▪ Abstract Research in the past decade has revealed that many cytosolic proteins are recruited to different cellular membranes to form protein-protein and lipid-protein interactions during cell signaling and membrane trafficking. Membrane recruitment of these peripheral proteins is mediated by a growing number of modular membrane-targeting domains, including C1, C2, PH, FYVE, PX, ENTH, ANTH, BAR, FERM, and tubby domains, that recognize specific lipid molecules in the membranes. Structural studies of these membrane-targeting domains demonstrate how they specifically recognize their cognate lipid ligands. However, the mechanisms by which these domains and their host proteins are recruited to and interact with various cell membranes are only beginning to unravel with recent computational studies, in vitro membrane binding studies using model membranes, and cellular translocation studies using fluorescent protein-tagged proteins. This review summarizes the recent progress in our understanding of how the kinetics and energetics of membrane-protein interactions are regulated during the cellular membrane targeting and activation of peripheral proteins.

Artemisinins are key anti-malarial drugs, but artemisinin resistance has been increasing; this study identifies the molecular target of artemisinins as phosphatidylinositol-3-kinase and increase of the lipid product phosphatidylinositol-3-phosphate induces resistance in Plasmodium falciparum. The emergence of artemisinin resistance is a major threat to world-wide malaria treatment and control and although artemisinins have been linked with a variety of cellular factors, there has been no consensus on the relevant biochemical targets or mechanisms underpinning resistance. Here Kasturi Haldar and colleagues show that artemisinins target the parasite phosphatidylinositol-3-kinase (PfPI3K) to inhibit the production of phosphatidylinositol 3-phosphate (PI3P). Mutation in PfKelch13, a previously identified resistance marker, increases levels of PfPI3K in both clinically derived strains and in engineered laboratory parasites. This work points to PfPI3K as the key mediator of artemisinin resistance and a target for malaria elimination. Artemisinins are the cornerstone of anti-malarial drugs1. Emergence and spread of resistance to them2,3,4 raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance5,6,7,8,9,10. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.

P47 phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X‐ray structure of the p47 phox PX domain revealed two distinct basic pockets on the membrane‐binding surface, each occupied by a sulfate. These two pockets have different specificities: one preferentially binds phosphatidylinositol 3,4‐bisphosphate [PtdIns(3,4)P2] and is analogous to the phophatidylinositol 3‐phosphate (PtdIns3P)‐binding pocket of p40 phox , while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipid‐binding pockets radically increases membrane affinity. Strikingly, measurements for full‐length p47 phox show that membrane interaction by the PX domain is masked by an intramolecular association with the C‐terminal SH3 domain (C‐SH3). Either a site‐specific mutation in C‐SH3 (W263R) or a mimic of the phosphorylated form of p47 phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater affinity than the isolated PX domain.

Mechanisms that alter protein phosphatase 2A (PP2A)‐dependent lung tumour suppression via the I2PP2A/SET oncoprotein are unknown. We show here that the tumour suppressor ceramide binds I2PP2A/SET selectively in the nucleus and including its K209 and Y122 residues as determined by molecular modelling/simulations and site‐directed mutagenesis. Because I2PP2A/SET was found overexpressed, whereas ceramide was downregulated in lung tumours, a sphingolipid analogue drug, FTY720, was identified to mimick ceramide for binding and targeting I2PP2A/SET, leading to PP2A reactivation, lung cancer cell death, and tumour suppression in vivo . Accordingly, while molecular targeting of I2PP2A/SET by stable knockdown prevented further tumour suppression by FTY720, reconstitution of WT‐I2PP2A/SET expression restored this process. Mechanistically, targeting I2PP2A/SET by FTY720 mediated PP2A/RIPK1‐dependent programmed necrosis (necroptosis), but not by apoptosis. The RIPK1 inhibitor necrostatin and knockdown or genetic loss of RIPK1 prevented growth inhibition by FTY720. Expression of WT‐ or death‐domain‐deleted (DDD)‐RIPK1, but not the kinase‐domain‐deleted (KDD)‐RIPK1, restored FTY720‐mediated necroptosis in RIPK1 ^−/− MEFs. Thus, these data suggest that targeting I2PP2A/SET by FTY720 suppresses lung tumour growth, at least in part, via PP2A activation and necroptosis mediated by the kinase domain of RIPK1.

Epsin and AP180/CALM are endocytotic accessory proteins that have been implicated in the formation of clathrin-coated pits. Both proteins have phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂)-binding domains in their N termini, but these domains are structurally and functionally different. To understand the basis of their distinct properties, we measured the PtdIns(4,5)P₂-dependent membrane binding of the epsin N-terminal homology (ENTH) domain and the AP180 N-terminal homology (ANTH) domain by means of surface plasmon resonance and monolayer penetration techniques and also calculated the effect of PtdIns(4,5)P₂ on the electrostatic potential of these domains. PtdIns(4,5)P₂ enhances the electrostatic membrane association of both domains; however, PtdIns(4,5)P₂ binding exerts distinct effects on their membrane dissociation. Specifically, PtdIns(4,5)P₂ induces the membrane penetration of the N-terminal α-helix of the ENTH domain, which slows the membrane dissociation of the domain and triggers the membrane deformation. These results provide the biophysical explanation for the membrane bending activity of epsin and its ENTH domain.