Aliquots of late schizonts were incubated with F-ArgMy1 (10 M) and a buffer control for 3 h
Aliquots of late schizonts were incubated with F-ArgMy1 (10 M) and a buffer control for 3 h. contribute to the force required during an invasion event (Physique ?Physique11a).4 The anchor point for this invasion force is provided by the buried clamp-like conversation between the tail of the parasites myosin motor myosin A (MyoA) and its light chain, Myosin A tail interacting protein (MTIP; Physique ?Physique11b). This conversation has previously been studied using a variety of binding assays, NMR, and an alanine mutation scan, attributing tight binding to key amino acids on each face of the helical MyoA tail.5,6 Open in a separate window Determine 1 Binding of My1, F-My1, and F-My2 to PfMTIP. (a) Linear model of glideosome and motor complex, within the context of erythrocyte host cell invasion. Adapted from Cowman et al.7 (b) Annotated crystal structure of a truncated Myosin-A peptide [799C816] (gray) clamped by recombinant PfMTIP, highlighting the C- (red) and N-terminal regions (green).5 (c) Peptide sequences and N-/C-terminal modifications for synthesized peptides based on the truncated PfMyoA[799C816] sequence, with an additional N-terminal glycine spacer. Green star indicates addition of a 5(6)-carboxyfluorescein moiety. (d) Thermodynamic parameters for ITC experiment of binding between My1 and F-My1 peptides and PfMTIP (= 2). (e) My1 peptide ITC binding isotherm titrated into (-)-Epigallocatechin gallate PfMTIP. (f) F-My1 peptide ITC binding isotherm titrated into PfMTIP. (-)-Epigallocatechin gallate (g) Direct binding of F-My1 (red) and F-My2 (blue) to PfMTIP, measured by fluorescence anisotropy (= 3). Recent work has exhibited that this MyoA motor is essential for malaria parasite invasion of the human red blood cell in the most virulent species affecting people, parasites, meaning that although parasites completed the invasion process, the invasion event lasted 10 min rather than 30 s. 8 A truncated MyoA[803C817] peptide was previously claimed to inhibit the growth of a culture, with (-)-Epigallocatechin gallate IC50 = 84 M.10 However, the targets engaged and localization/uptake of the peptide were undetermined, and subsequent work has cast doubt on this conclusion.6 While the MyoA:MTIP PPI offers a potentially exciting therapeutic target, it presents a number of challenges, particularly the localization of the fully formed MyoA:MTIP complex behind three unique membranes: the host erythrocyte plasma membrane, the parasitophorous vacuole (PV) membrane, and (-)-Epigallocatechin gallate the parasite plasma membrane.4 Previous research has elucidated the binding potential of a truncated MyoA peptide consisting of the C-terminal residues 799C816 with recombinant asexual cycle transitions through three Rabbit Polyclonal to ARHGEF19 developmental stages of growth over 48 h: rings, trophozoites, and schizonts. The ring stage initiates immediately postinvasion (PI) and is a relatively dormant phase; it is followed at ca. 12 h PI by the trophozoite stage, a period of intense growth for the parasite. An increased demand for nutrients during this rapid growth necessitates the formation of membrane channels, termed new permeability pathways (NPPs).11 Peptides are known to be brought in these NPPs, potentially providing a mechanism for delivery of a MyoA peptide.12 Finally, at ca. 36 h PI, the parasite transitions to a schizont and transforms into many discrete merozoites, preparing for egress at 48 h PI and subsequent invasion of new host erythrocytes. = 2).5,6,10 The observed binding affinity was concurrent with previously published values for the F-My1 peptide, parasite, an N-terminal 5(6)-carboxyfluorescein (FAM) moiety was added to the My1 peptide separated by a glycine spacer; this peptide was termed F-My1. A weaker-binding control was synthesized with a double mutation exchanging two residues from the hydrophilic and hydrophobic faces of the buried MTIP:MyoA conversation: F-My1 (R806A/A809R), termed F-My2 (sequences shown in Physique ?Physique11c).6 This approach was favored oversimple alanine mutation in order to keep the overall charge of both peptides the same and enable pairwise comparisons for parasite uptake. F-My1 and F-My2 were assayed by ITC and fluorescence anisotropy (FA). ITC showed that incorporation of N-terminal FAM was well tolerated in F-My1 (Physique ?Figure11d,f) with binding affinities remaining in the low nanomolar range ((3D7 strain) for 3 h during each of three life stages (ring, trophozoite, and schizont). Flow cytometry exhibited that cell permeability of F-My1 and F-My2 was generally low and heavily dependent on the lifecycle stage (Physique ?Physique22a), peaking at 13 1% for F-My1 in schizonts. A possible explanation for the increased uptake in late stage parasites is usually peptide entry through an NPP present only at late stages of schizogony. Alternatively, it may be due to increased leakiness of red blood cell and parasitophorous vacuole (PV) membranes when the cytoskeleton is usually broken down as the parasite prepares to egress.15 Regardless, it was necessary to optimize the peptide sequence to improve its cell permeability properties to enable uptake in earlier life stages, before the formation of the analyzed by flow cytometry. (a) Uptake of F-My1 and (-)-Epigallocatechin gallate F-My2 peptides over three stages of Pf3D7 lifecycle, percentage uptake calculated as.