Research

Wider research context Of the five human heme peroxidases, evolved to perform widely divergent functions, thyroid peroxidase (TPO) is the least well studied. The multidomain membrane protein catalyses the biosynthesis of thyroid hormones, which are essential for metabolism, growth and development of the human body. With the use of hydrogen peroxide TPO catalyses the iodination and coupling of tyrosine residues on the surface of thyroglobulin (TG) in the thyroid gland. However, there are open questions about substrate selectivity, biochemical characteristics, the coupling mechanism and the roles of the individual domains. Critically, TPO is at the core of two autoimmune thyroid diseases that combined afflict nearly 150 million people but the only two clinically approved TPO inhibitors are not specific. Objectives This project will elucidate the biochemical and structural characteristics of TPO. The main goals are (I) understanding the kinetics and substrate specificity of TPO, (II) provide structural data of TPO alone and of relevant ligated states and (III) clarify the mode of action of TPO inhibitors. Approach In preliminary work an expression and purification protocol for truncated TPO variants was established. Importantly, it was found that it is possible to reconstitute and link the heme cofactor even after purification, yielding highly pure and enzymatically active recombinant TPO. This allows the first detailed spectroscopic, thermodynamic and structural study of the enzyme. This will include an analysis of the reaction kinetics of TPO using a range of methods, including stopped-flow UV-vis spectroscopy for pre-steady kinetics, analysis of the interaction with small molecules (i.e. inhibitors, ligands) and TG with thermodynamic and mass spectroscopic assays. Finally, this project aims to solve the X-ray crystal structure of TPO alone and in complex with biologically relevant substrates, ligands and inhibitors. Level of originality TPO is crucial in thyroid hormone biosynthesis. However, to date the available biochemical and structural data is scarce and does not allow (I) a clear rationalization of the structure-function relationship of the active site architecture and TPO reactivity, (II) the role of the additional TPO domains and (III) the design of new more specific inhibitors. This project aims to provide a complete enzymatic and structural characterization of TPO to address these issues. Primary researchers involved Vera Pfanzagl graduated from the international PhD program BioToP at BOKU in 2019. She worked primarily on structure-function relationships of heme enzymes and during her postdoc focused on human heme peroxidases. Within this Fellowship she aims to obtain her habilitation to advance her academic career. She will be supported and coached by Kristina Djinovic-Carugo, a highly recognized expert in structural biology and head of EMBLE Grenoble and Chris Oostenbrink (professor at BOKU), an expert in molecular modelling and simulation.

The mammalian immune system possesses a remarkable ability to discern self from non-self, a critical function in safeguarding against infections. At the molecular level, this discrimination is facilitated by pattern recognition receptors present on eukaryotic cells, which can identify conserved non-self molecules characteristic of microorganisms. Among these molecules, lipopolysaccharide (LPS) stands out as a complex glycolipid abundantly present in Gram-negative bacterial cell wall, playing a central role in host-pathogen interaction. LPS is universally recognised by specific innate immune proteins that elicit a beneficial pro-inflammatory defense response to infection while maintaining immune homeostasis. However, bacterial pathogens possess various mechanisms to adapt their cell membranes in response to transmission between the environment, vectors, and human hosts, often altering LPS composition to modulate the host immune response. In particular, modifications to the phosphate groups of lipid A, the major immunostimulatory component of LPS, can shield bacteria from recognition by host cationic antimicrobial peptides. Yet, the impact of such modifications on LPS-specific pattern recognition receptors of the host innate immune system remains largely unexplored, particularly with regard to the recently identified cytosolic LPS-sensing proteins crucial for anti-tumor immunity. Due to the high heterogeneity of bacterial glycans and the inherent instability of modified phosphate groups, the isolation of structurally defined intact LPS fragments from bacterial sources is not feasible. Chemical synthesis, however, is a reliable method for providing molecularly defined immunomodulatory LPS motifs to study the effects of unique phosphate group modifications on the interaction with host immune receptors involved in antitumour defence. Carbohydrate chemistry, or glycochemistry, offers versatile tools for the synthesis of complex glycans, providing structurally defined, homogeneous molecules of high purity suitable for biological studies. Leveraging the glycochemistry toolbox, our project aims to develop innovative synthetic strategies for the assembly of complex phosphorylated glycans, culminating in a library of bacterial LPS motifs with phosphate group modifications reflecting those found in different bacterial species. In collaboration with international research groups in immunology and structural biology, we will investigate the immunobiological activity and interaction of our synthetic phosphorylated glycolipid-glycan library with corresponding proteins. By developing a collection of synthetic bacterial lipid A variants and LPS epitopes with uniquely modified phosphate groups, our research aims to elucidate the structural and molecular basis of their interaction with host innate immune receptors, thereby advancing our understanding of LPS-induced antibacterial defense and antitumor immunity mechanisms.

The final sensorial quality of a wine is the result of a multitude of interactions between all the chemical components within the wine and specific environmental factors such as the temperature of the wine. Since influenced by numerous factors such as grape varieties, growing conditions, climate change, yeast strains, wine making technologies, human experiences, the evaluation and preservation of wine quality – in terms of reproducibility from year to year - is nowadays the main challenge for both wine producers and wine science community. Viticultural practices aim primarily at producing high quality grapes that would reflect varietal flavours and aromas and/or characters typical for a specific region or terroir. In Austria, Districtus Austriae Controllatus (DAC) is a classification for regionally typical quality wine that provides products of distinction in wine market. An accurate evaluation and assessment of the wine quality, identity and typicity is of high significance for vintners to perform proper wine classification and target marketing. The aim of this project is on grape and wine quality evaluation, and regional typical quality characterization and prediction using elemental and sensory analysis, non-targeted and targeted metabolomics, spectroscopic approaches, and artificial intelligence. Grape quality is the most important factor for making high quality wine and some grape metabolites can have a strong relation to the wine quality. The relationship between the grape metabolites and the wine quality will be explored using non-targeted metabolomics and spectroscopic approaches and wine quality prediction models generated by artificial intelligence and machine learning algorithms. Of particular focus in this project is providing detailed chemical characterization that elucidates the influence of the Viennese wine growing region (origin) on Viennese Gemischter Satz DAC and Grüner Veltliner. As final output of the project, software, apps and a unique quality mark tag will be developed, for wine quality prediction and authenticity assessment based on established databases. This solution will be designed and developed to prove the identity and authenticity of each bottle and trace them. In turn, the outcomes of this project aim to both support origin marketing and future maintenance of wine production processes and wine quality in Vienna.