Endocytosis in Malaria Parasites: Investigating the Function and Mechanism of the Endocytic Protein PfKelch13

Abstract

Malaria remains a major global health burden, with Plasmodium falciparum responsible for the majority of severe cases and mortality. Artemisinin-based combination therapies have significantly reduced malaria-related deaths, but the emergence of drug resistance threatens these advances. Mutations in the Kelch13 protein are the primary molecular markers of artemisinin resistance, yet the normal function Kelch13 remains poorly understood. This thesis investigates the structural stability, membrane association, and potential protein–protein interactions of Kelch13. Using a combination of varied protein constructs and expression techniques, lipid-binding assays, and pull-down experiments, this study aimed to define how Kelch13 domain architecture contributes to its biochemical function in cytostome-mediated endocytosis. Multiple constructs were expressed in either bacterial or mammalian systems to assess the impact of domain content and expression context on protein stability. Truncated Kelch13 expressed in Escherichia coli and full-length Kelch13 expressed in HEK293-F cells exhibited substantial contamination, instability, and degradation, while a shorter BTB/KRP construct expressed in HEK293-F cells yielded a more homogeneous and stable preparation. Lipid overlay assays suggested that Kelch13 may associate with negatively charged phospholipids, although interpretation is limited by protein instability and assay constraints. Notably, the BTB/Kelch construct did not display detectable lipid binding, indicating that the N-terminal and/or CCD regions absent from this construct may contribute to membrane association. Pull-down assays did not provide conclusive evidence for interactions between Kelch13 and dynamin-related proteins, due to protein heterogeneity and non-specific binding. Overall, this thesis provides preliminary evidence for the domain-dependent membrane association of Kelch13.