359 research outputs found

    The compatible solutes ectoine and 5-hydroxyectoine: Catabolism and regulatory mechanisms

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    To cope with osmotic stress many microorganisms make use of short, osmotically active, organic compounds, the so-called compatible solutes. Examples for especially effective members of this type of molecules are the tetrahydropyrimidines ectoine and 5-hydroxyectoine. Both molecules are produced by a large number of microorganisms, not only to fend-off osmotic stress, but also for example low and high temperature challenges. The biosynthetic pathway used by these organisms to synthesize ectoines has already been studied intensively and the enzymes used therein are characterized quite well, both biochemically as well as structurally. However, synthesis of ectoines is only half the story. Inevitably, ectoines are frequently released from the producer cells in different environmental settings. Especially in highly competitive habitats like the upper ocean layers some bacteria specialized on a niche like this. The model organism used in this work is such a species. It is the marine bacterium Ruegeria pomeroyi DSS-3 which belongs to the Roseobacter-clade. Roseobacter species are heterotrophic Proteobacteria which can live in symbiosis with phytoplankton as well as turning against them in a bacterial warfare fashion to scavenge valuable nutrients. Ectoines can be imported by R. pomeroyi DSS-3 in a high-affinity fashion and be used as energy as well as carbon- and nitrogen-sources. To achieve this, both ectoines rings are degraded by the hydrolase EutD and deacetylated by the deacetylase EutE. The first hydrolysis products α-ADABA (from ectoine) and hydroxy-α-ADABA (from hydroxyectoine) are deacetylated to DABA and hydroxy-DABA which are in additional biochemical reactions transformed to aspartate to fuel the cell’s central metabolism. The role and functioning of the EutDE enzymes which work in a concerted fashion are a central aspect of this work. Both enzymes could be biochemically and structurally characterized, and the architecture of the metabolic pathway could be illuminated. α-ADABA and hydroxy-α-ADABA are not only central to ectoine catabolism, but also to the regulatory mechanisms associated with it. Both molecules serve as inducers of the central regulatory protein of this pathway, the MocR-/GabR-type regulator protein EnuR. In the framework of this dissertation molecular details could be clarified which enable the EnuR repressor molecule to sense both molecules with high affinity to subsequently derepress the genes for the import and catabolism of ectoines

    Mining the secretome of the lignocellulose degrading fungus Parascedosporium putredinis NO1

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    The demand for sustainable and renewable alternatives to finite and environmentally damaging fossil fuel resources is growing. Lignocellulosic biomass is available in vast amounts, at low cost, and could provide fuels, chemicals, and materials if deconstructed effectively. However, its recalcitrant nature makes the cost-effective utilisation of this substrate difficult to achieve. Despite this, wood-degrading fungi have evolved an array of powerful enzymes for the deconstruction of all components of lignocellulose including the polysaccharides and the aromatic polymer lignin. In this work, the lignocellulose-degrading capacity of the ascomycete fungus Parascedosporium putredinis NO1 was explored in detail. New bioinformatic strategies were developed and employed to probe the genome of P. putredinis NO1, the first genome of its genus, and to isolate an in silico secretome to allow clearer characterisation of the biomass-degrading response. Proteomic investigations of the growth of P. putredinis NO1 on multiple industrially relevant lignocellulosic substrates demonstrated remarkable variation in the P. putredinis NO1 secretome depending on growth substrate. Molecular techniques were used to expand the demonstration of the varied secretome temporally and support the hypothesis of a tailored enzymatic response to different lignocellulosic substrates, an understanding of which will be important for the development of efficient biorefinery technology. The tailored secretome was exploited by investigating the enzymatic response of P. putredinis NO1 when grown on substrates with varying lignin contents. This allowed identification of proteins with patterns of abundance suggesting roles in the breakdown of lignin. The characterisation of lignocellulose-degrading organisms from underexplored branches of the tree of life, and the identification and characterisation of new enzymes with unclear or unknown roles in lignocellulose breakdown will be vital to improving our understanding of lignocellulose deconstruction and to achieve this efficiently in biorefineries

    Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry

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    Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics

    Aiding the conservation of two wooden Buddhist sculptures with 3D imaging and spectroscopic techniques

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    The conservation of Buddhist sculptures that were transferred to Europe at some point during their lifetime raises numerous questions: while these objects historically served a religious, devotional purpose, many of them currently belong to museums or private collections, where they are detached from their original context and often adapted to western taste. A scientific study was carried out to address questions from Museo d'Arte Orientale of Turin curators in terms of whether these artifacts might be forgeries or replicas, and how they may have transformed over time. Several analytical techniques were used for materials identification and to study the production technique, ultimately aiming to discriminate the original materials from those added within later interventions

    Exploring Humoral Immune Responses by Mass Spectrometry: Resolving Structures, Interactions, and Clonal Repertoires of Antibodies

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    In his thesis “Exploring Humoral Immune Responses by Mass Spectrometry”, Maurits den Boer uses mass spectrometry to shed new light on antibody responses. Antibodies play a crucial role in the immune protection against threats like bacteria, viruses, and cancers. When valuable antibodies are discovered, they can therefore be reproduced for use as a medicine. A better understanding of their structures, interactions, and repertoires is therefore key to finding novel treatments for many diseases. In the first part of his thesis, Maurits and coworkers used mass spectrometry to study antibody structures and interactions, leading to two major findings. They first uncovered a mechanism by which Staphylococcus aureus bacteria can evade antibody responses, and how this mechanism may be circumvented in future therapies. Second, he redefined the textbook structure of circulating IgM antibodies by showing that they are universally attached to an extra protein. This may have major implications for how these antibodies function, and their use as therapeutics. In a second line of research, Maurits focused on the development of innovative techniques for antibody repertoire analysis and discovery. Together with coworkers, he explored the use of electron-based fragmentation mass spectrometry, developing methods to obtain valuable pieces of antibody sequence information. Finally, he combined multiple layers of mass spectrometry analysis to discover and fully determine the sequence of a malignant patient antibody. Combined, this demonstrates the promise of mass spectrometry as a compelling new approach for therapeutic antibody discovery

    Enabling Structural Proteomics with High Efficiency Protein Enrichment Technology

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    The functional state of proteins is inherently flexible, which allows them to interact with other biomolecules, including other proteins, to carry out many of their cellular functions. Understanding the structural dynamics of proteins and their network of associations is key to understanding their role in biology. Proteomics, the collection of mass spectrometry (MS)-based techniques to study proteins, provides a broad view of the organization of protein structure, from an individual dynamic unit to large-scale multiprotein assemblies, enabled by the application of labelling chemistries. This dissertation presents novel analytical workflows and data analysis routines to overcome current challenges in proteomics methods for the identification of protein-protein interactions (PPIs) and the study of protein conformation and dynamics. Affinity purification followed by mass spectrometry (AP-MS) is a prominent approach in the study of PPIs. However, the conventional workflow suffers from low enrichment efficiencies. I present and evaluate a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing non-specific binding. It enables the study of protein conformational analysis directly from cells as I demonstrate first by describing new concepts in data analysis for hydrogen/deuterium exchange mass spectrometry (HX-MS) and second by applying them to proteins isolated directly from cells

    Regulatory mechanisms of the TRPM7 channel-kinase

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    The transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a bifunctional protein containing a transmembrane channel segment and a serine/threonine-protein kinase domain. The TRPM7 channel is selective to divalent cations. TRPM7 regulates many biological processes such as the organismal balance of Zn2+, Ca2+ and Mg2+, immunity, embryonic development and cell signalling. The widely accepted view is that cytosolic Mg2+ and Mg·ATP inhibit the TRPM7 channel to link the metabolic state of the cell to the uptake of divalent cations. More recently, independent studies focusing on the identification of native TRPM7 channel complexes revealed the association of TRPM7 with the metal transporters CNNM1-4. However, the mechanisms underlying TRPM7 sensitivity to intracellular Mg2+ and the regulatory role of CNNM1-4 remain poorly understood. Therefore, we used site-directed mutagenesis, patch-clamp measurements and other techniques to demonstrate that a regulatory Mg2+ binding site is formed by the side chains of N1097 in mouse TRPM7. Our results suggest a direct interaction between Mg2+ and N1097 leading to a stabilization of the TRPM7 in the closed conformation. In line with these findings, we showed that the sensitivity of TRPM7 to physiological concentrations of intracellular Mg2+ was selectively affected by an introduction of point mutation (N1097Q). In addition, our results indicate that the CNNM3 protein has no effect on the TRPM7 channel but rather negatively regulates the kinase activity of TRPM7. Thus, our study suggests new mechanistic insights into the regulatory characteristics of the kinase-coupled TRPM7 channel.Der Transient-Rezeptor-Potential-Kationenkanal, Unterfamilie M, Mitglied 7 (TRPM7) ist ein bifunktionelles Protein, das ein Transmembrankanalsegment umfasst, was mit einer Serin/Threonin-Proteinkinase-Domäne fusioniert ist. Der TRPM7-Kanal ist selektiv für Zn2+, Ca2+ und Mg2+. TRPM7 reguliert zahlreiche biologische Prozesse wie die Zn2+-, Ca2+- und Mg2+-Homöostase im Organismus, die Embryonalentwicklung, Immunreaktionen und Signaltransduktion. Eine weit verbreitete Ansicht ist, dass zytosolisches Mg2+ und Mg·ATP als negative Regulatoren des TRPM7-Kanals wirken, um die Aufnahme zweiwertiger Kationen auf den Stoffwechselzustand der Zelle anzupassen. Kürzlich haben unabhängige Studien, die sich auf die Identifizierung nativer TRPM7-Kanalkomplexe konzentrierten, die Verbindung von TRPM7 mit den Metalltransportern CNNM1-4 aufgezeigt. Die Mechanismen, die der Sensitivität von TRPM7 gegenüber intrazellulärem Mg2+ und der regulatorischen Rolle von CNNM1-4 zugrunde liegen, sind jedoch nach wie vor kaum verstanden. Daher haben wir eine Kombination aus ortsgerichteter Mutagenese, Patch-Clamp-Techniken, Western-Blotting und 3DProteinmodelierung eingesetzt, um zu zeigen, dass die Seitenketten von N1097 in TRPM7 in der Maus zwischen den Untereinheiten eine Mg2+-regulierende Stelle bilden. Unsere Ergebnisse legen nahe, dass Mg2+ direkt mit diesem Proteinsegment interagiert und dadurch TRPM7 im geschlossenen Zustand stabilisiert. In Übereinstimmung mit diesem Modell haben wir festgestellt, dass Punktmutationen in der Mg2+-Regulationsstelle (N1097Q und N1098Q) die Sensitivität von TRPM7 gegenüber physiologischen Konzentrationen von intrazellulärem Mg2+ aufhebt. Darüber hinaus deuten unsere Ergebnisse darauf hin, dass das CNNM3-Protein keine Auswirkungen auf den TRPM7-Kanal hat, sondern vielmehr die Kinaseaktivität von TRPM7 negativ reguliert. Unsere Studie liefert somit neue mechanistische Erkenntnisse über die regulatorischen Eigenschaften des Kinase-gekoppelten TRPM7-Kanals
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