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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 phospho-residues, S268, which when de-phosphorylated 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 S268, and describe a functional interaction between residues S268 and S270. 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.
Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
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Sonja Reiland,
Giovanni Finazzi,
Anne Endler,
Adrian Willig,
Katja Baerenfaller,
Jonas Grossmann, Bertran Gerrits,
Dorothea Rutishauser,
Wilhelm Gruissem,
Jean-David Rochaix,
Sacha Baginsky
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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.
Proceedings of the National Academy of Sciences 08/2011; 108(31):12955-60. · 9.68 Impact Factor
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Shiva K. Tyagarajan,
Himanish Ghosh,
Gonzalo E. Yévenes,
Irina Nikonenko,
Claire Ebeling,
Cornelia Schwerdel,
Corinne Sidler,
Hanns Ulrich Zeilhofer, Bertran Gerrits,
Dominique Muller,
Jean-Marc Fritschy
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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 Ca2+-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.
Proceedings of the National Academy of Sciences 01/2011; 108(1):379-384. · 9.68 Impact Factor
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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.
Blood 09/2010; 116(13):e26-34. · 9.90 Impact Factor
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Martin Steuble, Bertran Gerrits,
Alexander Ludwig,
José María Mateos,
Tu-My Diep,
Mitsuo Tagaya,
Alexander Stephan,
Philipp Schätzle,
Beat Kunz,
Peter Streit,
Peter Sonderegger
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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.
Proteomics 09/2010; 10(21):3775-88. · 4.43 Impact Factor
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ABSTRACT: The composition of organic matter in natural ecosystems is strongly
influenced by the microorganisms present. Conversely, bacteria and fungi
are limited by the amount and type of organic matter available in a
given environment, most of which is ultimately derived from plants.
Changes in the stoichiometry and biochemical constituents of plant
litter may therefore alter species composition and elicit changes in the
activities of microbial communities and their component parts. The
identification of the microbial proteins of a given habitat together
with the analysis of their phylogenetic origin and their spatial and
temporal distribution are expected to provide fundamentally new insights
into the role of microbial diversity in biogeochemical processes. To
relate structure and functionality of microbial communities involved in
leaf-litter decomposition we determined biogeochemistry, community
structure by phospholipid fatty acid (PLFA)-analyses, enzymatic
activities, and analysed the protein complement of different litter
types, which were collected in winter and spring at various Austrian
sampling sites, in a semi-quantitative proteomics approach by one
dimensional polyacrylamide gel electrophoresis (1-D-SDS-PAGE) combined
with liquid chromatography/tandem mass-spectrometry (LC-MS/MS). Protein
abundances were determined by counting the number of MS/MS spectra
assigned to each protein. In samples with high manganese and phosphor
content a significant increase of fungal proteins from February to May
was observed, which was in good agreement with the PLFA-analyses showing
similar trends towards an increase of the fungal community. In contrast,
the PLFA analysis revealed no temporal changes in the community at
Achenkirch and even a decrease in the fungal/bacterial ratio at
Klausen-Leopoldsdorf, two sampling sites low in P and Mn; similar trends
are reflected in our spectral counts. In conclusion, semi-quantitative
proteome- and PLFA-analyses suggest that fungal and bacterial abundance
positively correlates with the total amount of P and Mn within the
different litter types. Spectral counts of extracellular enzymes
demonstrated a significant increase of these enzymes in the May, which
was also mirrored by measurements of total enzymatic activities. The
finding that almost all hydrolytic enzymes identified from litter were
of fungal origin suggests a prominent role of fungi during aerobic
litter decomposition.
04/2010; 12:4164.
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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.
Proteomics 03/2010; 10(9):1819-30. · 4.43 Impact Factor
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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.
Methods in molecular biology (Clifton, N.J.) 01/2010; 658:127-36.
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Bernd Bodenmiller,
Stefanie Wanka,
Claudine Kraft,
Jörg Urban,
David Campbell,
Patrick G Pedrioli, Bertran Gerrits,
Paola Picotti,
Henry Lam,
Olga Vitek, [......],
Chao Zhang,
Kevan M Shokat,
Ralph Schlapbach,
Alejandro Colman-Lerner,
Garry P Nolan,
Alexey I Nesvizhskii,
Matthias Peter,
Robbie Loewith,
Christian von Mering,
Ruedi Aebersold
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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.
Science Signaling 01/2010; 3(153):rs4. · 7.50 Impact Factor
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Sandra Goetze,
Ermir Qeli,
Christian Mosimann,
An Staes, Bertran Gerrits,
Bernd Roschitzki,
Sonali Mohanty,
Eva M Niederer,
Endre Laczko,
Evy Timmerman,
Vinzenz Lange,
Ernst Hafen,
Ruedi Aebersold,
Joël Vandekerckhove,
Konrad Basler,
Christian H Ahrens,
Kris Gevaert,
Erich Brunner
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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.
PLoS Biology 11/2009; 7(11):e1000236. · 11.45 Impact Factor
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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.
Genes & development 09/2009; 23(16):1929-43. · 12.08 Impact Factor
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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.
Plant physiology 05/2009; 150(2):889-903. · 6.53 Impact Factor
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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.
Journal of Biological Chemistry 03/2009; 284(16):10793-807. · 4.77 Impact Factor
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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.
Proteomics 02/2009; 9(2):310-21. · 4.43 Impact Factor
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Nature Biotechnology 01/2009; 26(12):1339-40. · 29.50 Impact Factor
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Frank Potthast, Bertran Gerrits,
Jari Häkkinen,
Dorothea Rutishauser,
Christian H Ahrens,
Bernd Roschitzki,
Katja Baerenfaller,
Richard P Munton,
Pascal Walther,
Peter Gehrig,
Philipp Seif,
Peter H Seeberger,
Ralph Schlapbach
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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.
Journal of Chromatography B 08/2007; 854(1-2):173-82. · 2.89 Impact Factor
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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.
Analytical Chemistry 06/2007; 79(11):4074-82. · 5.86 Impact Factor
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ABSTRACT: In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hERalpha LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERalpha LBD. The relative gas-phase stability of selected hERalpha LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately diethylstilbestrol > E2 > genistein > bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K(d)) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.
Protein Science 05/2007; 16(5):938-46. · 2.80 Impact Factor
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ABSTRACT: Postreplicative mismatch repair (MMR) involves the concerted action of at least 20 polypeptides. Although the minimal human MMR system has recently been reconstituted in vitro, genetic evidence from different eukaryotic organisms suggests that some steps of the MMR process may be carried out by more than one protein. Moreover, MMR proteins are involved also in other pathways of DNA metabolism, but their exact role in these processes is unknown. In an attempt to gain novel insights into the function of MMR proteins in human cells, we searched for interacting partners of the MutL homologues MLH1 and PMS2 by tandem affinity purification and of PMS1 by large scale immunoprecipitation. In addition to proteins known to interact with the MutL homologues during MMR, mass spectrometric analyses identified a number of other polypeptides, some of which bound to the above proteins with very high affinity. Whereas some of these interactors may represent novel members of the mismatch repairosome, others appear to implicate the MutL homologues in biological processes ranging from intracellular transport through cell signaling to cell morphology, recombination, and ubiquitylation.
Journal of Biological Chemistry 03/2007; 282(5):2976-86. · 4.77 Impact Factor
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Richard P Munton,
Ry Tweedie-Cullen,
Magdalena Livingstone-Zatchej,
Franziska Weinandy,
Marc Waidelich,
Davide Longo,
Peter Gehrig,
Frank Potthast,
Dorothea Rutishauser, Bertran Gerrits,
Christian Panse,
Ralph Schlapbach,
Isabelle M Mansuy
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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.
Molecular & Cellular Proteomics 03/2007; 6(2):283-93. · 7.40 Impact Factor
