A single amino acid substitution in the pleckstrin homology domain of phospholipase C delta1 enhances the rate of substrate hydrolysis.

The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC delta1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 +/- 0.2 and 2.0 +/- 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4, 5)P2)-stimulated PI(4,5)P2 hydrolysis (interfacial Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P2 with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.Peer reviewe

Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy

BACKGROUND: Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte G(s) signaling blocked invasion by the human malaria parasite Plasmodium falciparum. METHODS AND FINDINGS: We investigated the erythrocyte guanine nucleotide regulatory protein G(s) as a novel antimalarial target. Erythrocyte “ghosts” loaded with a G(s) peptide designed to block G(s) interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte G(s) is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens G(s) activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host G(s) to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β(2)-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection. CONCLUSIONS: Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte G(s) antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials

Lipid rafts and malaria parasite infection of erythrocytes.

<p>Infection of human erythrocytes by the malarial parasite, Plasmodium falciparum, results in complex membrane sorting and signaling events in the mature erythrocyte. These events appear to rely heavily on proteins resident in erythrocyte lipid rafts. Over the past five years, we and others have undertaken a comprehensive characterization of major proteins present in erythrocyte detergent-resistant membrane lipid rafts and determined which of these proteins traffic to the host-derived membrane that bounds the intraerythrocytic parasite. The data suggest that raft association is necessary but not sufficient for vacuolar recruitment, and that there is likely a mechanism of active uptake of a subset of erythrocyte detergent-resistant membrane proteins. Of the ten internalized proteins, few have been evaluated for a role in malarial entry. The beta(2)-adrenergic receptor and heterotrimeric G protein G(s) signaling pathway proteins regulate invasion. The implications of these differences are discussed. In addition, the latter finding indicates that erythrocytes possess important signaling pathways. These signaling cascades may have important influences on in vivo malarial infection, as well as on erythrocyte membrane flexibility and adhesiveness in sickle cell anemia. With respect to malarial infection, host signaling components alone are not sufficient to induce formation of the malarial vacuole. Parasite proteins are likely to have a major role in making the intraerythrocytic environment conducive for vacuole formation. Such interactions should be the focus of future efforts to understand malarial infection of erythrocytes since host- and parasite-targeted interventions are urgently needed to combat this terrible disease.</p

Lipid rafts and malaria parasite infection of erythrocytes.

Infection of human erythrocytes by the malarial parasite, Plasmodium falciparum, results in complex membrane sorting and signaling events in the mature erythrocyte. These events appear to rely heavily on proteins resident in erythrocyte lipid rafts. Over the past five years, we and others have undertaken a comprehensive characterization of major proteins present in erythrocyte detergent-resistant membrane lipid rafts and determined which of these proteins traffic to the host-derived membrane that bounds the intraerythrocytic parasite. The data suggest that raft association is necessary but not sufficient for vacuolar recruitment, and that there is likely a mechanism of active uptake of a subset of erythrocyte detergent-resistant membrane proteins. Of the ten internalized proteins, few have been evaluated for a role in malarial entry. The beta(2)-adrenergic receptor and heterotrimeric G protein G(s) signaling pathway proteins regulate invasion. The implications of these differences are discussed. In addition, the latter finding indicates that erythrocytes possess important signaling pathways. These signaling cascades may have important influences on in vivo malarial infection, as well as on erythrocyte membrane flexibility and adhesiveness in sickle cell anemia. With respect to malarial infection, host signaling components alone are not sufficient to induce formation of the malarial vacuole. Parasite proteins are likely to have a major role in making the intraerythrocytic environment conducive for vacuole formation. Such interactions should be the focus of future efforts to understand malarial infection of erythrocytes since host- and parasite-targeted interventions are urgently needed to combat this terrible disease.</p

Erythrocyte Ghosts Support Malarial Invasion and Growth

<div><p>(A) LY-D–loaded erythrocyte ghosts (yellow) were infected with P. falciparum (detected by blue DNA stain Hoechst 33342; indicated by arrow). Bar represents 5 μm.</p> <p>(B) Ghosts (white bars) or erythrocytes (grey bars) infected at low (left) or high (right) parasitemias were monitored by Giemsa staining of thin blood smears at the indicated times of infection; mean values are shown. Error bars show standard deviation of triplicate measurements of a representative experiment.</p> <p>(C) An infected culture containing 50% ghosts and 50% erythrocytes was monitored for cell type-specific parasitemias by counting the number of Hoechst 33342–stained parasite nuclei in fluorescent ghosts (white bars) versus non-fluorescent erythrocytes (grey bars) using DIC and fluorescence microscopy of live cells (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#st2" target="_blank">Methods</a>). Error bars show standard deviation of triplicate measurements of a representative experiment.</p> <p>(D) Giemsa-stained thin blood smears showing ring-, trophozoite-, and schizont-stage parasites in normal (upper) and ghosted (lower) erythrocytes. Bar represents 5 μm.</p> <p>(E) Growth of P. falciparum in ghosts (Ghs) or in erythrocytes (RBCs) in culture over multiple life cycles. Parasites were cultured in ghosts (indicated by dashed line) or in normal erythrocytes (indicated by solid line) for 60 h to ~25% parasitemia, and then were diluted into normal erythrocytes and grown for an additional 50 h. Parasitemia was assessed by counting Giemsa-stained thin blood smears. Error bars show 95% CIs of duplicate measurements at each time point.</p></div

Hematological and Signaling Characteristics and Cargo Loading of Erythrocyte Ghosts

<div><p>(A) Ghosts (left) were biconcave but retained less pigment than intact erythrocytes (right); bar represents 10 μm.</p> <p>(B) Ghost MCH (normal range for erythrocytes 27.5–33.5 pg/cell), MCV (normal range for erythrocytes 80–100 fl), and RDW (normal range for erythrocytes 11.0%–15.0%) were determined using a Coulter counter (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#st2" target="_blank">Methods</a>). Intracellular ATP levels were determined by luciferase-based ATP assays (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#st2" target="_blank">Methods</a>); the 95% confidence interval (CI) for ATP levels is indicated; two experiments.</p> <p>(C) Ghosts loaded with high molecular weight rhodamine-labeled antibody were imaged without fixation by fluorescence microscopy; bar represents 25 μm.</p> <p>(D) Flow cytometry of unloaded (white fill) and FITC-dextran–loaded ghosts (green fill) 2 h post-resealing, showed homogenous loading of cargo. For each cell type, 100,000 gated events were captured.</p> <p>(E) Western blot of GST-loaded ghosts after incubation in culture for 24 h and subsequent treatment in the presence or absence of proteinase K (Prot K) and/or Triton X-100 (TX100); see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#st2" target="_blank">Methods</a>. GST was detected by GST-specific immunoblotting.</p> <p>(F) cAMP production in normal, intact erythrocytes treated with isopreterenol (iso) and/or propranolol (prop) as measured by enzyme-linked immunosorbent assay (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#st2" target="_blank">Methods</a><i>)</i>. Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control erythrocytes was 0.61 pmol cAMP (95% CI 0.20–1.01 pmol; three experiments) per 1 × 10⁸ cells. Induced cAMP levels can vary—the maximal value obtained (once) was 14.58 pmol cAMP (95% CI 13.11–16.03 pmol) per 1 × 10⁸ cells.</p> <p>(G) cAMP production in ghosts measured and depicted as described in (F). Error bars show 95% CI of triplicate measurements in a representative experiment. The mean baseline cAMP concentration in control ghosts was 1.12 pmol cAMP (95% CI 0.59–1.64 pmol; three experiments) per 1 × 10⁸ cells. The maximal isoproterenol-induced value obtained in a single ghost cAMP assay was 9.00 pmol cAMP (95% CI 7.37–10.62 pmol) per 1 × 10⁸ cells.</p></div

Schematic for the Production of Ghosts with G_s Signaling and Malarial Infection that Closely Mimic Normal Erythrocytes

<p>Erythrocytes were washed and resuspended to 50% Hct in PBS-glucose containing proteins of interest (“cargo,” yellow spheres) as described in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#st2" target="_blank">Methods</a>. The cell suspension was dialyzed against ATP-supplemented hypotonic potassium buffer, lysing cells, and allowing exogenous cargoes to enter and endogenous cytoplasmic proteins to exit lysed cells (depicted over time as increasing extracellular hemoglobin, change in cell color from red to pink, and loss of membrane integrity). Lysed cells were removed to cold test tubes and resealed at 37 °C for 1 h and washed three times in RPMI and once in RPMI supplemented with 10% human serum (cRPMI) to eliminate extracellular hemoglobin, cargo, and buffer (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#st2" target="_blank">Methods</a>). Ghosts prepared in this manner were subsequently characterized in hematological, signaling, and malarial infection assays, as described in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030528#pmed-0030528-g002" target="_blank">Figure 2</a>.</p