Thiol-based regulation of protein modifications in a host-pathogen setting

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During the first funding period of the priority program SPP1710, our group has studied the redox state of thiol groups in the proteins of bacteria exposed to cells in our immune system. We recognized that hypochlorous acid (HOCl) is the major reactive species influencig the thiol redox state of proteins in phagocytized bacteria. The release of HOCl in the phagolysosome of these neutrophils leads to the complete breakdown of the cellular thiol-disulfide redox state in the phagocytized bacteria.In this funding period, we propose to elucidate how the release of HOCl and other reactive species in the phagolysosome affects the immune cells themselves. We propose to study the distribution of reactive species in the host cell using the roGFP2-based redox probes that we already successfully established to determine the redox state of phagocytized bacteria. Additionally, we intend to study, which protein thiols in the phagocytic cell are affected by the reactive species, in order to uncover potential redox regulation mechanisms. In a third part of our project we want to study the involvement of thioredoxin in the removal of N-chlorinations from proteins. N-chlorination of lysine and arginine sidechains is a post-translational modification caused by HOCl, and, during the first funding period, we found out that it can be reduced by thioredoxin, linking this HOCl-induced oxidative protein modification to the thiol-redox system of the cell.To test our hypotheses, we plan to:1. Express roGFP2-based probes in the neutrophil-like cell line PLB-985 to real-time monitor redox changes in relevant cellular compartments in response to the exposition to opsonized E. coli cells.2. Monitor changes in the thiol redox proteome state of different compartments of the neutrophil-like cell line PLB-985 when exposed to bacteria.3. Elucidate the mechanism of the NADPH-dependent removal of N-Chlorination modifications in proteins by thioredoxin.