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

IDH-mutant glioma specific association of rs55705857 located at 8q24.21 involves MYC deregulation

The single nucleotide polymorphism rs55705857, located in a non-coding but evolutionarily conserved region at 8q24.21, is strongly associated with IDH-mutant glioma development and was suggested to be a causal variant. However, the molecular mechanism underlying this association has remained unknown. With a case control study in 285 gliomas, 316 healthy controls, 380 systemic cancers, 31 other CNS-tumors, and 120 IDH-mutant cartilaginous tumors, we identified that the association was specific to IDH-mutant gliomas. Odds-ratios were 9.25 (5.17–16.52; 95% CI) for IDH-mutated gliomas and 12.85 (5.94–27.83; 95% CI) for IDH-mutated, 1p/19q co-deleted gliomas. Decreasing strength with increasing anaplasia implied a modulatory effect. No somatic mutations were noted at this locus in 114 blood-tumor pairs, nor was there a copy number difference between risk-allele and only-ancestral allele carriers. CCDC26 RNA-expression was rare and not different between the two groups. There were only minor subtype-specific differences in common glioma driver genes. RNA sequencing and LC-MS/MS comparisons pointed to significantly altered MYC-signaling. Baseline enhancer activity of the conserved region specifically on the MYC promoter and its further positive modulation by the SNP risk-allele was shown in vitro. Our findings implicate MYC deregulation as the underlying cause of the observed association

Functional proteomics outlines the complexity of breast cancer molecular subtypes

Breast cancer is a heterogeneous disease comprising a variety of entities with various genetic backgrounds. Estrogen receptor-positive, human epidermal growth factor receptor 2-negative tumors typically have a favorable outcome; however, some patients eventually relapse, which suggests some heterogeneity within this category. In the present study, we used proteomics and miRNA profiling techniques to characterize a set of 102 either estrogen receptor-positive (ER+)/progesterone receptorpositive (PR+) or triple-negative formalin-fixed, paraffin-embedded breast tumors. Protein expressionbased probabilistic graphical models and flux balance analyses revealed that some ER+/PR+ samples had a protein expression profile similar to that of triple-negative samples and had a clinical outcome similar to those with triple-negative disease. This probabilistic graphical model-based classification had prognostic value in patients with luminal A breast cancer. This prognostic information was independent of that provided by standard genomic tests for breast cancer, such as MammaPrint, OncoType Dx and the 8-gene Score

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography-tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

Post-translational modification of proteins with glycosylation is of key importance in many biological systems in eukaryotes, influencing fundamental biological processes and regulating protein function. Changes in glycosylation are therefore of interest in understanding these processes and are also useful as clinical biomarkers of disease. The presence of glycosylation can also inhibit protease digestion and lower the quality and confidence of protein identification by mass spectrometry. While deglycosylation can improve the efficiency of subsequent protease digest and increase protein coverage, this step is often excluded from proteomic workflows. Here, we performed a systematic analysis that showed that deglycosylation with peptide-N-glycosidase F (PNGase F) prior to protease digestion with AspN or trypsin improved the quality of identification of the yeast cell wall proteome. The improvement in the confidence of identification of glycoproteins following PNGase F deglycosylation correlated with a higher density of glycosylation sites. Optimal identification across the proteome was achieved with PNGase F deglycosylation and complementary proteolysis with either AspN or trypsin. We used this combination of deglycosylation and complementary protease digest to identify changes in the yeast cell wall proteome caused by lack of the Alg3p protein, a key component of the biosynthetic pathway of protein N-glycosylation. The cell wall of yeast lacking Alg3p showed specifically increased levels of Cis3p, a protein important for cell wall integrity. Our results showed that deglycosylation prior to protease digestion improved the quality of proteomic analyses even if protein glycosylation is not of direct relevance to the study at hand

Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the bla[subscript NDM] gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Department of Chemistr

New targeted approaches for the quantification of dataâ independent acquisition mass spectrometry

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136721/1/pmic12608.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136721/2/pmic12608_am.pd

A model-based assay design to reproduce in vivo patterns of acute drug-induced toxicity

For more than a decade pharmaceutical R&D has been hampered by considerable attrition rates during clinical trials. The main reasons for drug failure is related to the lack of efficacy, limitations with respect to ADME (absorption, distribution, metabolism and excretion) properties, and—in approximately 30% of the cases—unforeseen toxicity (Kola and Landis 2004). The majority of adverse drug reactions observed in the clinical phase refer to organ injuries, e.g. of the cardiovascular system, the liver, the central nervous system and skeletal muscle (Cook et al. 2014). This clearly demonstrates the limited predictive accuracy of current preclinical models such as the rodent bioassay in evaluating repeated dose toxicity for predicting human toxic risks. It has been argued that overall, only 43% of toxic effects in humans may be correctly predicted by applying rodent-based safety evaluation protocols due to the fact that these assays tend to generate relatively large numbers of false negative as well as false positive read outs (Hartung 2009)

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

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