MRE11 complex links RECQ5 helicase to sites of DNA damage
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Zheng, L, Kanagaraj, R, Mihaljevic, B, Schwendener, S, Sartori, Alessandro A; https://orcid.org/0000-0003-2770-0333, Gerrits, B, Shevelev, I, Janscak, Pavel; https://orcid.org/0000-0002-1748-7789, Zheng, L, Kanagaraj, R, Mihaljevic, B, Schwendener, S, Sartori, Alessandro A; https://orcid.org/0000-0003-2770-0333, Gerrits, B, Shevelev, I, and Janscak, Pavel; https://orcid.org/0000-0002-1748-7789
- Abstract
RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11-RAD50-NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3'-->5' exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.
- Published
- 2009
12. PhosphoPep - A phosphoproteome resource for systems biology research in Drosophila Kc167 cells
- Author
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Bodenmiller, B., Malmstrom, J., Gerrits, B., Campbell, D., Lam, Henry H N, Schmidt, A., Rinner, O., Mueller, L.N., Shannon, P.T., Pedrioli, P.G., Panse, C., Lee, H.K., Schlapbach, R., Aebersold, R., Bodenmiller, B., Malmstrom, J., Gerrits, B., Campbell, D., Lam, Henry H N, Schmidt, A., Rinner, O., Mueller, L.N., Shannon, P.T., Pedrioli, P.G., Panse, C., Lee, H.K., Schlapbach, R., and Aebersold, R.
- Abstract
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. © 2007 EMBO and Nature Publishing Group All rights reserved.
- Published
- 2007
13. PhosphoPep--a phosphoproteome resource for systems biology research in Drosophila Kc167 cells
- Author
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Bodenmiller, B, Malmstrom, J, Gerrits, B, Campbell, D, Lam, H, Schmidt, A, Rinner, O, Mueller, L N, Shannon, P T, Pedrioli, P G, Panse, C, Lee, H K, Schlapbach, R, Aebersold, R, Bodenmiller, B, Malmstrom, J, Gerrits, B, Campbell, D, Lam, H, Schmidt, A, Rinner, O, Mueller, L N, Shannon, P T, Pedrioli, P G, Panse, C, Lee, H K, Schlapbach, R, and Aebersold, R
- Abstract
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.
- Published
- 2007
14. Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations
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Munton, R P, Tweedie-Cullen, R, Livingstone-Zatchej, M, Weinandy, F, Waidelich, M, Longo, D, Gehrig, P, Potthast, F, Rutishauser, D, Gerrits, B, Panse, C, Schlapbach, R, Mansuy, I M, Munton, R P, Tweedie-Cullen, R, Livingstone-Zatchej, M, Weinandy, F, Waidelich, M, Longo, D, Gehrig, P, Potthast, F, Rutishauser, D, Gerrits, B, Panse, C, Schlapbach, R, and Mansuy, I M
- Abstract
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.
- Published
- 2007
15. Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity
- Author
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Pani, E., primary, Menigatti, M., additional, Schubert, S., additional, Hess, D., additional, Gerrits, B., additional, Klempnauer, K-H., additional, and Ferrari, S., additional
- Published
- 2008
- Full Text
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16. Design and Experimental Verification of an Exit Manifold for Improved Current Generation in an Offshore Engineering Basin
- Author
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Chin, S. N., additional, Gerrits, B., additional, and Colboume, B., additional
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- 2001
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17. Proteomic analysis in aortic media of patients with Marfan syndrome reveals increased activity of calpain 2 in aortic aneurysms.
- Author
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Pilop C, Aregger F, Gorman RC, Brunisholz R, Gerrits B, Schaffner T, Gorman JH 3rd, Matyas G, Carrel T, Frey BM, Pilop, Christiane, Aregger, Fabienne, Gorman, Robert C, Brunisholz, Rene, Gerrits, Bertran, Schaffner, Thomas, Gorman, Joseph H 3rd, Matyas, Gabor, Carrel, Thierry, and Frey, Brigitte M
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- 2009
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18. Proteomic analysis in aortic media of patients with Marfan syndrome reveals increased activity of calpain 2 in aortic aneurysms
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Pilop, C, Aregger, F, Gorman, R C, Brunisholz, R, Gerrits, B, Schaffner, T, Gorman III, J H, Mátyás, G, Carrel, T, and Frey, Brigitte M
- Subjects
3. Good health
19. PhosphoPep--a phosphoproteome resource for systems biology research in Drosophila Kc167 cells
- Author
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Bodenmiller B, Malmstrom J, Gerrits B, Campbell D, Lam H, Schmidt A, Rinner O, Ln, Mueller, Pt, Shannon, Pg, Pedrioli, Panse C, Hk, Lee, Ralph Schlapbach, Aebersold R, University of Zurich, and Aebersold, R
- Subjects
General Immunology and Microbiology ,Applied Mathematics ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,Genetics and Molecular Biology ,1100 General Agricultural and Biological Sciences ,10124 Institute of Molecular Life Sciences ,2604 Applied Mathematics ,Computational Theory and Mathematics ,1300 General Biochemistry, Genetics and Molecular Biology ,2400 General Immunology and Microbiology ,General Biochemistry ,570 Life sciences ,biology ,U7 Systems Biology / Functional Genomics ,General Agricultural and Biological Sciences ,Information Systems
20. Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster
- Author
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Goetze, S, Qeli, E, Mosimann, C, Staes, A, Gerrits, B, Roschitzki, B, Mohanty, S, Niederer, E M, Laczko, E, Timmerman, E, Lange, V, Hafen, E, Aebersold, R, Vandekerckhove, J, Basler, K, Ahrens, C H, Gevaert, K, and Brunner, E
- Subjects
3. Good health
21. Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells - interplay of ErbB and IGF1 signalling through IGFBP3 regulation
- Author
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Worthington Jenny, Bertani Mariana, Chan Hong-Lin, Gerrits Bertran, and Timms John F
- Subjects
Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Abstract Background Members of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression. Methods Gene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression. Results 775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells. Conclusions These data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3.
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- 2010
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22. Prolyl Isomerase PIN1 Regulates DNA Double-Strand Break Repair by Counteracting DNA End Resection
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Oliver Zerbe, Martin Steger, Christine Neugebauer, Bertran Gerrits, Daniela Hühn, Lorenza P. Ferretti, Shreya Paliwal, Olga Murina, Alessandro A. Sartori, Pavel Janscak, Kay Hänggi, Lorenzo Lafranchi, Giannino Del Sal, Reto Walser, University of Zurich, Sartori, Alessandro A, Steger, M, Murina, O, Hühn, D, Ferretti, Lp, Walser, R, Hänggi, K, Lafranchi, L, Neugebauer, C, Paliwal, S, Janscak, P, Gerrits, B, DEL SAL, Giannino, Zerbe, O, and Sartori, Aa
- Subjects
Genome instability ,DNA damage ,Pin1 ,1309 Developmental Biology ,1307 Cell Biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Ubiquitin ,1312 Molecular Biology ,Prolyl isomerase ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,10061 Institute of Molecular Cancer Research ,fungi ,Cell Biology ,Double Strand Break Repair ,DNA End-Joining Repair ,enzymes and coenzymes (carbohydrates) ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,PIN1 ,Cancer research ,570 Life sciences ,Homologous recombination ,DNA - Abstract
The regulation of DNA double strand break (DSB) repair by phosphorylation dependent signaling pathways is crucial for the maintenance of genome stability; however remarkably little is known about the molecular mechanisms by which phosphoryla tion controls DSB repair. Here we show that PIN1 a phosphorylation specific prolyl isomerase inter acts with key DSB repair factors and affects the rela tive contributions of homologous recombination (HR) and nonhomologous end joining (NHEJ) to DSB repair. We find that PIN1 deficient cells display reduced NHEJ due to increased DNA end resection whereas resection and HR are compromised in PIN1 overexpressing cells. Moreover we identify CtIP as a substrate of PIN1 and show that DSBs become hyperresected in cells expressing a CtIP mutant refractory to PIN1 recognition. Mechanisti cally we provide evidence that PIN1 impinges on CtIP stability by promoting its ubiquitylation and sub sequent proteasomal degradation. Collectively these data uncover PIN1 mediated isomerization as a regulatory mechanism coordinating DSB repair.
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23. Probing the "Default Network Interference Hypothesis" With EEG: An RDoC Approach Focused on Attention.
- Author
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Gerrits B, Vollebregt MA, Olbrich S, van Dijk H, Palmer D, Gordon E, Pascual-Marqui R, Kessels RPC, and Arns M
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- Adult, Brain physiopathology, Brain Waves, Data Interpretation, Statistical, Female, Humans, Male, Middle Aged, Neural Pathways physiology, Signal Processing, Computer-Assisted, Attention physiology, Attention Deficit Disorder with Hyperactivity physiopathology, Brain physiology, Electroencephalography
- Abstract
Studies have shown that specific networks (default mode network [DMN] and task positive network [TPN]) activate in an anticorrelated manner when sustaining attention. Related EEG studies are scarce and often lack behavioral validation. We performed independent component analysis (ICA) across different frequencies (source-level), using eLORETA-ICA, to extract brain-network activity during resting-state and sustained attention. We applied ICA to the voxel domain , similar to functional magnetic resonance imaging methods of analyses. The obtained components were contrasted and correlated to attentional performance (omission errors) in a large sample of healthy subjects (N = 1397). We identified one component that robustly correlated with inattention and reflected an anticorrelation of delta activity in the anterior cingulate and precuneus, and delta and theta activity in the medial prefrontal cortex and with alpha and gamma activity in medial frontal regions. We then compared this component between optimal and suboptimal attentional performers. For the latter group, we observed a greater change in component loading between resting-state and sustained attention than for the optimal performers. Following the National Institute of Mental Health Research Domain Criteria (RDoC) approach, we prospectively replicated and validated these findings in subjects with attention deficit/hyperactivity disorder. Our results provide further support for the "default mode interference hypothesis."
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- 2019
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24. Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells.
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Estoppey D, Hewett JW, Guy CT, Harrington E, Thomas JR, Schirle M, Cuttat R, Waldt A, Gerrits B, Yang Z, Schuierer S, Pan X, Xie K, Carbone W, Knehr J, Lindeman A, Russ C, Frias E, Hoffman GR, Varadarajan M, Ramadan N, Reece-Hoyes JS, Wang Q, Chen X, McAllister G, Roma G, Bouwmeester T, and Hoepfner D
- Subjects
- Cells, Cultured, Enzyme Inhibitors chemistry, Gene Deletion, Humans, Induced Pluripotent Stem Cells drug effects, Induced Pluripotent Stem Cells metabolism, Nicotinamide Phosphoribosyltransferase genetics, Pharmacogenomic Testing methods, CRISPR-Cas Systems, Drug Discovery methods, Enzyme Inhibitors pharmacology, Nicotinamide Phosphoribosyltransferase antagonists & inhibitors
- Abstract
Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.
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- 2017
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25. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.
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Ginster S, Bardet M, Unterreiner A, Malinverni C, Renner F, Lam S, Freuler F, Gerrits B, Voshol J, Calzascia T, Régnier CH, Renatus M, Nikolay R, Israël L, and Bornancin F
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, B-Cell CLL-Lymphoma 10 Protein, Blotting, Western, CARD Signaling Adaptor Proteins genetics, CARD Signaling Adaptor Proteins metabolism, Caspases genetics, Cell Line, Cells, Cultured, Electrophoresis, Polyacrylamide Gel, Guanylate Cyclase genetics, Guanylate Cyclase metabolism, HEK293 Cells, Humans, Immunoblotting, Jurkat Cells, Lymphocytes metabolism, Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein, Mutagenesis, Neoplasm Proteins genetics, Protein Isoforms genetics, Signal Transduction genetics, Signal Transduction physiology, TNF Receptor-Associated Factor 6 genetics, Ubiquitination genetics, Ubiquitination physiology, Caspases metabolism, Neoplasm Proteins metabolism, Protein Isoforms metabolism, T-Lymphocytes metabolism, TNF Receptor-Associated Factor 6 metabolism
- Abstract
The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A., Competing Interests: All co-authors work or used to work for Novartis. However, this does not alter our adherence to PLoS ONE policies on sharing data and materials.
- Published
- 2017
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26. Exploring Glucocorticoid Receptor Agonists Mechanism of Action Through Mass Cytometry and Radial Visualizations.
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Abraham Y, Gerrits B, Ludwig MG, Rebhan M, and Gubser Keller C
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- Antigens, CD metabolism, Antigens, Differentiation, Myelomonocytic metabolism, Cell Line, Humans, Leukocytes, Mononuclear drug effects, Macrophages cytology, Macrophages drug effects, Macrophages immunology, Receptors, Cell Surface metabolism, Dexamethasone pharmacology, Flow Cytometry methods, Receptors, Glucocorticoid agonists, Receptors, Glucocorticoid immunology
- Abstract
Recent advances in combining flow cytometry and mass spectrometry have led to the development of mass cytometry, allowing for the interrogation of complex cell populations on an unprecedented scale. The volumes and high dimensionality of mass cytometry data pose significant challenges in terms of analysis and visualization. We implement a method called Radviz, where multidimensional single cell data can be visualized as a projection that maintains the original dimensions and data complexity whilst facilitating analysis and visualization. This enables identification of changes in populations, focusing the analysis on the most relevant aspect of large multidimensional datasets. To highlight the potential of Radviz, we profiled peripheral mononuclear blood cells (PBMCs) from three healthy donors and showed donor-specific differences in the number and composition of cell populations. In a second study, we explored the anti-inflammatory effects of two glucocorticoid receptor (GR) ligands (cpd6 and cpd11) compared to dexamethasone (Dex) on human primary macrophages. Standard analysis at the population level showed that cpd6 and cpd11 have an overall anti-inflammatory profile similar to that of Dex. CyTOF profiling and Radviz-driven analysis at the single cell level confirmed this observation, and identified a concentration-dependent effect of cpd6 that was not detected at the population level. Altogether, Radviz combines the strengths of a projection method, reducing the dimensionality of datasets, with that of a scatter plot, where the identity of each point can be inferred from the distance to the axis. This enables the visual exploration, analysis, and interpretation of complex, high dimensional data. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)
- Published
- 2017
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27. Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.
- Author
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Geering B, Zokouri Z, Hürlemann S, Gerrits B, Ausländer D, Britschgi A, Tschan MP, Simon HU, and Fussenegger M
- Subjects
- Autophagy physiology, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Cells, Cultured, Humans, Protein Binding physiology, Apoptosis physiology, Death-Associated Protein Kinases metabolism, Fluorescence, Proteomics methods
- Abstract
Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
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- 2015
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28. Surfaceome of classical Hodgkin and non-Hodgkin lymphoma.
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Hofmann A, Thiesler T, Gerrits B, Behnke S, Sobotzki N, Omasits U, Bausch-Fluck D, Bock T, Aebersold R, Moch H, Tinguely M, and Wollscheid B
- Subjects
- Antigens, CD metabolism, Cell Line, Hodgkin Disease pathology, Humans, Immunohistochemistry, Lymphoma, Non-Hodgkin pathology, Neoplasm Proteins metabolism, Phenotype, Proteomics, Tissue Array Analysis, Hodgkin Disease metabolism, Lymphoma, Non-Hodgkin metabolism, Membrane Proteins metabolism, Proteome metabolism
- Abstract
Purpose: Classical Hodgkin lymphoma (cHL) is characterized by a low percentage of tumor cells in a background of diverse, reactive immune cells. cHL cells commonly derive from preapoptotic germinal-center B cells and are characterized by the loss of B-cell markers and the varying expression of other hematopoietic lineage markers. This phenotypic variability and the scarcity of currently available cHL-specific cell surface markers can prevent clear distinction of cHL from related lymphomas., Experimental Design: We applied the cell surface capture technology to directly measure the pool of cell surface exposed proteins in four cHL and four non-Hodgkin lymphoma (NHL) cell lines., Results: More than 1000 membrane proteins, including 178 cluster of differentiation annotated proteins, were identified and allowed the generation of lymphoma surfaceome maps. The functional properties of identified cell surface proteins enable, but also limit the information exchange of lymphoma cells with their microenvironment., Conclusion and Clinical Relevance: Selected candidate proteins with potential diagnostic value were evaluated on a tissue microarray (TMA). Primary lymphoma tissues of 126 different B cell-derived lymphoma cases were included in the TMA analysis. The TMA analysis indicated gamma-glutamyltranspeptidase 1 as a potential additional marker that can be included in a panel of markers for differential diagnosis of cHL versus NHL., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
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- 2015
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29. Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate.
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Krumova P, Reyniers L, Meyer M, Lobbestael E, Stauffer D, Gerrits B, Muller L, Hoving S, Kaupmann K, Voshol J, Fabbro D, Bauer A, Rovelli G, Taymans JM, Bouwmeester T, and Baekelandt V
- Subjects
- Amino Acid Sequence, Animals, Brain metabolism, HEK293 Cells, Humans, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2, Mice, Mice, Knockout, Microtubules metabolism, Molecular Sequence Data, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins metabolism, Parkinson Disease genetics, Parkinson Disease metabolism, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases deficiency, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism
- Abstract
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant forms of Parkinson's disease. LRRK2 is a modular, multidomain protein containing 2 enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling. Although enormous efforts have been made, the exact pathophysiologic mechanisms of LRRK2 are still not completely known. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. This approach allows the identification of substrates of 1 particular kinase in a complex cellular environment. Several of the identified peptides are involved in the regulation of microtubule (MT) dynamics, including microtubule-associating protein (MAP)/microtubule affinity-regulating kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate MT-binding proteins such as Tau, MAP2, and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic-labeling experiments in living cells confirmed MARK1 as an LRRK2 substrate. Moreover, we also showed that LRRK2 and MARK1 are interacting in eukaryotic cells. Our findings contribute to the identification of physiologic LRRK2 substrates and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology., (© FASEB.)
- Published
- 2015
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30. Scalable production in human cells and biochemical characterization of full-length normal and mutant huntingtin.
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Huang B, Lucas T, Kueppers C, Dong X, Krause M, Bepperling A, Buchner J, Voshol H, Weiss A, Gerrits B, and Kochanek S
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- Cell Line, Circular Dichroism, Doxycycline pharmacology, Humans, Huntingtin Protein, Mutation, Nerve Tissue Proteins chemistry, Phosphorylation, Protein Processing, Post-Translational, Protein Structure, Secondary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism
- Abstract
Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.
- Published
- 2015
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31. Quantitative Lys-ϵ-Gly-Gly (diGly) proteomics coupled with inducible RNAi reveals ubiquitin-mediated proteolysis of DNA damage-inducible transcript 4 (DDIT4) by the E3 ligase HUWE1.
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Thompson JW, Nagel J, Hoving S, Gerrits B, Bauer A, Thomas JR, Kirschner MW, Schirle M, and Luchansky SJ
- Subjects
- HEK293 Cells, HeLa Cells, Humans, Lysine chemistry, Mass Spectrometry, Neoplasms metabolism, Proteasome Endopeptidase Complex metabolism, Proteolysis, Proteomics, RNA Interference, Tumor Suppressor Proteins, Ubiquitin chemistry, Ubiquitin Thiolesterase metabolism, Ubiquitin-Specific Peptidase 7, Gene Expression Regulation, Neoplastic, Oligopeptides chemistry, Transcription Factors metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2014
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32. Prolyl isomerase PIN1 regulates DNA double-strand break repair by counteracting DNA end resection.
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Steger M, Murina O, Hühn D, Ferretti LP, Walser R, Hänggi K, Lafranchi L, Neugebauer C, Paliwal S, Janscak P, Gerrits B, Del Sal G, Zerbe O, and Sartori AA
- Subjects
- Carrier Proteins genetics, Carrier Proteins metabolism, Cell Line, Cyclin-Dependent Kinase 2 genetics, Cyclin-Dependent Kinase 2 metabolism, DNA Breaks, Double-Stranded, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Endodeoxyribonucleases, Genomic Instability, HEK293 Cells, Homologous Recombination, Humans, NIMA-Interacting Peptidylprolyl Isomerase, Nuclear Proteins genetics, Nuclear Proteins metabolism, Phosphorylation, Ubiquitination, DNA genetics, DNA End-Joining Repair, Peptidylprolyl Isomerase genetics, Peptidylprolyl Isomerase metabolism
- Abstract
The regulation of DNA double-strand break (DSB) repair by phosphorylation-dependent signaling pathways is crucial for the maintenance of genome stability; however, remarkably little is known about the molecular mechanisms by which phosphorylation controls DSB repair. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, interacts with key DSB repair factors and affects the relative contributions of homologous recombination (HR) and nonhomologous end-joining (NHEJ) to DSB repair. We find that PIN1-deficient cells display reduced NHEJ due to increased DNA end resection, whereas resection and HR are compromised in PIN1-overexpressing cells. Moreover, we identify CtIP as a substrate of PIN1 and show that DSBs become hyperresected in cells expressing a CtIP mutant refractory to PIN1 recognition. Mechanistically, we provide evidence that PIN1 impinges on CtIP stability by promoting its ubiquitylation and subsequent proteasomal degradation. Collectively, these data uncover PIN1-mediated isomerization as a regulatory mechanism coordinating DSB repair., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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33. Protein complex interactor analysis and differential activity of KDM3 subfamily members towards H3K9 methylation.
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Brauchle M, Yao Z, Arora R, Thigale S, Clay I, Inverardi B, Fletcher J, Taslimi P, Acker MG, Gerrits B, Voshol J, Bauer A, Schübeler D, Bouwmeester T, and Ruffner H
- Subjects
- Blotting, Western, DNA Primers genetics, HEK293 Cells, Humans, Immunoprecipitation, Mass Spectrometry, Methylation, Microscopy, Fluorescence, Oxidoreductases, N-Demethylating metabolism, Epigenesis, Genetic physiology, Histones metabolism, Jumonji Domain-Containing Histone Demethylases metabolism, Transcription Factors metabolism
- Abstract
Histone modifications play an important role in chromatin organization and gene regulation, and their interpretation is referred to as epigenetic control. The methylation levels of several lysine residues in histone tails are tightly controlled, and JmjC domain-containing proteins are one class of broadly expressed enzymes catalyzing methyl group removal. However, several JmjC proteins remain uncharacterized, gaps persist in understanding substrate recognition, and the integration of JmjC proteins into signaling pathways is just emerging. The KDM3 subfamily is an evolutionarily conserved group of histone demethylase proteins, thought to share lysine substrate specificity. Here we use a systematic approach to compare KDM3 subfamily members. We show that full-length KDM3A and KDM3B are H3K9me1/2 histone demethylases whereas we fail to observe histone demethylase activity for JMJD1C using immunocytochemical and biochemical approaches. Structure-function analyses revealed the importance of a single amino acid in KDM3A implicated in the catalytic activity towards H3K9me1/2 that is not conserved in JMJD1C. Moreover, we use quantitative proteomic analyses to identify subsets of the interactomes of the 3 proteins. Specific interactor candidates were identified for each of the three KDM3 subfamily members. Importantly, we find that SCAI, a known transcriptional repressor, interacts specifically with KDM3B. Taken together, we identify substantial differences in the biology of KDM3 histone demethylases, namely enzymatic activity and protein-protein interactions. Such comparative approaches pave the way to a better understanding of histone demethylase specificity and protein function at a systems level and are instrumental in identifying the more subtle differences between closely related proteins.
- Published
- 2013
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34. Extracellular signal-regulated kinase and glycogen synthase kinase 3β regulate gephyrin postsynaptic aggregation and GABAergic synaptic function in a calpain-dependent mechanism.
- Author
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Tyagarajan SK, Ghosh H, Yévenes GE, Imanishi SY, Zeilhofer HU, Gerrits B, and Fritschy JM
- Subjects
- Animals, Brain metabolism, Electrophysiology methods, Glycogen Synthase Kinase 3 beta, HEK293 Cells, Hippocampus metabolism, Humans, Immunoprecipitation methods, Mass Spectrometry methods, Models, Biological, Mutagenesis, Site-Directed, Neurons metabolism, Patch-Clamp Techniques, Phenotype, Plasmids metabolism, Rats, Synapses metabolism, Calpain metabolism, Carrier Proteins metabolism, Glycogen Synthase Kinase 3 metabolism, Membrane Proteins metabolism, Mitogen-Activated Protein Kinase 3 metabolism
- Abstract
Molecular mechanisms of plasticity at GABAergic synapses are currently poorly understood. To identify signaling cascades that converge onto GABAergic postsynaptic density proteins, we performed MS analysis using gephyrin isolated from rat brain and identified multiple novel phosphorylation and acetylation residues on gephyrin. Here, we report the characterization of one of these phosphoresidues, Ser-268, which when dephosphorylated leads to the formation of larger postsynaptic scaffolds. Using a combination of mutagenesis, pharmacological treatment, and biochemical assays, we identify ERK as the kinase phosphorylating Ser-268 and describe a functional interaction between residues Ser-268 and Ser-270. We further demonstrate that alterations in gephyrin clustering via ERK modulation are reflected by amplitude and frequency changes in miniature GABAergic postsynaptic currents. We unravel novel mechanisms for activity- and ERK-dependent calpain action on gephyrin, which are likely relevant in the context of cellular signaling affecting GABAergic transmission and homeostatic synaptic plasticity in pathology.
- Published
- 2013
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35. Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF).
- Author
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Reiland S, Finazzi G, Endler A, Willig A, Baerenfaller K, Grossmann J, Gerrits B, Rutishauser D, Gruissem W, Rochaix JD, and Baginsky S
- Subjects
- Amino Acid Sequence, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins analysis, Arabidopsis Proteins genetics, Chromatography, Ion Exchange, Chromatography, Liquid, Electron Transport, Light, Membrane Proteins genetics, Membrane Proteins metabolism, Molecular Sequence Data, Mutation, Phosphoproteins analysis, Phosphoproteins genetics, Phosphorylation, Photosynthesis genetics, Photosynthesis radiation effects, Photosystem I Protein Complex genetics, Photosystem I Protein Complex metabolism, Photosystem II Protein Complex genetics, Photosystem II Protein Complex metabolism, Protein Kinases genetics, Protein Serine-Threonine Kinases, Sequence Homology, Amino Acid, Spectrometry, Mass, Electrospray Ionization, Arabidopsis Proteins metabolism, Phosphoproteins metabolism, Protein Kinases metabolism, Proteomics methods
- Abstract
Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast.
- Published
- 2011
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36. Proteome profiling suggests a pro-inflammatory role for plasma cells through release of high-mobility group box 1 protein.
- Author
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Vettermann C, Castor D, Mekker A, Gerrits B, Karas M, and Jäck HM
- Subjects
- Animals, Antibody Formation genetics, Autoantigens genetics, Autoantigens metabolism, Blotting, Western, Cell Differentiation genetics, Cell Differentiation immunology, Cells, Cultured, Electrophoresis, Gel, Two-Dimensional, Eukaryotic Initiation Factors genetics, Eukaryotic Initiation Factors immunology, Eukaryotic Initiation Factors metabolism, Gene Expression Profiling, HMGB1 Protein genetics, HMGB1 Protein metabolism, Immunity, Humoral genetics, Inflammation genetics, Inflammation immunology, Inflammation metabolism, Lipid Metabolism genetics, Lipid Metabolism immunology, Lipopolysaccharides pharmacology, Mass Spectrometry, Mice, Mice, Inbred C57BL, Molecular Chaperones genetics, Molecular Chaperones immunology, Molecular Chaperones metabolism, Plasma Cells cytology, Plasma Cells metabolism, Proteasome Endopeptidase Complex genetics, Proteasome Endopeptidase Complex metabolism, Proteome metabolism, RNA, Messenger analysis, Autoantigens immunology, HMGB1 Protein immunology, Plasma Cells immunology, Proteasome Endopeptidase Complex immunology, Proteome genetics, Proteome immunology, RNA, Messenger biosynthesis
- Abstract
The final step of B-cell maturation is to differentiate into plasma cells, a process that is accompanied by gross changes in subcellular organization to enable antibody secretion. To better understand this critical step in mounting a humoral immune response, we analyzed proteome dynamics during plasma cell differentiation with combined 2-DE/MS. Thirty-two identified protein spots changed in relative abundance when lipopolysaccharide (LPS)-stimulated primary B cells differentiated into antibody-secreting plasma cells. A correlative analysis of protein and transcript abundance suggested that one third of these proteins are post-transcriptionally regulated. Apart from ER-resident chaperones, lipid metabolic enzymes, and translation initiation factors, we identified several proteins that had not been previously studied in plasma cells. Among them is the transiently upregulated proteasome activator (PA) 28γ, a component of the putative nuclear proteasome. Additionally, we discovered that the non-canonical inflammatory cytokine high-mobility group box 1 (HMG1) was released from plasma cells into the extracellular milieu. This suggests a novel role for plasma cells as pro-inflammatory mediators, which has important implications for various autoimmune diseases and chronic inflammation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2011
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37. Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin.
- Author
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Tyagarajan SK, Ghosh H, Yévenes GE, Nikonenko I, Ebeling C, Schwerdel C, Sidler C, Zeilhofer HU, Gerrits B, Muller D, and Fritschy JM
- Subjects
- Animals, Calpain metabolism, Cells, Cultured, Electrophysiology, Glycogen Synthase Kinase 3 antagonists & inhibitors, Glycogen Synthase Kinase 3 beta, Immunohistochemistry, Lithium Chloride pharmacology, Neurons metabolism, Phosphorylation, Rats, Tandem Mass Spectrometry, Carrier Proteins metabolism, Glycogen Synthase Kinase 3 metabolism, Hippocampus cytology, Membrane Proteins metabolism, Neuronal Plasticity physiology, Neurons physiology, Synapses physiology
- Abstract
Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca(2+)-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.
- Published
- 2011
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38. Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.
- Author
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Bodenmiller B, Wanka S, Kraft C, Urban J, Campbell D, Pedrioli PG, Gerrits B, Picotti P, Lam H, Vitek O, Brusniak MY, Roschitzki B, Zhang C, Shokat KM, Schlapbach R, Colman-Lerner A, Nolan GP, Nesvizhskii AI, Peter M, Loewith R, von Mering C, and Aebersold R
- Subjects
- Bayes Theorem, Chromatography, Liquid, Gene Deletion, Models, Biological, Phosphoric Monoester Hydrolases genetics, Phosphorylation, Phosphotransferases genetics, Saccharomyces cerevisiae, Species Specificity, Tandem Mass Spectrometry, Metabolic Networks and Pathways physiology, Phosphoproteins metabolism, Phosphoric Monoester Hydrolases metabolism, Phosphotransferases metabolism, Proteomics methods, Signal Transduction physiology
- Abstract
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.
- Published
- 2010
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39. Molecular characterization of a trafficking organelle: dissecting the axonal paths of calsyntenin-1 transport vesicles.
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Steuble M, Gerrits B, Ludwig A, Mateos JM, Diep TM, Tagaya M, Stephan A, Schätzle P, Kunz B, Streit P, and Sonderegger P
- Subjects
- Amyloid beta-Protein Precursor chemistry, Amyloid beta-Protein Precursor metabolism, Animals, Calcium-Binding Proteins chemistry, Electrophoresis, Polyacrylamide Gel, Endocytosis physiology, Endosomes metabolism, Hippocampus cytology, Hippocampus metabolism, Immunohistochemistry, Kinesins chemistry, Kinesins metabolism, Mice, Prosencephalon cytology, Prosencephalon metabolism, Axons metabolism, Biological Transport physiology, Calcium-Binding Proteins metabolism, Endosomes chemistry, Proteomics methods
- Abstract
Kinesin motors play crucial roles in the delivery of membranous cargo to its destination and thus for the establishment and maintenance of cellular polarization. Recently, calsyntenin-1 was identified as a cargo-docking protein for Kinesin-1-mediated axonal transport of tubulovesicular organelles along axons of central nervous system neurons. To further define the function of calsyntenin-1, we immunoisolated calsyntenin-1 organelles from murine brain homogenates and determined their proteome by MS. We found that calsyntenin-1 organelles are endowed with components of the endosomal trafficking machinery and contained the β-amyloid precursor protein (APP). Detailed biochemical analyses of calsyntenin-1 immunoisolates in conjunction with immunocytochemical colocalization studies with cultured hippocampal neurons, using endosomal marker proteins for distinct subcompartments of the endosomal pathways, indicated that neuronal axons contain at least two distinct, nonoverlapping calsyntenin-1-containing transport packages: one characterized as early-endosomal, APP positive, the other as recycling-endosomal, APP negative. We postulate that calsyntenin-1 acts as a general mediator of anterograde axonal transportation of endosomal vesicles. In this role, calsyntenin-1 may actively contribute to axonal growth and pathfinding in the developing as well as to the maintenance of neuronal polarity in the adult nervous system; further, it may actively contribute to the stabilization of APP during its anterograde axonal trajectory.
- Published
- 2010
- Full Text
- View/download PDF
40. Proteomic cell surface phenotyping of differentiating acute myeloid leukemia cells.
- Author
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Hofmann A, Gerrits B, Schmidt A, Bock T, Bausch-Fluck D, Aebersold R, and Wollscheid B
- Subjects
- Antigens, CD metabolism, Antineoplastic Agents pharmacology, Cell Differentiation drug effects, Cell Line, Tumor, Flow Cytometry, HL-60 Cells, Humans, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute pathology, Membrane Proteins metabolism, Neoplasm Proteins metabolism, Phenotype, Tandem Mass Spectrometry, Tretinoin pharmacology, Leukemia, Myeloid, Acute metabolism, Proteome metabolism, Proteomics methods
- Abstract
Immunophenotyping by flow cytometry or immunohistochemistry is a clinical standard procedure for diagnosis, classification, and monitoring of hematologic malignancies. Antibody-based cell surface phenotyping is commonly limited to cell surface proteins for which specific antibodies are available and the number of parallel measurements is limited. The resulting limited knowledge about cell surface protein markers hampers early clinical diagnosis and subclassification of hematologic malignancies. Here, we describe the mass spectrometry based phenotyping of 2 all-trans retinoic acid treated acute myeloid leukemia model systems at an unprecedented level to a depth of more than 500 membrane proteins, including 137 bona fide cell surface exposed CD proteins. This extensive view of the leukemia surface proteome was achieved by developing and applying new implementations of the Cell Surface Capturing (CSC) technology. Bioinformatic and hierarchical cluster analysis showed that the applied strategy reliably revealed known differentiation-induced abundance changes of cell surface proteins in HL60 and NB4 cells and it also identified cell surface proteins with very little prior information. The extensive and quantitative analysis of the cell surface protein landscape from a systems biology perspective will be most useful in the clinic for the improved subclassification of hematologic malignancies and the identification of new drug targets.
- Published
- 2010
- Full Text
- View/download PDF
41. Proteome analysis of fungal and bacterial involvement in leaf litter decomposition.
- Author
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Schneider T, Gerrits B, Gassmann R, Schmid E, Gessner MO, Richter A, Battin T, Eberl L, and Riedel K
- Subjects
- Cellulases metabolism, Pectobacterium carotovorum growth & development, Peptide Hydrolases metabolism, Polygalacturonase metabolism, Aspergillus nidulans enzymology, Pectobacterium carotovorum enzymology, Plant Leaves chemistry, Plant Leaves metabolism, Plant Proteins analysis, Proteome analysis
- Abstract
Fungi and bacteria are key players in the decomposition of leaf litter, but their individual contributions to the process and their interactions are still poorly known. We combined semi-quantitative proteome analyses (1-D PAGE-LC-MS/MS) with qualitative and quantitative analyses of extracellular degradative enzyme activities to unravel the respective roles of a fungus and a bacterium during litter decomposition. Two model organisms, a mesophilic Gram-negative bacterium (Pectobacterium carotovorum) and an ascomycete (Aspergillus nidulans), were grown in both, pure culture and co-culture on minimal medium containing either glucose or beech leaf litter as sole carbon source. P. carotovorum grew best in co-culture with the fungus, whereas growth of A. nidulans was significantly reduced when the bacterium was present. This observation suggests that P. carotovorum has only limited capabilities to degrade leaf litter and profits from the degradation products of A. nidulans at the expense of fungal growth. In accordance with this interpretation, our proteome analysis revealed that most of the extracellular biodegradative enzymes (i.e. proteases, pectinases, and cellulases) in the cultures with beech litter were expressed by the fungus, the bacterium producing only low levels of pectinases.
- Published
- 2010
- Full Text
- View/download PDF
42. Mapping of phosphorylation sites by LC-MS/MS.
- Author
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Gerrits B and Bodenmiller B
- Subjects
- Analytic Sample Preparation Methods, Binding Sites, Chromatography, Affinity, Databases, Protein, Dendrimers chemistry, Microspheres, Organophosphorus Compounds chemistry, Peptide Fragments analysis, Peptide Fragments isolation & purification, Phosphoproteins chemistry, Phosphorylation, Proteins chemistry, Titanium chemistry, Chromatography, Liquid methods, Proteins metabolism, Tandem Mass Spectrometry methods
- Abstract
Reversible protein phosphorylation ranks among the most important post-translational modifications that occurs in the cell. It is therefore highly relevant to elucidate the phosphorylation states of a given biological system, albeit challenging. Most notably the often low stoichiometry of phosphorylation is inherently incompatible with standard LC-MS analysis of a complex protein digest mixture, primarily due to the relative low dynamic range of current mass analyzers. Therefore a need for specific enrichment of phosphorylated peptides or proteins exists. Significant progress surrounding the biochemical analysis of reversible protein phosphorylation in the past years has led to the development of several new techniques to isolate or enrich phosphopeptides, particularly in large-scale analyses. This chapter deals with three such examples.
- Published
- 2010
- Full Text
- View/download PDF
43. Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster.
- Author
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Goetze S, Qeli E, Mosimann C, Staes A, Gerrits B, Roschitzki B, Mohanty S, Niederer EM, Laczko E, Timmerman E, Lange V, Hafen E, Aebersold R, Vandekerckhove J, Basler K, Ahrens CH, Gevaert K, and Brunner E
- Subjects
- Acetylation, Alanine genetics, Alanine metabolism, Animals, Animals, Genetically Modified, Binding Sites genetics, Blotting, Western, Carboxy-Lyases genetics, Carboxy-Lyases metabolism, Cell Line, Databases, Protein, Drosophila Proteins genetics, Drosophila melanogaster cytology, Drosophila melanogaster genetics, HeLa Cells, Humans, Immunoprecipitation, Mass Spectrometry, Mutation, Protein Biosynthesis, Serine genetics, Serine metabolism, Threonine genetics, Threonine metabolism, Transgenes genetics, Drosophila Proteins metabolism, Drosophila melanogaster metabolism, Protein Processing, Post-Translational physiology
- Abstract
Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2009
- Full Text
- View/download PDF
44. Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis.
- Author
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Huber A, Bodenmiller B, Uotila A, Stahl M, Wanka S, Gerrits B, Aebersold R, and Loewith R
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Antifungal Agents pharmacology, Cycloheximide pharmacology, Phosphorylation drug effects, Protein Binding, Protein Synthesis Inhibitors pharmacology, Protein Transport, RNA Polymerase I metabolism, RNA Polymerase III metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae metabolism, Signal Transduction, Sirolimus pharmacology, Transcription Factors metabolism, Protein Biosynthesis physiology, Protein Serine-Threonine Kinases metabolism, Proteome drug effects, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The target of rapamycin complex 1 (TORC1) is an essential multiprotein complex conserved from yeast to humans. Under favorable growth conditions, and in the absence of the macrolide rapamycin, TORC1 is active, and influences virtually all aspects of cell growth. Although two direct effectors of yeast TORC1 have been reported (Tap42, a regulator of PP2A phosphatases and Sch9, an AGC family kinase), the signaling pathways that couple TORC1 to its distal effectors were not well understood. To elucidate these pathways we developed and employed a quantitative, label-free mass spectrometry approach. Analyses of the rapamycin-sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between Tap42 and Sch9. Follow-up detailed characterization shows that Sch9 regulates RNA polymerases I and III, the latter via Maf1, in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis genes. This demonstrates that Sch9 is a master regulator of protein synthesis.
- Published
- 2009
- Full Text
- View/download PDF
45. Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks.
- Author
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Reiland S, Messerli G, Baerenfaller K, Gerrits B, Endler A, Grossmann J, Gruissem W, and Baginsky S
- Subjects
- Amino Acid Sequence, Arabidopsis Proteins chemistry, Databases, Protein, Models, Biological, Molecular Sequence Data, Phosphoproteins chemistry, Phosphorylation, Proteome chemistry, Sequence Alignment, Substrate Specificity, Arabidopsis metabolism, Arabidopsis Proteins analysis, Chloroplasts enzymology, Phosphoproteins analysis, Protein Kinases metabolism, Proteome analysis
- Abstract
We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as well as two new motifs at the carboxyl terminus of ribosomal proteins. Using the phosphorylation motifs as a footprint for the activity of a specific kinase class, we connected the phosphoproteins with their putative kinases and constructed a chloroplast CKII phosphorylation network. The network topology suggests that CKII is a central regulator of different chloroplast functions. To provide insights into the dynamic regulation of protein phosphorylation, we analyzed the phosphoproteome at the end of day and end of night. The results revealed only minor changes in chloroplast kinase activities and phosphorylation site utilization. A notable exception was ATP synthase beta-subunit, which is found phosphorylated at CKII phosphorylation sites preferentially in the dark. We propose that ATP synthase is regulated in cooperation with 14-3-3 proteins by CKII-mediated phosphorylation of ATP synthase beta-subunit in the dark.
- Published
- 2009
- Full Text
- View/download PDF
46. MRE11 complex links RECQ5 helicase to sites of DNA damage.
- Author
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Zheng L, Kanagaraj R, Mihaljevic B, Schwendener S, Sartori AA, Gerrits B, Shevelev I, and Janscak P
- Subjects
- Cell Line, DNA Breaks, Double-Stranded, DNA Replication, DNA-Binding Proteins analysis, Exodeoxyribonucleases metabolism, Humans, MRE11 Homologue Protein, Nuclear Proteins metabolism, RecQ Helicases analysis, DNA Damage, DNA-Binding Proteins metabolism, RecQ Helicases metabolism
- Abstract
RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11-RAD50-NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3'-->5' exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.
- Published
- 2009
- Full Text
- View/download PDF
47. Neuronal Nogo-A modulates growth cone motility via Rho-GTP/LIMK1/cofilin in the unlesioned adult nervous system.
- Author
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Montani L, Gerrits B, Gehrig P, Kempf A, Dimou L, Wollscheid B, and Schwab ME
- Subjects
- Actin Depolymerizing Factors genetics, Animals, Cells, Cultured, Electrophoresis, Gel, Two-Dimensional, Growth Cones ultrastructure, Isotope Labeling, Lim Kinases genetics, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Myelin Proteins genetics, Neurons cytology, Nogo Proteins, PC12 Cells, Protein Array Analysis, Rats, Retinal Ganglion Cells cytology, Retinal Ganglion Cells physiology, Signal Transduction physiology, Spinal Cord chemistry, Spinal Cord metabolism, rho GTP-Binding Proteins genetics, Actin Depolymerizing Factors metabolism, Growth Cones metabolism, Lim Kinases metabolism, Myelin Proteins metabolism, Neurons physiology, rho GTP-Binding Proteins metabolism
- Abstract
Nogo-A has been extensively studied as a myelin-associated neurite outgrowth inhibitor in the lesioned adult central nervous system. However, its role in the intact central nervous system has not yet been clarified. Analysis of the intact adult nervous system of C57BL/6 Nogo-A knock-out (KO) versus wild-type (WT) mice by a combined two-dimensional gel electrophoresis and isotope-coded affinity tagging approach revealed regulation of cytoskeleton-, transport-, and signaling growth-related proteins, pointing to regulation of the actin cytoskeleton, the neuronal growth machinery, and in particular the Rho-GTPase/LIMK1/cofilin pathway. Nogo-A KO adult neurons showed enlarged, more motile growth cones compared with WT neurons. The phenotype was reproduced by acute in vitro neutralization of neuronal Nogo-A. LIMK1 phosphorylation was increased in Nogo-A KO growth cones, and its reduction caused the decrease of KO growth cone motility to WT levels. Our study suggests that in the unlesioned adult nervous system, neuronal Nogo-A can restrict neuronal growth through negative modulation of growth cone motility.
- Published
- 2009
- Full Text
- View/download PDF
48. In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach.
- Author
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Endler A, Reiland S, Gerrits B, Schmidt UG, Baginsky S, and Martinoia E
- Subjects
- Amino Acid Sequence, Amino Acids chemistry, Arabidopsis genetics, Arabidopsis metabolism, Hordeum genetics, Hordeum ultrastructure, Intracellular Membranes chemistry, Microsomes chemistry, Microsomes metabolism, Molecular Sequence Data, Phosphopeptides genetics, Phosphopeptides metabolism, Phosphorylation genetics, Plant Leaves chemistry, Plant Leaves ultrastructure, Plant Proteins genetics, Plant Proteins metabolism, Plastids metabolism, Proteomics, Vacuoles chemistry, Vacuoles metabolism, Hordeum metabolism, Phosphopeptides isolation & purification, Plant Proteins isolation & purification, Plastids chemistry
- Abstract
In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast-enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO(2)) affinity chromatography and were subsequently analysed using LC-ESI-MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na(+)/H(+) antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.
- Published
- 2009
- Full Text
- View/download PDF
49. The Mass Distance Fingerprint: a statistical framework for de novo detection of predominant modifications using high-accuracy mass spectrometry.
- Author
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Potthast F, Gerrits B, Häkkinen J, Rutishauser D, Ahrens CH, Roschitzki B, Baerenfaller K, Munton RP, Walther P, Gehrig P, Seif P, Seeberger PH, and Schlapbach R
- Subjects
- Algorithms, Peptides chemistry, Mass Spectrometry methods
- Abstract
We describe a statistical measure, Mass Distance Fingerprint, for automatic de novo detection of predominant peptide mass distances, i.e., putative protein modifications. The method's focus is to globally detect mass differences, not to assign peptide sequences or modifications to individual spectra. The Mass Distance Fingerprint is calculated from high accuracy measured peptide masses. For the data sets used in this study, known mass differences are detected at electron mass accuracy or better. The proposed method is novel because it works independently of protein sequence databases and without any prior knowledge about modifications. Both modified and unmodified peptides have to be present in the sample to be detected. The method can be used for automated detection of chemical/post-translational modifications, quality control of experiments and labeling approaches, and to control the modification settings of protein identification tools. The algorithm is implemented as a web application and is distributed as open source software.
- Published
- 2007
- Full Text
- View/download PDF
50. Ultrahigh mass resolution and accurate mass measurements as a tool to characterize oligomers in secondary organic aerosols.
- Author
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Reinhardt A, Emmenegger C, Gerrits B, Panse C, Dommen J, Baltensperger U, Zenobi R, and Kalberer M
- Subjects
- Aerosols chemistry, Molecular Weight, Sensitivity and Specificity, Spectroscopy, Fourier Transform Infrared, Mass Spectrometry methods, Organic Chemicals chemistry
- Abstract
Organic aerosols are a major fraction, often more than 50%, of the total atmospheric aerosol mass. The chemical composition of the total organic aerosol mass is poorly understood, although hundreds of compounds have been identified in the literature. High molecular weight compounds have recently gained much attention because this class of compounds potentially represents a major fraction of the unexplained organic aerosol mass. Here we analyze secondary organic aerosols, generated in a smog chamber from alpha-pinene ozonolysis with ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). About 450 compounds are detected in the mass range of m/z 200-700. The mass spectrum is clearly divided into a low molecular weight range (monomer) and a high molecular weight range, where dimers and trimers are distinguishable. Using the Kendrick mass analysis, the elemental composition of about 60% of all peaks could be determined throughout the whole mass range. Most compounds have high O:C ratios between 0.4 and 0.6. Small compounds (i.e., monomers) have a higher maximum O:C ratio than dimers and trimers, suggesting that condensation reactions with, for example, the loss of water are important in the oligomer formation process. A program developed in-house was used to determine exact mass differences between peaks in the monomer, dimer, and trimer mass range to identify potential monomer building blocks, which form the co-oligomers observed in the mass spectrum. A majority of the peaks measured in the low mass region of the spectrum (m/z < 300) is also found in the calculated results. For the first time the elemental composition of the majority of peaks over a wide mass range was determined using advanced data analysis methods for the analysis of ultra-high-resolution MS data. Possible oligomer formation mechanisms in secondary organic aerosols were investigated.
- Published
- 2007
- Full Text
- View/download PDF
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