Metal and inhibitor binding studies on metallo-beta-lactamases

The heterogeneity of the metal content observed in Metallo-ß-Lactamases (MBLs) hampers the design of potential inhibitors. In the first part of the work, three representative members of the MBLs, namely BcII, CphA and L1 were investigated using mass spectrometric and spectroscopic methods. Experimental parameters for the detection of the metal-protein and ternary metalloprotein-inhibitor complexes using ESI-MS1 were evaluated and optimized. SAR1 determined in the gas phase were in agreement with kinetic assays performed in solution. This demonstrates the suitability of this technique for the screening for new inhibitors of MBLs and for the detection of metal:enzyme:inhibitor ratios. Competition-titrations in combination with ESI-MS, revealed that for different subclasses of the MBL, the inhibition by (R,S)-thiomandelate and D-captopril is strongly influenced by the nature of the metal ion and the metal content of the protein. In the second part of the work, the metal ion dependent flexibility of different parts of the BcII protein was investigated using HDX-MS1. It was shown that the metal-free enzyme was the least ordered structure and that the high flexibility at the metal binding site and the domain interface region in the Cd1-enzyme might facilitate the transfer of the metal between the two binding sites. These findings deliver important parameters for future development of efficient inhibitors for these enzymes.Die heterogene Metallbesetzung in Metallo-ß-Lactamasen (MBLs) ist einer der Hauptgründe für den bislang geringen Erfolg bei der Entwicklung effizienter Inhibitoren für diese Enzymklasse. Im ersten Teil der Arbeit wurden drei repräsentative Vertreter der MBLs (BcII, CphA und L1) mit massenspektrometrischen und spektroskopischen Methoden untersucht. Es wurden Methoden der "nicht denaturierenden" ESI-MS1 für den Nachweis von Metall-Protein- sowie ternärer Metallprotein-Inhibitor-Interaktionen entwickelt.Die mittels ESI-MS in der Gasphase ermittelten SAR1 stimmten sehr gut mit den zuvor in Lösung ermittelten überein. Somit konnte gezeigt werden, dass ESI-MS eine geeignete Methode für die Bestimmung von Metall-Enzym-Inhibitor-Stöchiometrien und damit für die Identifizierung neuer effizienter Inhibitoren darstellt. Durch die Kombination von ESI-MS Experimenten mit Konkurrenztitrationen zeigte sich, dass die Hemmung verschiedener MBL-Subklassen mittels (R,S)-Thiomandelsäure und D-Captopril stark von der Art des gebundenen Metalls sowie von der Metall-Protein-Stöchiometrie beeinflusst wird. Im zweiten Teil der Arbeit konnte mittels HDX-MS1 gezeigt werden, dass beim metallfreien Enzym die Sekundärstruktur am wenigsten ausgeprägt ist und dass das Cd1-BcII Enzym der metal-freien BcII Spezies sehr zu ähneln scheint, wenn nur das aktive Zentrum und die Interdomainen-Region betrachtet werden. Dies liefert ein tiefergehendes Verständnis der MBL sowie Grundlagen zur Entwicklung neuer Inhibitoren

Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify \u3e4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories

Untersuchungen zur Bindung von Metall und Inhibitor an Metallo-beta-Lactamasen

The heterogeneity of the metal content observed in Metallo-ß-Lactamases (MBLs) hampers the design of potential inhibitors. In the first part of the work, three representative members of the MBLs, namely BcII, CphA and L1 were investigated using mass spectrometric and spectroscopic methods. Experimental parameters for the detection of the metal-protein and ternary metalloprotein-inhibitor complexes using ESI-MS1 were evaluated and optimized. SAR1 determined in the gas phase were in agreement with kinetic assays performed in solution. This demonstrates the suitability of this technique for the screening for new inhibitors of MBLs and for the detection of metal:enzyme:inhibitor ratios. Competition-titrations in combination with ESI-MS, revealed that for different subclasses of the MBL, the inhibition by (R,S)-thiomandelate and D-captopril is strongly influenced by the nature of the metal ion and the metal content of the protein. In the second part of the work, the metal ion dependent flexibility of different parts of the BcII protein was investigated using HDX-MS1. It was shown that the metal-free enzyme was the least ordered structure and that the high flexibility at the metal binding site and the domain interface region in the Cd1-enzyme might facilitate the transfer of the metal between the two binding sites. These findings deliver important parameters for future development of efficient inhibitors for these enzymes.Die heterogene Metallbesetzung in Metallo-ß-Lactamasen (MBLs) ist einer der Hauptgründe für den bislang geringen Erfolg bei der Entwicklung effizienter Inhibitoren für diese Enzymklasse. Im ersten Teil der Arbeit wurden drei repräsentative Vertreter der MBLs (BcII, CphA und L1) mit massenspektrometrischen und spektroskopischen Methoden untersucht. Es wurden Methoden der "nicht denaturierenden" ESI-MS1 für den Nachweis von Metall-Protein- sowie ternärer Metallprotein-Inhibitor-Interaktionen entwickelt.Die mittels ESI-MS in der Gasphase ermittelten SAR1 stimmten sehr gut mit den zuvor in Lösung ermittelten überein. Somit konnte gezeigt werden, dass ESI-MS eine geeignete Methode für die Bestimmung von Metall-Enzym-Inhibitor-Stöchiometrien und damit für die Identifizierung neuer effizienter Inhibitoren darstellt. Durch die Kombination von ESI-MS Experimenten mit Konkurrenztitrationen zeigte sich, dass die Hemmung verschiedener MBL-Subklassen mittels (R,S)-Thiomandelsäure und D-Captopril stark von der Art des gebundenen Metalls sowie von der Metall-Protein-Stöchiometrie beeinflusst wird. Im zweiten Teil der Arbeit konnte mittels HDX-MS1 gezeigt werden, dass beim metallfreien Enzym die Sekundärstruktur am wenigsten ausgeprägt ist und dass das Cd1-BcII Enzym der metal-freien BcII Spezies sehr zu ähneln scheint, wenn nur das aktive Zentrum und die Interdomainen-Region betrachtet werden. Dies liefert ein tiefergehendes Verständnis der MBL sowie Grundlagen zur Entwicklung neuer Inhibitoren

zur Erlangung des Grades

This dissertation is the result of three and a half years of work during which many people have supported me. I would like to thank all of them in the next lines: First of all, special thanks go to my supervisor Priv. Doz. Dr. Hans-Werner Adolph for its helpful and stimulating discussions during this work and for the reviewing of the thesis. I would also like to sincerely thank Prof. Dipl. Ing. Dr. tech. Elmar Heinzle, who gave me th

Network integration and modelling of dynamic drug responses at multi-omics levels

Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data.ISSN:2399-364

New quantitative mass spectrometry approaches reveal different ADP-ribosylation phases dependent on the levels of oxidative stress

Oxidative stress is a potent inducer of protein ADP-ribosylation. Although individual oxidative stress-induced ADP-ribosylated proteins have been identified, it is so far not clear to which extent different degrees of stress severity quantitatively and qualitatively alter ADP-ribosylation. Here, we investigated both quantitative and qualitative changes of the hydrogen peroxide (H2O2)-induced ADP-ribosylome using a label-free shotgun quantification and a parallel reaction monitoring (PRM) mass spectrometry approach for a selected number of identified ADP-ribosylated peptides. Although the major part of the basal HeLa ADP-ribosylome remained unchanged upon all tested H2O2 concentrations, some selected peptides change the extent of ADP-ribosylation depending on the degree of the applied oxidative stress. Low oxidative stress (i.e. 4 μm and 16 μm H2O2) caused a reduction in ADP-ribosylation of modified proteins detected under untreated conditions. In contrast, mid to strong oxidative stress (62 μm to 1 mm H2O2) induced a significant increase in ADP-ribosylation of oxidative stress-targeted proteins. The application of the PRM approach to SKOV3 and A2780, ovarian cancer cells displaying different sensitivities to PARP inhibitors, revealed that the basal and the H2O2-induced ADP-ribosylomes of SKOV3 and A2780 differed significantly and that the sensitivity to PARP inhibitors correlated with the level of ARTD1 expression in these cells. Overall, this new PRM-MS approach has proven to be sensitive in monitoring alterations of the ADP-ribosylome and has revealed unexpected alterations in proteins ADP-ribosylation depending on the degree of oxidative stress

Automated Workflow for Large-Scale Selected Reaction Monitoring Experiments

Targeted proteomics allows researchers to study proteins of interest without being drowned in data from other, less interesting proteins or from redundant or uninformative peptides. While the technique is mostly used for smaller, focused studies, there are several reasons to conduct larger targeted experiments. Automated, highly robust software becomes more important in such experiments. In addition, larger experiments are carried out over longer periods of time, requiring strategies to handle the sometimes large shift in retention time often observed. We present a complete proof-of-principle software stack that automates most aspects of selected reaction monitoring workflows, a targeted proteomics technology. The soft-ware allows experiments to be easily designed and carried out. The steps automated are the generation of assays, generation of mass spectrometry driver files and methods files, and the import. and analysis of the data. All data are normalized to a common retention time scale, the data are then scored using a novel score model, and the error is subsequently estimated. We also show that selected reaction monitoring can be used for label-free quantification. All data generated are stored in a relational database, and the growing resource further facilitates the design of new experiments. We apply the technology to a large-scale experiment studying how Streptococcus pyogenes remodels its proteome under stimulation of human plasma

Quantitative Proteomics Reveal Distinct Protein Regulations Caused by Aggregatibacter actinomycetemcomitans within Subgingival Biofilms

Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species "subgingival" biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute protein numbers of the other biofilm species, it caused qualitative changes in their overall protein expression profile. These molecular shifts within the biofilm warrant further investigation on their potential impact on its virulence properties, and association with periodontal pathogenesis