Research

Science communication is a rapidly developing field and of great importance, especially for young researchers. In this project, scientists (Postdoc Level) will be trained in different forms of science communcation. The coaching involves workshops coaching sessions in small groups. Visibility will be enhanced by featuring the researchers on BOKU social media channels. In addition, a podcast will be launched to highlight the work of young, inspiring researchers that serve as role models for future scientific generations.

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.