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ItemMolecular mechanisms of artemisinin action and resistance in the malaria parasite Plasmodium falciparum( 2018)Malaria is the disease caused by infection of red blood cells with the protozoan parasite, Plasmodium. In 2016 alone, 216 million people suffered from malaria, leading to 445 000 deaths. Artemisinin-based combination therapies are the first-line treatment currently recommended by the World Health Organisation, for uncomplicated Plasmodium falciparum malaria. Youyou Tu, the scientist who led the team that discovered artemisinin, was awarded a share in The Nobel Prize for Medicine in 2015, in recognition of the importance of this discovery. Yet the mechanism of action of this life saving drug remains largely unknown. Artemisinin and its derivatives (ARTs) are sesquiterpene lactones that contain a 1,2,4- trioxane core and endoperoxide bridge. ARTs are widely accepted to be pro-drugs that are activated inside the cell by iron-catalysed reductive scission of the endoperoxide bridge. The resultant radical species is thought to rapidly react with accessible nucleophiles, including free thiols of cysteine residues in nearby proteins. Several studies have demonstrated that ARTs form adducts with hundreds of different parasite proteins in different compartments of the cell. However, the critical event leading to parasite death has not yet been elucidated. We hypothesised that ART-induced parasite killing is triggered by a lethal accumulation of cellular and protein damage. Here we show, using the novel cell-permeable thiol probe, tetraphenylethene maleimide (TPE-MI), that treatment of P. falciparum cultures with the clinically relevant ART derivative dihydroartemisinin (DHA), causes an increase in the level of unfolded proteins in the cell, indicating protein damage. We further show that DHA activates the Unfolded Protein Response (UPR), a well-conserved eukaryotic signalling pathway triggered by accumulation of unfolded proteins in the endoplasmic reticulum (ER). The UPR works to restore by protein homeostasis by arresting protein translation, thereby halting the influx of newly synthesised unfolded protein into the ER and thus preventing further increases in unfolded protein and protecting the cell from unfolded/misfolded protein damage. The UPR of protozoan parasites primarily involves stalling of protein translation via eIF (eukaryotic initiation factor)-2a phosphorylation. The P. falciparum kinase responsible Abstract ii for regulation of the UPR has not yet been identified. Here we identify, using genetic knockdown and small molecule inhibition, that Protein Kinase 4 (PK4) phosphorylates eIF2a in response to DHA and 1, 4-dithiothreitol (DTT), a reducing agent well known to induce ER stress and activation of the UPR. The ER also works to restore protein homeostasis by disposing of terminally misfolded proteins via the ubiquitin-proteasome pathway, a process termed ER-associated degradation. We show that DHA treatment actually results in a reduced capacity of the proteasome to degrade protein, thereby resulting in a build-up of polyubiquitylated proteins. Furthermore, we find that co-treatment of parasites with DHA and chemical inhibitors of polyubiquitylation, prevents polyubiquitylated proteins from accumulating, reduces the level of ER stress and strongly antagonises DHA-induced killing. These findings lead us to conclude that accumulation of polyubiquitylated proteins is the critical event that underlies DHA-induced parasite killing. Furthermore, we show that ART resistant parasites appear to accumulate a lower level of polyubiquitylated proteins following ART treatment. Thus, a defect in protein ubiquitylation may underlie ART resistance. Altogether, we propose a mechanism for ART action, whereby ARTs kill parasites via a two-pronged approach: inducing protein misfolding/unfolding and preventing protein degradation.