Faaiz, M. | Search bioRxiv

bioRxiv2025

A Plasmodium falciparum molecular mechanism of heme binding and sensitivity to artemisinins

Mutations in Plasmodium falciparum Kelch13 (K13) confer artemisinin resistance (ART-R) which threatens global malaria control, but known K13 functions fail to explain clinical ART-R. We reported that K13 binds the oxidant heme in vitro, however, its functions in redox-stress, cell survival and death remained unknown. Since taut control of free heme is not feasible in infected erythrocytes, we utilized a non-erythroid cell model to show that K13 directly binds and is stabilized by nanomolar heme levels. K13 also binds and regulates a major redox transcription factor, which is displaced by heme into the nucleus, to raise redox-stress responses that become suppressed during artemisinin-induced death (ART-death). K13s evolutionarily conserved kelch domain confers heme-binding and ART-death characteristics to its mammalian orthologue KEAP1. Chemical or genetic elevation of K13, fuels ART-death proportionate to K13 levels even in vast excess of heme, suggesting a novel plasmodial redox-survival mechanism licenses ART-death in clinical ART-R.

cell biology↗

bioRxiv2024

Artemisinin-resistant Plasmodium falciparum Kelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation

The rapid emergence of artemisinin resistance (ART-R) poses a challenge to global malaria control efforts. ART potency is triggered by ferrous iron- and/or heme-mediated cleavage of the endoperoxide bond to generate reactive heme-ART alkoxy radicals and covalent heme-ART adducts that alkylate parasite targets or inhibit the detoxification of heme into {beta}-hematin crystals; both of which lead to parasite death. Mutations in the P. falciparum Kelch-containing protein Kelch13 (PfKekch13) confer clinical ART-R, in which the resistant parasites exhibit impaired hemoglobin uptake, reduced heme yield, and thus decreased ART activation. However, a more direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that recombinant, purified PfKelch13 wild-type (WT) protein displays measurable binding affinity for both iron and heme, the main effectors for ART activation. Comparative biochemical analyses further indicate weaker heme-binding affinities in the two Southeast Asian ART-R PfKelch13 mutants C580Y and R539T compared to the ART-sensitive WT and A578S mutant proteins, which ultimately translates into reduced yield of heme-ART derivatives. In conclusion, this study provides the first evidence for regulated ART activation via the heme-binding propensity of PfKelch13, which may contribute towards modulating the level of ART-R in malaria parasites with PfKelch13 mutations.

biochemistry↗