The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in higher eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the Ring1B/Bmi1/UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with multiple nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome.

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of Plasmodium falciparum carrying variants of the pfk13 locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, ubp1 and ap2μ, are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic P. falciparum clones, each carrying orthologues of mutations in pfap2μ and pfubp1 associated with artemisinin resistance in Plasmodium chabaudi, were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce in vitro artemisinin susceptibility at the early ring stage in P. falciparum. These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite’s adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in Plasmodium parasites should now be elucidated.

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13. Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent. IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum. In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

Studies of the susceptibility of Plasmodium falciparum to the artemisinin family of antimalarial drugs provide a complex picture of partial resistance (tolerance) associated with increased parasite survival in vitro and in vivo. We present an overview of the genetic loci that, in mutant form, can independently elicit parasite tolerance. These encode kelch propeller domain protein PfK13, ubiquitin hydrolase UBP-1, actin filament-organising protein Coronin, also carrying a propeller domain, and the trafficking adaptor subunit AP-2μ. Detailed studies of these proteins and the functional basis of artemisinin tolerance in blood stage parasites are enabling a new synthesis of our understanding to date. To guide further experimental work, we present two major conclusions. Firstly, we propose a dual-component model of artemisinin tolerance in P. falciparum comprising suppression of artemisinin activation in early ring-stage by reducing endocytic haemoglobin capture from host cytosol, coupled with enhancement of cellular healing mechanisms in surviving cells. Secondly, these two independent requirements limit the likelihood of development of complete artemisinin resistance by P. falciparum, favouring deployment of existing drugs in new schedules designed to exploit these biological limits, thus extending the useful life of current combination therapies.