Relevance of the abundance and redox state of Kelch13 for the artemisinin susceptibility of Plasmodium falciparum

  • Malaria is still a big problem globally causing more than 400,000 deaths each year. Although very effective antimalarial drugs are on the market, their mode of action is still not fully understood. In the last years some patients showed an increased clearance time from Plasmodium falciparum malaria parasites after a therapy with the most effective antimalarial compound artemisinin. It was shown that mutations in the propeller domain of a protein called PfKelch13 are directly linked to this decreased susceptibility towards the drug. To gain insights into the protein function, I produced different mutants of a truncated version of this protein containing the BTB and propeller domain in high yield and purity in insect cells. I showed a positive correlation between the solubility of the recombinant protein and the artemisinin susceptibility. Prominent PfKelch13 mutants from the field with decreased artemisinin susceptibility (I543T, R539T, C580Y) were insoluble when recombinantly expressed suggesting that improper folding of PfKelch13 leads to this decrease in sensitivity. The mutation C580Y is the most frequent mutation in South East Asia. The substitution of this cysteine residue does not allow the formation of an intramolecular disulfide bond with cysteine C532 according to an existing crystal structure. Interestingly, substitution of these cysteines to serines did not show improper folding in insect cells, arguing that rather specific substitutions than the residue position itself are responsible for the alteration of drug sensitivity. To test the impact of the disulfide bond on artemisinin susceptibility, I generated stable transgenic parasites expressing the corresponding serine mutations. Neither C580S nor C532S showed decreased artemisinin susceptibility. Therefore, it could be excluded that the formation of the intramolecular disulfide bond has an influence on artemisinin susceptibility. We further asked, if the protein abundance has an influence on the sensitivity towards the drug. I successfully generated stable transgenic parasites expressing His8-PFKELCH13 fused to the glmS riboswitch. I showed that protein levels could be efficiently down-regulated by more than 90% resulting in a very low parasite susceptibility towards artemisinin. This strain offers a basis for future experiments in the understanding of the impact of PfKelch13 protein levels on biochemical pathways in malaria parasites. Peroxiredoxins (Prxs) play an important role in protecting the cell from high amounts hydroperoxides. Among the five known Prxs in P. falciparum our group took PfAOP as a model enzyme to study the catalytic cycle. It has been shown that PfAOP reduces hydroperoxides like H2O2 or tBuOOH with fast kinetics, and that reduction of the protein is linked to the GSH/Grx system (Djuika et al. 2013). However, no direct kinetic data was available for the reductive half-reaction of PfAOP and GSH. In this thesis, I qualitatively showed that oxidized PfAOP can be glutathionylated and that in a next step glutathione can be transferred to PfGrx. I further determined the rate constants of the glutathionylation of PfAOP by stopped-flow measurements. Rate constants of around 10^5 M-1s-1 indicate a fast kinetic that is able to protect the protein from hyperoxidation and inactivation. Furthermore, I determined the activation energy, entropy and enthalpy for this reaction of 41.1 kJ/mol, -0.79 J/mol and 39.8 kJ/mol, respectively. Hence, the activation energy of the glutathionylation of oxidized PfAOP suggests the break of two to three hydrogen bonds and is rather temperature-independent.

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Author:Robin Schumann
Advisor:Marcel Deponte
Document Type:Doctoral Thesis
Language of publication:English
Publication Date:2022/04/13
Year of Publication:2021
Publishing Institute:Technische Universität Kaiserslautern
Granting Institute:Technische Universität Kaiserslautern
Acceptance Date of the Thesis:2021/03/31
Date of the Publication (Server):2023/02/15
Number of page:XIII, 110
Faculties / Organisational entities:Kaiserslautern - Fachbereich Chemie
DDC-Cassification:5 Naturwissenschaften und Mathematik / 540 Chemie
Licence (German):Creative Commons 4.0 - Namensnennung (CC BY 4.0)