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Plasmodium falciparum Antioxidant Protein: Kinetic Characterization and Physiological Relevance as a Potential Redox Sensor in Malaria Parasites

Staudacher, Verena

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Abstract

Peroxiredoxins (Prx) are ubiquitous thiol-dependent hydroperoxidases and belong to the most abundant enzymes in a variety of organisms from all kingdoms of life. Furthermore, Prx act as redox sensors in hydrogen peroxide signaling. The Antioxidant Protein (PfAOP), a Prx5-type enzyme from the malaria parasite Plasmodium falciparum, was recently shown to belong to a special subclass of Prx using glutaredoxin (Grx) and reduced glutathione (GSH) as electron donors. In this work, two modulatory residues of PfAOP were characterized that influence the enzyme parameters and the inactivation susceptibility. Gain- and loss-of-function mutants were generated in which the size of residue 109 was altered and the catalytically relevant but nonessential cysteine residue 143 was present or absent. Using steady-state kinetic measurements with recombinant PfAOP, the overall reactivity of these mutant enzymes was analyzed and the mechanism of the reduction of the oxidized enzyme was unraveled. Rate constants for the oxidation and over-oxidation of the peroxidatic cysteine residues were determined by stopped-flow kinetic measurements. A mutant enzyme was identified that is catalytically more active and more durable regarding hydrogen peroxide dependent inactivation compared to the wild-type enzyme. This suggests that PfAOP not only acts as a hydroperoxidase but might also have an additional function as part of a redox relay in vivo. Fusion constructs between redox-sensitive green fluorescent protein 2 (roGFP2) and Prx are valuable tools for redox measurements in living cells. Using the in vitro well characterized gain- and loss-of-function mutants of PfAOP, different fusion constructs with roGFP2 were generated and the in vivo roGFP2 readouts were analyzed in yeast. We showed that the ratiometrically measured degree of oxidation of the roGFP2 fusion proteins correlates with the corresponding in vitro enzyme properties of the attached Prx. Additionally, the roGFP2 signal can be used to map the over-oxidation based inactivation of the Prx. This will allow the assessment of protein structure-function relationships, like post-translational protein modifications, in vivo. Further future applications are the estimation of absolute intracellular peroxide concentrations and the improvement of redox sensors. To gain more insight into the physiological relevance of PfAOP, knockout parasites were generated in the P. falciparum strain 3D7 using the CRSIPR/Cas9 system. The 3D7Δpfaop knockout lines were viable and showed no significant growth phenotype under standard cell culture conditions. Furthermore, the IC50 values for external oxidants remained unchanged. In a previous conducted quantitative trait locus analysis a locus on chromosome 7, encoding 49 genes including PFAOP, was associated with altered artemisinin susceptibility in malaria parasites. Here, we showed that the deletion of the gene encoding PfAOP does neither affect IC50 values nor ring stage survival rates for artemisinin. Thus, the correlation between chromosome 7 and the artemisinin susceptibility is probably based on one of the other genes within the identified locus. Western blot analyses of PFAOP over-expressing P. falciparum strains in combination with peroxide challenges revealed a band corresponding to a probable interaction partner of PfAOP in the cytosol. However, this protein could not be identified so far and it remains to be shown if it is part of a peroxide dependent redox relay involved in redox signaling. In summary, the findings of this thesis lead to a better understanding of the kinetic mechanism of peroxiredoxins. Furthermore, it could be shown that in vitro kinetic properties of peroxidases correlate with the roGFP2 readout of corresponding fusion constructs inside living cells. So far, the physiological relevance of PfAOP remains unknown, but our results suggest that PfAOP might exert an additional function as a redox sensor in vivo.

Item Type: Dissertation
Supervisor: Lanzer, Prof. Dr. Michael
Date of thesis defense: 16 March 2018
Date Deposited: 26 Mar 2018 12:35
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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