Interrogation of Ubiquitin-Conjugating Enzyme 13 in Plasmodium Parasites

Abstract

Nearly half of the global population is at risk of malaria infection, a disease caused by unicellular Plasmodium parasites. The evolution and persistence of these intracellular pathogens spans millions of years and has resulted in robust mechanisms of survival. Plasmodium falciparum, the deadliest parasite species, undergoes a complex lifecycle involving transmission from the mosquito vector to the human host where it first develops in the liver before invading red blood cells (RBCs). The majority of our therapeutic arsenal targets the asexual blood stage (ABS) of infection during which parasites cyclically replicate in RBCs, causing the clinical symptoms of malaria. However, rising resistance to the frontline antimalarial drug artemisinin (ART) continues to thwart eradication efforts. Hence, a greater understanding of essential pathways that support Plasmodium development and survival will aid in the discovery of novel therapeutic strategies.The diverse enzyme machinery that mediates the post-translational modification (PTM) of proteins with the small protein modifier ubiquitin (Ub) is a principal orchestrator of nearly all eukaryotic processes, yet knowledge of these molecular players in Plasmodium remains elusive. The wide array of Ub linkage types enables a dynamic Ub code that coordinates multitudes of critical cellular functions. Therefore, efforts to untangle and decipher this enigmatic code in Plasmodium will provide fundamental insight into parasite physiology, ultimately offering potential malaria drug targets.Herein, we interrogate the essential P. falciparum ubiquitin-conjugating enzyme 13 (PfUbc13), which coordinates noncanonical lysine 63-linked ubiquitin (K63-Ub) protein modifications. The regulation and consequences of K63-Ub modifications in Plasmodium are largely uncharted, providing vast areas for exploration. Orthologs of Ubc13, the central mediator of K63-Ub, have well-defined roles in the immune and stress responses of higher eukaryotes. However, the widely divergent and poorly annotated Plasmodium proteome requires focused investigations to resolve the roles of PfUbc13 and K63-Ub in supporting these pathogenic parasites.First, we identify and characterize the small molecule NSC697923 to be an irreversible, covalent inhibitor that targets the PfUbc13 catalytic cysteine. We then deploy NSC697923 to evaluate pharmacological targeting of Ubc13 against multiple Plasmodium life stages. We observe synergism against parasites in co-treatments of NSC697923 with the clinical ART derivative dihydroartemisinin (DHA), implicating PfUbc13 in the mode of action. Furthermore, we employ a chemoproteomic strategy, involving the isolation of K63-Ub-modified proteins from parasites treated with NSC697923, to unveil putative PfUbc13 substrate proteins. Our proteomic hits and subsequent biochemical studies support a role for PfUbc13 and K63-Ub modifications in maintaining global protein synthesis. We also propose additional enzymes that may be involved in the regulation of K63-Ub modifications in Plasmodium. Collectively, these findings broaden our understanding of Ub-dependent pathways that impact Plasmodium protein synthesis and establish important value for the development of innovative malaria therapies that target PfUbc13.