The glutathione redox couple as a thiol switch operator in the malaria parasite Plasmodium falciparum

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Due to its crucial functions in rapidly growing and multiplying cells, the redox metabolism of many protozoan parasites including the malaria parasite Plasmodium is considered as a major target for chemotherapeutic prophylaxis and intervention. Notably, redox networks in malaria parasites have unique features such as the absence of a genuine catalase and glutathione peroxidase and the presence of plasmoredoxin and four distinct selenoproteins. Furthermore, redox signaling is a major regulatory principle in the parasite-host cell unit, it is involved in the pathophysiology of malaria, and several antimalarial drugs mediate their effects at least partially by redox-based mechanisms. Over the last years we have intensely studied redox-related enzymes in malaria parasites with respect to structure, function, localization, interaction, and (glutathione dependent) redox regulation. Within the SPP 1710 project proposed here, we aim to further elucidate the function and regulation of central redox switches in P. falciparum by focusing on thiol switch-based redox sensing and thiol switch-based interactions of proteins.1. Thiol switch-based redox sensing in P. falciparum. Within this first focus, we aim for genomic integration and systematic comparison of the redox probes hGrx1roGFP2 and sfroGFP2 as well as the H2O2 probe roGFP2Orp1 and novel H2O2 probes currently being developed. This shall result in robust detection systems in cytosol and organelles of the parasite. Wherever possible, the readout shall be transferred from the confocal laser scanning microscope to a plate reader. The genetically encoded redox probes shall be employed to study mechanisms of drug action and drug resistance in malaria parasites. We further plan to determine sulfenylation levels in unstressed and stressed parasites. Then, in a SILAC approach, the quantitative and qualitative changes in glutathionylation and, in direct comparison, sulfenylation under oxidative stress shall be elucidated. As in the first funding period, the impact of redox-regulatory thiol modifications on structure and function of selected enzymes shall be studied.2. Thiol switch-based interaction of proteins. In this second focus, using a mixed-disulfide fishing approach with mutants of PfPrx1a and PfPrx1m, we plan to identify the redox interactome of these peroxiredoxins. Selected interactions will be verified using recombinant proteins. The role of distinct cysteine residues in the interaction between the two Prx and their redoxins will be studied by surface plasmon resonance (SPR). The suitability of SPR in analyzing redox interactions and modifications shall be systematically compared with ITC and microscale thermophoresis. In cooperation with other groups of the SPP consortium, SPR will be employed to study protein interactions, target specificity and reactivity.