Dr. Bengt Christian Hedberg received his Ph.D. in organic chemistry, Uppsala University, Sweden, in 2005. From 2008 to 2014, he served as the research leader of Max Planck, Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Germany, and from 2014 to now, he has served as a professor of bioorganic chemistry, Institute of Chemistry, Umeå University, Sweden. In 2013, he was selected as a Wallenberg Academy Fellow of the Royal Swedish Academy of Sciences. In the past five years, he has published more than 15 papers in PNAS, Angelwandte Chemie, Biological Journal, Journal of Organizational Chemistry, Organizational Letters and other journals.
This presentation covers a new method denotedReactiveProtein -ProteomeProfiling“RP3”. We have created a robust workflow for absolute substrate profiling of bacterial toxins against cellular target proteins employing covalent nucleotide co-substrates resulting in covalent ternary complexes. During infection, pathogenic bacteria tweak their eukaryotic hosts by translocating toxins. Toxins display catalytic activity against host cell proteins, targeting key functions, often using an abundant metabolite as co-substrate. No tools exist for the proteomic evaluation of the absolute substrate profile of a given toxin. Non-covalent affinity enrichment gives biased results depending on the reagent and relative target abundance. Our concept is based on covalent and subsequently cleavable capture of the target proteins by the reactive co-substrate of the toxin. Mutants (X to C) in the nucleotide-binding pocket of the toxin IbpA (H. somni)were modified with ATP-analogues carrying an electrophile. The binary complex forms the ternary complex with the IbpA-substrate cdc42in vitro. A number of other toxins belonging to the Fic-class have been profiled. For ADP-ribosylation, Pertussis toxin captures Giain vitrousing NAD+analogues. Altogether, this suggests that the RP3concept is valid and practicable.The project is highly significant for the infection biology field in terms of profiling toxins, but also to the chemical biology community, by moving ABPP to macromolecules via RP3.