241 million cases of human malaria disease and 627,000 deaths were reported in 2021. Malaria eradication is hindered by the complex dual-host life cycle of the causative Plasmodium spp parasite and the rapid evolution of parasite-derived antimalarial resistance. The best-in-class antimalarials target parasite proteins expressed in the symptomatic asexual blood stage of disease. These protein virulence factors and their homeostasis, encompassing protein synthesis, activity, and degradation, are critical for parasite survival within the human host.
Ubiquitination is the key post-translational modification in eukaryotic cells that governs protein degradation, localisation, and activity. This process results in the tagging of the 7.6 kDa ubiquitin molecule to lysine or methionine residues on protein substrates by a concerted enzyme cascade of E1, E2 and E3 enzymes. The E3 enzymes mediate the final key ubiquitin ligation step in this reaction and govern substrate selection. To accommodate the discriminate selection of specific substrates, most eukaryotic cells encode a diverse repertoire of E3 ligases with about 700 members expressed in human cells. Of these enzymes, the largest superfamily includes the Cullin-Ring-Ligase (CRL) complexes of E3 ligases. A single modular CRL complex contains one of 7 Cullin scaffold proteins which simultaneously engages a ubiquitin-binding RING-type protein, a substrate adaptor protein, and one of multiple substrate receptors.
Plasmodium falciparum, the most deadly human malaria parasite, encodes for the critical proteins required for ubiquitination, but we currently lack functional characterization of the key players in this pathway.
In this study, we characterised the P. falciparum CRL E3 ligases using CRISPR/Cas9 conditional gene knockouts, high-resolution imaging, and whole-cell mass spectrometry. We discovered an essential but minimal CRL repertoire in parasites, controlled by only two Cullin scaffolds. We identified a PfCullin1-linked CRL complex involved in parasite inner-membrane biogenesis and DNA replication, which remarkably recruits only one substrate receptor, compared to the ~70 receptors used interchangeably by human cells. A second CRL complex functioning through a PfCullin4 scaffold utilises a previously unidentified adaptor protein and receptors to support correct DNA replication. Collectively these results identify for the first time, the essential role of human malaria parasite E3 ubiquitin ligases.