Unraveling the role of HECT E3 ubiquitin ligases in Plasmodium egress

Malaria is a deadly disease that significantly increases the risk of heart disease. It is caused by infection with Plasmodium parasites which have a life cycle with three main phases. The clinical symptoms of malaria manifest during the blood stage of infection, where parasites undergo asexual replication inside red blood cells (RBCs). Plasmodium falciparum is the deadliest species causing the disease, responsible for over 90% of malaria-related deaths worldwide. A greater understanding of the molecular pathways that regulate asexual proliferation in RBCs is crucial for identifying new drug targets to combat antimalarial resistance. Protein ubiquitylation is a prevalent post-translational modification that regulates a wide variety of cellular processes in eukaryotes, such as cell cycle progression and protein homeostasis. Plasmodium encode their own ubiquitin system, consisting of ubiquitin, a proteasome, and over 50 putative ubiquitin ligases (E3s), that is essential for parasite replication in RBCs. It was recently found that HECT E3 activity is required during parasite egress from RBCs, a necessary step for the re-invasion of RBCs, yet the specific HECT-type E3s involved in this process remain unclear. Aim 1 will utilize a reverse-genetics approach targeting HECT-containing E3s in P. falciparum. Genetic perturbation will be achieved using a rapamycin-inducible knockout system, enabling stage-specific characterization of the HECT E3s. Aim 2 will perform proteomic profiling of ubiquitylated proteins utilizing a novel ubiquitin enrichment reagent, both with and without chemical and genetic perturbation, to identify candidate substrates of the HECT E3s. Finally, candidate substrates will be validated using biochemical and genetic approaches in P. falciparum. In summary, this study will define specific HECT E3s involved in regulating parasite egress from RBCs as well as their target substrates. It will also provide mechanistic insights into how HECT-mediated ubiquitylation modifies protein function in Plasmodium. Overall, this proposal will significantly advance our understanding of ubiquitylation in Plasmodium and improve our ability to develop antimalarials targeting this pathway.

Basic Science

Jan 2024 — Dec 2025

$144,580

United States