Quantitative Assessment of Protein Interaction with Methyl-Lysine Analogues by Hybrid Computational and Experimental Approaches

ABSTRACT: In cases where binding ligands of proteins are not easily available, structural analogues are often used. For example, in the analysis of proteins recognizing different methyl-lysine residues in histones, methyl-lysine analogues based on methyl-amino-alkylated cysteine residues have been introduced. Whether these are close enough to justify quantitative interpretation of binding experiments is however questionable. To systematically address this issue, we developed, applied, and assessed a hybrid computational/experimental approach that extracts the binding free energy difference between the native ligand (methyl-lysine) and the analogue (methyl-amino-alkylated cysteine) from a thermodynamic cycle. Our results indicate that measured and calculated binding differences are in very good agreement and therefore allow the correction of measured affinities of the analogues. We suggest that quantitative binding parameters for defined ligands in general can be derived by this method with remarkable accuracy. Fine-tuned regulation of gene expression in eukaryotic cellsrelies on packaging of DNA into different chromatin contexts.1 The repetitive unit of chromatin, the nucleosome, is formed by wrapping short stretches of DNA around

Impact of axial active magnetic bearing stiffness coefficient on resonance frequencies of reaction wheel rotor

Разработана математическая модель системы «ротор - электромагнитные подшипники» для электродвигателя-маховика системы ориентации и стабилизации космического аппарата. Модель учитывает собственные частоты изгибных колебаний ротора и коэффициенты жесткости электромагнитных подшипников. Предложен способ повышения угловой жесткости системы путем применения многополюсного осевого электромагнитного подшипника и рассмотрено влияние его коэффициента жесткости на собственные частоты системы.The paper presents the mathematical model of «rotor - active magnetic bearings» system for reaction wheel used in spacecraft attitude control system. Developed model consider the natural frequencies of rotor bending oscillations and stiffness parameters of electromagnetic bearing. Method of angular stiffness increasing by using multipolar axial magnetic bearing is suggested and the results of impact analysis of multipolar axial magnetic bearing stiffness on resonance frequencies of system is considered

Evaluating Depressive Symptoms in Schizophrenia: A Psychometric Comparison of the Calgary Depression Scale for Schizophrenia and the Hamilton Depression Rating Scale

Background: The aim of this study was to compare two measures of depression in patients with schizophrenia and schizophrenia spectrum disorder, including patients with delusional and schizoaffective disorder, to conclude implications for their application. Sampling and Methods: A total of 278 patients were assessed using the Calgary Depression Scale for Schizophrenia (CDSS) and the Hamilton Depression Rating Scale (HAMD-17). The Positive and Negative Syndrome Scale (PANSS) was also applied. At admission and discharge, a principal component analysis was performed with each depression scale. The two depression rating scales were furthermore compared using correlation and regression analyses. Results: Three factors were revealed for the CDSS and HAMD-17 factor component analysis. A very similar item loading was found for the CDSS at admission and discharge, whereas results of the loadings of the HAMD-17 items were less stable. The first two factors of the CDSS revealed correlations with positive, negative and general psychopathology. In contrast, multiple significant correlations were found for the HAMD-17 factors and the PANSS sub-scores. Multiple regression analyses demonstrated that the HAMD-17 accounted more for the positive and negative symptom domains than the CDSS. Conclusions:The present results suggest that compared to the HAMD-17, the CDSS is a more specific instrument to measure depressive symptoms in schizophrenia and schizophrenia spectrum disorder, especially in acutely ill patients. Copyright (c) 2012 S. Karger AG, Base

Failure of Working Memory Training to Enhance Cognition or Intelligence

Fluid intelligence is important for successful functioning in the modern world, but much evidence suggests that fluid intelligence is largely immutable after childhood. Recently, however, researchers have reported gains in fluid intelligence after multiple sessions of adaptive working memory training in adults. The current study attempted to replicate and expand those results by administering a broad assessment of cognitive abilities and personality traits to young adults who underwent 20 sessions of an adaptive dual n-back working memory training program and comparing their post-training performance on those tests to a matched set of young adults who underwent 20 sessions of an adaptive attentional tracking program. Pre- and post-training measurements of fluid intelligence, standardized intelligence tests, speed of processing, reading skills, and other tests of working memory were assessed. Both training groups exhibited substantial and specific improvements on the trained tasks that persisted for at least 6 months post-training, but no transfer of improvement was observed to any of the non-trained measurements when compared to a third untrained group serving as a passive control. These findings fail to support the idea that adaptive working memory training in healthy young adults enhances working memory capacity in non-trained tasks, fluid intelligence, or other measures of cognitive abilities.National Institutes of Health (U.S.) (Blueprint for Neuroscience Research (T90DA022759/R90DA023427)United States. Defense Advanced Research Projects Agency (government contract no. NBCHC070105)United States. Dept. of Defense (National Defense Science and Engineering Fellowship)Massachusetts Institute of Technology (Sheldon Razin (1959) Fellowship

Quantitative proteomic analysis of the binding proteins of histone H3 and the Four and a Half LIM-3 (FHL3) protein

Die Aufklärung spezifischer Protein-Protein-Interaktionen ist eine Grundvoraussetzung für das Verständnis vieler zellulärer Vorgänge. Durch massenspektrometrische Proteomanalysen wurde eine Vielzahl von posttranslationalen Proteinmodifikation, wie beispielsweise Phosphorylierungen, entdeckt, die Protein-Protein-Interaktionen regulieren können. Diese regulatorischen Proteinmodifikationen erschweren die Analyse der Protein-Protein-Interaktionen weiterhin, da sie in manchen Fällen Interaktionen erst ermöglichen, in anderen Fällen diese jedoch unterdrücken. Ziel dieser Arbeit war es, mit den Mitteln der Chemischen Biologie und massenspektrometrischer Proteomanalysen die Protein-Protein-Wechselwirkungen von Histon H3 in Abhängigkeit von Phosphorylierung zu untersuchen sowie die Bindungspartner des Adapterproteins Four-and-a-Half-LIM 3 (FHL3) zu identifizieren. Das erste Projekt dieser Arbeit widmete sich den phosphorylierungs-abhängigen Interaktionsproteinen von Histon H3. Histone verpacken DNA in Form von Chromatin und können Genregulation über posttranslationale Modifikationen steuern. Die Phosphorylierung von Serin-10 in Histon H3 ist eine dieser Modifikationen. In diesem Projekt sollten Proteine identifiziert werden, die vermittelt durch diese Modifikation an Histon H3 binden. Des Weiteren war geplant Bindeprotein von H3 zu identifizieren, die durch die Serin-10-Phosphorylierung von Histone H3 unterdrückt werden sowie die zugehörigen Phosphatasen, die diese Modifikation entfernen. Dazu wurde Phosphonomethylenalanin (Pma), ein Mimetikum von Phosphoserin, dass nicht durch Phosphatasen gespalten werden kann, an Position 10 in ein Peptid inkorporiert, das vom N-Terminus von H3 abgeleitet wurde. Nach Funktionsüberprüfung dieses Pma-modifizierten Peptids wurde eine Proteomanalyse basierend auf der SILAC-Methode (Stable isotope labeling by amino acids in cell culture) durchgeführt. Durch diese Analyse wurden diverse 14-3-3-Isoformen als Bindeproteine von Serin-10-phosphorylierten Histon H3 identifiziert. Phosphatasen konnten hingegen nicht gefunden werden. Differenzielle SILAC–Analysen mit H3-Peptiden synthetisiert aus L- oder D-Aminosäuren erlaubten weiterhin die Identifikation vieler Proteine, die spezifisch an unmodifiziertes H3 binden. Darunter waren Komponenten eines Deacetylase-Komplexes (NuRD), Hitzeschockproteine (HSC70, APG2), transkriptionellen Aktivatoren (WDR5, MYST2) und Repressoren (EHMT2, KDM5B). Mit HAT1 und RBBP7 wurden ferner zwei Bindeproteine vom unmodifizierten H3 identifiziert, deren Bindung durch die Phosphorylierung von Serin-10 unterdrückt wurde. In nachfolgenden Validierungsexperimenten konnten diese Beobachtungen bestätigt werden. Für ausgewählte Proteine wurde des Weiteren die Beeinflussung des Bindungsvermögens in Abhängigkeit von allen bekannten Phosphorylierungen in H3-Tail untersucht. In einem zweiten Projekt wurde das Four-and-a-Half-LIM 3 (FHL3) Protein einer quantitativen SILAC-Proteomanalyse unterzogen, mit dem Ziel, die bislang noch größtenteils unbekannten Interaktionspartner dieses Adapterproteins zu identifizieren und eine vermutete Interaktion mit Chromatinkomponenten experimentell zu überprüfen. Zunächst konnte gezeigt werden, dass FHL3 sowohl im Zellkern als auch im Cytosol, der für die Proteomanalyse verwendeten Fibroblastenzelline (Swiss 3T3) lokalisiert ist. Die nachfolgenden Proteomanalysen erlaubten die reproduzierbare Identifikation von 484 FHL3-Bindeproteinen in Zellkernextrakten und 244 in den cytosolischen Fraktionen dieser Zelllinie, wobei die meisten der identifizierten Protein bis jetzt nicht als Interaktionspartner von FHL3 beschrieben wurden. Eine direkte Interaktion mit Histonen konnte nicht beobachtet werden, wohl aber Proteine, die an Chromatinprozessierungen beteiligt sind. Ausgewählte FHL3-Interaktionspartner wurden durch Pull-Down Experimente und Western Blot Analyse bestätigt. Darunter befanden sich der transkriptionelle Coaktivator CBP, Schlüsselkomponenten der eukaryotischen DNA-Replikation (MCM3 und POLD1) und die mitotische Serin/Threonin-Kinase TLK1. Neben diesen kernlokalisierten Proteinen konnte die FHL3-Interaktion der Ubiquitin-Ligase DTX3L im nuklearen Extrakt und der cytosolischen Fraktion bestätigt werden sowie die FHL3-Bindung der cytosolischen Polyphosphoinositide-Phosphatase FIG4. Durch diese Proteomanalyse und die nachgeschalteten Validierungsexperimente konnte FHL3 mit einer Vielzahl essentieller sowie pathologischer zellulärer Prozesse in Verbindung gebracht werden.The identification of specific protein-protein interactions is a basic requirement for a comprehensive understanding of many cellular processes. Mass spectrometric proteome analyses have uncovered a multitude of posttranslational protein modifications, such as protein phosphorylation, which regulate protein-protein interactions. These regulatory modifications add a new layer of complexity because they can either facilitate or block protein-protein interactions. The aim of this thesis was the investigation of protein-protein interaction network of histone H3 and its modulation by protein phosphorylation as well as the identification of binding partners of the adapter protein Four-and-a-Half-Lim 3 (FHL3) by means of chemical biology and mass spectrometric proteomic analysis. The first project of this thesis focused on the phosphorylation-depended interaction proteins of histone H3. Histones package DNA in form of chromatin and control gene activity via posttranslational modifications. The phosphorylation of serine-10 is one of those modifications and in this project proteins should be identified that bind to histone H3 in response to this modification. Furthermore, it was planned to identify binding proteins of unmodified H3, which are suppressed by serine-10 phosphorylation and to find associated phosphatases which remove this modification. Therefore, phosphonomethylenalanin (Pma), a mimetic of phosphoserine that cannot be cleaved by phosphatases was incorporated at position 10 in a peptide derived from the N-terminus of H3. After testing the functionality of the Pma-modified peptide, a proteome analysis was performed based on the SILAC method (stable isotope labeling by amino acids in cell culture). The analysis uncovered various 14 3 3 isoforms as binding proteins of serin-10 phosphorylated histone H3. However, phosphatases could not be identified. Differential SILAC analyses with H3 peptides synthesized from L or D amino acids identified many proteins that specifically bind to unmodified H3. Among them are components of a deacetylase complex (NuRD), heat shock proteins (HSC70, APG2), transcriptional activators (WDR5, MYST2) and repressors (EHMT2, KDM5B). Furthermore with HAT1 and RBBP7 two binding proteins of the unmodified H3 were identified, whose binding activity was suppressed by the phosphorylation of serine-10. These observations were confirmed in subsequent validation experiments. In addition the modulation of binding capability of selected proteins was examined in dependency of all known phosphorylation sites in H3. In the second project the Four-and-a-Half- LIM protein 3 was subjected to a quantitative proteome analysis with the aim to identify the interaction partners of this adapter protein and to verify a putative interaction with chromatin components. Initially, it has been demonstrated that FHL3 is localized in both the nucleus and the cytosol of Swiss 3T3 mouse fibroblasts used in this project. The following 484 FHL3 binding proteins were identified in nuclear extracts and 244 in the cytosolic fractions of this cell line. Most of these reproducibly identified proteins have not been described as interaction partner of FHL3. A direct interaction with histones could not be observed but several proteins involved in chromatin processing could be identified. Selected FHL3 interaction partners were confirmed by pull-down experiments and western blot analysis. These proteins include the transcriptional coactivator CBP, key components of the eukaryotic DNA replication (MCM3 and POLD1) and the mitotic serine/threonine kinase TLK1. Beside these nuclear localized proteins, an interaction between FHL3 and the ubiquitin ligase DTX3L could be confirmed in the nuclear and the cytosolic fraction of Swiss 3T3 cells as well as the interaction of FHL3 with the cytosolic polyphosphoinositide phosphatase FIG4. Based on this proteome analysis and the downstream validation experiments FHL3 could be connected to a variety of essential, as well as pathological cellular processes

Analysis of Phosphorylation-Dependent Protein–Protein Interactions of Histone H3

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails

Analysis of Phosphorylation-Dependent Protein–Protein Interactions of Histone H3

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails

Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells.

BACKGROUND: Polycomb group (PcG) genes code for chromatin multiprotein complexes that are responsible for maintaining gene silencing of transcriptional programs during differentiation and in adult tissues. Despite the large amount of information on PcG function during development and cell identity homeostasis, little is known regarding the dynamics of PcG complexes and their role during terminal differentiation. RESULTS: We show that two distinct polycomb repressive complex (PRC)2 complexes contribute to skeletal muscle cell differentiation: the PRC2-Ezh2 complex, which is bound to the myogenin (MyoG) promoter and muscle creatine kinase (mCK) enhancer in proliferating myoblasts, and the PRC2-Ezh1 complex, which replaces PRC2-Ezh2 on MyoG promoter in post-mitotic myotubes. Interestingly, the opposing dynamics of PRC2-Ezh2 and PRC2-Ezh1 at these muscle regulatory regions is differentially regulated at the chromatin level by Msk1 dependent methyl/phospho switch mechanism involving phosphorylation of serine 28 of the H3 histone (H3S28ph). While Msk1/H3S28ph is critical for the displacement of the PRC2-Ezh2 complex, this pathway does not influence the binding of PRC2-Ezh1 on the chromatin. Importantly, depletion of Ezh1 impairs muscle differentiation and the chromatin recruitment of MyoD to the MyoG promoter in differentiating myotubes. We propose that PRC2-Ezh1 is necessary for controlling the proper timing of MyoG transcriptional activation and thus, in contrast to PRC2-Ezh2, is required for myogenic differentiation. CONCLUSIONS: Our data reveal another important layer of epigenetic control orchestrating skeletal muscle cell terminal differentiation, and introduce a novel function of the PRC2-Ezh1 complex in promoter setting.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Анализ эффективного применения гидравлического разрыва пласта на Приобском нефтяном месторождении (ХМАО)

Объектом исследования является продуктивный пласт АС12 X месторождения. Цель работы – определить эффективность применения гидравлического разрыва пласта на X месторождении.The object of study is the productive layer AC12 X Place of Birth. The purpose of the work is to determine the effectiveness of hydraulic fracturing in the X field