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Research project (§ 26 & § 27)
Duration : 2020-11-02 - 2023-11-01

Human heme peroxidases figure prominently in human biology by contributing to tissue development and architecture, thyroid hormone biosynthesis and innate immunity. Myeloperoxidase (MPO), eosinophil peroxidase (EPO) and lactoperoxidase (LPO) exhibit an indispensable role in microbial killing by releasing potent antimicrobial oxidants. However, these reaction products may also adversely affect tissues and cause acute and chronic inflammatory diseases. Consequently, there is a need for highly selective inhibitors for MPO, EPO and LPO with no risk of off-target effects, i.e. interference with thyroid peroxidase and peroxidasin 1, which share a similar heme cavity architecture. It has been found that pathogenic bacteria like Staphylococcus aureus have evolved a broad repertoire of strategies to resist microbial killing including SPIN (Staphylococcal Peroxidase INhibitor) that binds tightly to MPO and inhibits its enzymatic activity. SPIN shares no sequence homology to other known proteins and consists of two functionally distinct domains, i.e. a small N-terminal domain which acts as a molecular plug of the access channel and a C-terminal domain which mediates the specific binding to human MPO. This project will characterize the structural basis as well as the thermodynamics and kinetics of binding of SPIN-aureus to and inhibition of MPO including its interference with the individual reaction steps in the halogenation and peroxidase cycle of MPO. We further aim to understand the structure and function of recently identified SPIN-aureus homologs from other staphylococcal species and of an artificially designed SPIN-consensus protein that binds to both MPO and EPO. These investigations will include both comprehensive biochemical and biophysical investigations as well as molecular dynamics simulations. These studies will provide the basis for the design of specific binders and inhibitors for human peroxidases. In detail, we aim to design specific inhibitors for MPO, EPO and LPO employing (i) a rational design including saturation mutagenesis and chemical engineering of the N-terminal plug and (ii) directed evolution of the binding domain for specific interaction with MPO, EPO and LPO using yeast surface display combined with fluorescence activated cell sorting of newly generated SPIN libraries. Summing up, this project will provide (i) the fundamental biochemical understanding of the interaction of SPIN proteins with the human heme peroxidases as well as (ii) will design and select SPIN-based inhibitors of MPO, EPO and LPO. Future studies will focus on the application and further development of these lead candidates as potential drugs in in vitro and in vivo studies.
Research project (§ 26 & § 27)
Duration : 2021-01-01 - 2023-12-31

Wider research context / theoretical framework: Glycans cover the surfaces of all cells and are vary between species and phyla. Hypotheses/research questions /objectives: Based on previous work by us and others, the major underlying hypotheses are that: (i) anionic/zwitterionic modifications of N-glycans vary between orders and species of arthropods and these structures are either (ii) bound by mammalian immune lectins involved, e.g., in recognition of arthropod-derived viruses, (iii) involved in interactions of pathogens with arthropods (e.g., mosquitoes) as intermediate or final hosts or (iv) have innate function, e.g., in development of the arthropod species itself. Approach/methods Enzymatic remodelling of saccharides, preparation/analysis of natural glycans from selected insects and probing saccharides/glycans in an microarray format with lectins, pentraxins and antibodies. Level of originality / innovation The project has the goal of delivering new information about insect glycomes.
Research project (§ 26 & § 27)
Duration : 2020-07-01 - 2023-06-30

Theoretical framework: Prokaryotic heme biosynthesis has been studied extensively over several decades. In 2015 this topic came back into the focus due to the discovery of the so-called “coproporphyrin-dependent” (CPD) heme biosynthesis pathway by Dailey and co-workers, which is mainly utilized by Gram-positive bacteria. The CPD pathway differs from the protoporphyrin-dependent pathway in the sequence of uroporphyrinogen to heme b transformation and the involved enzymes (UroD, CgoX, CpfC ChdC). Studies on CpfC were performed until recently using protoporphyrin IX instead of coproporphyrin III. Objectives: In-depth knowledge on the biochemistry and molecular enzymology of CpfCs is very limited. We aim at studying the protein biochemistry and elucidating the enzymatic mechanisms of the coproporphyrin ferrochelatases from (i) Firmicutes and (ii) Actinobacteria in order to understand the mechanism of catalysis, including substrate binding of both substrates and the enzymatic insertion of ferrous iron into coproporphyrin III. Methods: Biochemical and biophysical characterization of the coproporphyrin ferrochelatases (CpfCs) will be performed using multiple high-end spectroscopic methods and steady-state and pre-steady state kinetic characterization of wild-type and mutated enzymes. Further we will employ state-of the art structural biology methods to gain profound knowledge of the enzyme’s structure to the very detail, such as protonation states (by neutron crystallography). Innovation: The CPD heme biosynthesis pathway was recently described in literature and many questions about the enzymes’ mechanisms and interactions are unanswered. It is essential for Gram-positive bacteria and a few intermediate and Gram-negative bacteria. Understanding the biochemistry of the involved enzymes, focussing on CpfCs, and the overall regulation of heme biosynthesis, uptake and degradation of these bacteria is a highly important basic research question. This project will provide a strong basis for the future development of new therapeutics against pathogenic Gram-positive bacteria.

Supervised Theses and Dissertations