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1.
Br J Pharmacol ; 166(8): 2263-76, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22471905

ABSTRACT

BACKGROUND AND PURPOSE: Choline analogues, a new type of antimalarials, exert potent in vitro and in vivo antimalarial activity. This has given rise to albitiazolium, which is currently in phase II clinical trials to cure severe malaria. Here we dissected its mechanism of action step by step from choline entry into the infected erythrocyte to its effect on phosphatidylcholine (PC) biosynthesis. EXPERIMENTAL APPROACH: We biochemically unravelled the transport and enzymatic steps that mediate de novo synthesis of PC and elucidated how albitiazolium enters the intracellular parasites and affects the PC biosynthesis. KEY RESULTS: Choline entry into Plasmodium falciparum-infected erythrocytes is achieved both by the remnant erythrocyte choline carrier and by parasite-induced new permeability pathways (NPP), while parasite entry involves a poly-specific cation transporter. Albitiazolium specifically prevented choline incorporation into its end-product PC, and its antimalarial activity was strongly antagonized by choline. Albitiazolium entered the infected erythrocyte mainly via a furosemide-sensitive NPP and was transported into the parasite by a poly-specific cation carrier. Albitiazolium competitively inhibited choline entry via the parasite-derived cation transporter and also, at a much higher concentration, affected each of the three enzymes conducting de novo synthesis of PC. CONCLUSIONS AND IMPLICATIONS: Inhibition of choline entry into the parasite appears to be the primary mechanism by which albitiazolium exerts its potent antimalarial effect. However, the pharmacological response to albitiazolium involves molecular interactions with different steps of the de novo PC biosynthesis pathway, which would help to delay the development of resistance to this drug.


Subject(s)
Antimalarials/metabolism , Erythrocytes/parasitology , Phosphatidylcholines/biosynthesis , Plasmodium falciparum/metabolism , Thiazoles/metabolism , Animals , Biological Transport/physiology , Choline/metabolism , Humans , Molecular Structure , Plasmodium falciparum/drug effects , Thiazoles/chemistry
2.
Infect Immun ; 69(9): 5329-34, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11500402

ABSTRACT

Pseudomonas aeruginosa exoenzyme S (ExoS) is an ADP-ribosyltransferase that modifies low-molecular-weight GTPases. Here we studied the effect of Rab5 ADP-ribosylation by ExoS on its cellular function, i.e., regulation of early endocytic events. Coculture of CHO cells with P. aeruginosa induced a marked decrease in horseradish peroxidase (HRP) uptake compared to noninfected cells, while coculture with a P. aeruginosa mutant strain that fails to produce ExoS did not lead to any change in HRP uptake. Microinjection of recombinant ExoS into Xenopus oocytes induced strong inhibition of basal HRP uptake by oocytes. Moreover, coinjection of recombinant ExoS with Rab5 abolished the typical stimulation of HRP uptake obtained after GTPase microinjection. Cytosols prepared from injected oocytes were used in an endosome-endosome fusion assay. Cytosol from ExoS-microinjected oocytes was ineffective in promoting endosome-endosome fusion. However, in these conditions, the addition of Rab5 to the assay led to fusion recovery. Finally, we found that the interaction of Rab5 with EEA1 was markedly diminished after Rab5 ADP-ribosylation by ExoS.


Subject(s)
ADP Ribose Transferases/metabolism , Adenosine Diphosphate Ribose/metabolism , Bacterial Toxins , Endocytosis , Pseudomonas aeruginosa/enzymology , rab5 GTP-Binding Proteins/metabolism , ADP Ribose Transferases/genetics , ADP Ribose Transferases/pharmacology , Animals , CHO Cells , Coculture Techniques , Cricetinae , Endosomes/physiology , Horseradish Peroxidase/metabolism , Membrane Fusion , Oocytes/metabolism , Pseudomonas aeruginosa/pathogenicity , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Xenopus , rab5 GTP-Binding Proteins/pharmacology
3.
Biochem Biophys Res Commun ; 244(2): 336-41, 1998 Mar 17.
Article in English | MEDLINE | ID: mdl-9514923

ABSTRACT

ADP-ribosylation of rab proteins by exoenzyme S (Exo S) of P. aeruginosa was studied using reticulocytes. 14-3-3 protein, the eukaryotic cofactor that is obligatory for Exo S activity, was found in association with reticulocyte endocytic vesicles and exosomes, vesicles previously shown to be enriched with rab4. Incubation of purified endocytic vesicles with Exo S triggered rab4 ADP-ribosylation. Transferrin recycling in SLO-permeabilized reticulocytes was highly impaired when Exo S was added to the cells, suggesting that ADP-ribosylation affected rab4 function. Moreover, in vitro ADP-ribosylation of different rab proteins was studied using the cofactor activity extracted from reticulocytes.


Subject(s)
ADP Ribose Transferases , Adenosine Diphosphate Ribose/metabolism , Bacterial Toxins , Poly(ADP-ribose) Polymerases/metabolism , Pseudomonas aeruginosa/enzymology , Reticulocytes/metabolism , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Amino Acid Sequence , Animals , Bacterial Proteins , Cell Membrane Permeability , Coenzymes/metabolism , Consensus Sequence , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , In Vitro Techniques , Molecular Sequence Data , Proteins/metabolism , Rats , Streptolysins , Transferrin/metabolism , rab4 GTP-Binding Proteins
4.
Eur J Biochem ; 228(1): 199-205, 1995 Feb 15.
Article in English | MEDLINE | ID: mdl-7883004

ABSTRACT

Electron spin resonance spectroscopy was used to investigate the presence of phospholipase A2 activity in endocytic vesicles prepared from reticulocytes and to define some of its characteristics. Using spin-labeled phospholipid analogues, we measured the hydrolysis rate of the ester bond at position 2 during incubation with reticulocyte endocytic vesicles. We have shown that this phospholipase A2 activity was membrane-associated, enriched in endocytic vesicles as compared to cytosol and plasma membrane. Enzymic activity was also observed in exosomes, vesicles coming from the endocytic compartment and released by reticulocytes during their maturation in erythrocytes. Neither the hydrolytic activity nor the membrane association was found to be Ca(2+)-dependent. Spin-labeled phospholipids with choline and serine polar heads were better substrates than glycerophosphoethanolamine analogues. Optimal pH was found to be close to neutral. 5,5'-Dithiobis(2-nitrobenzoic acid) and diisopropyl fluorophosphate very efficiently inhibited spin-labeled phospholipid hydrolysis. This phospholipase A2 activity was confirmed using a radioactive assay, although with much lower sensitivity. (E)-6-(Bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a specific mechanism-based inhibitor of calcium-independent phospholipases A2, was found to abolish the enzymic activity present in endocytic vesicles.


Subject(s)
Phospholipases A/metabolism , Reticulocytes/enzymology , Animals , Calcium/pharmacology , Electron Spin Resonance Spectroscopy , Hydrogen-Ion Concentration , Phospholipases A2 , Phospholipids/metabolism , Rats , Rats, Sprague-Dawley , Reticulocytes/ultrastructure
5.
Eur J Biochem ; 223(2): 403-10, 1994 Jul 15.
Article in English | MEDLINE | ID: mdl-8055909

ABSTRACT

Murine (2'-5')An-dependent RNase, a key enzyme of the interferon system, was purified from mouse spleen by affinity chromatography to immobilized (2'-5')An. Since the ribonuclease has high affinity to (2'-5')An, optimal non-denaturing conditions were obtained to disrupt the (2'-5')An-nuclease complex. Low-pH buffers in the presence of 0.1% Triton X-100 removed almost 80% of the enzyme from the (2'-5')An-agarose, preserving its (2'-5')An binding activity and RNA cleavage function. Purification was monitored using a classical radiobinding assay, ultraviolet covalent crosslinking method and denaturing-renaturing affinity blotting assay. The purified enzyme was a 160-kDa dimer that migrated with an apparent molecular mass of 78 kDa and was > 80% pure, as assessed by silver-stained SDS gels. Both a 160-kDa dimer and 78-kDa monomer were found in the cellular extract at a 5:1 ratio. Binding of radiolabeled (2'-5')An to (2'-5')An-dependent RNase either in crude extract or in purified form reached equilibrium by 5 h at 4 degrees C. 2-Mercaptoethanol was required to obtain (2-'5')An-binding activity but, interestingly, in the absence of this reducing agent, (2'-5')An-binding activity was initiated by preincubation with poly(U), a synthetic substrate of the nuclease. This new mechanistic feature indicates that interaction of poly(U) with nuclease induced a conformational modification allowing, in a second step, the binding of (2'-5')An. Furthermore, when activated by low amounts of (2'-5')An, the eluted purified enzyme degraded mRNA but there was still degradation in the absence of (2'-5')An. This suggested a loss of regulatory protein(s) during the purification step. Scatchard analysis showed that the purified enzyme had a Kd of 106 pM for (2'-5')An, similar to estimates obtained using crude spleen extracts (Kd 112 pM), indicating that the purified nuclease had almost identical (2'-5')An-binding properties to those identified in spleen extracts.


Subject(s)
Adenine Nucleotides/metabolism , Oligoribonucleotides/metabolism , Ribonucleases/isolation & purification , Spleen/enzymology , Adenine Nucleotides/pharmacology , Animals , Chromatography, Affinity , Kinetics , Mercaptoethanol/pharmacology , Mice , Molecular Weight , Oligoribonucleotides/pharmacology , Poly U/metabolism , Poly U/pharmacology , Radioligand Assay , Ribonucleases/metabolism
6.
Eur J Biochem ; 201(1): 295-302, 1991 Oct 01.
Article in English | MEDLINE | ID: mdl-1915375

ABSTRACT

The parameters regulating the internalization and recycling of transferrin-specific receptors were determined in guinea pig leukemic B lymphocytes, in the absence or presence of ligand. We show that after the cells were purified, 45-56% of the total receptors were on the cell surface. In the absence of transferrin, unoccupied receptors are quickly internalized (rate constant, 0.12 min-1) whereas their recycling is much slower (rate constant, 0.026 min-1). This difference between endocytosis and recycling rates leads to a balanced receptor distribution with only 22% of the total receptors outside after incubation of the cells for 20-30 min at 37 degrees C. The internalization rate of occupied receptors, measured in the presence of transferrin is faster (rate constant, 0.21 min-1) than that of unoccupied receptors calculated in the absence of transferrin (0.12 min-1; see above). On the other hand, mere binding of transferrin to its receptor, without internalization, arrested by cytoplasm acidification, is sufficient to induce a large increase (by a factor of seven) in the recycling rate of unoccupied internal receptors from 0.026 min-1 to 0.17 min-1. Thus, in these lymphocytes, transferrin mobilizes internal receptors by modifying the kinetic rates of internalization and recycling, leading to a new equilibrium between external and internal receptors.


Subject(s)
Endocytosis , Leukemia, Experimental/metabolism , Lymphocytes/metabolism , Receptors, Transferrin/metabolism , Transferrin/metabolism , Animals , Burkitt Lymphoma/metabolism , Female , Guinea Pigs , Hydrogen-Ion Concentration , Iodine Radioisotopes , Kinetics , Temperature , Tumor Cells, Cultured
7.
Eur J Biochem ; 177(3): 643-7, 1988 Nov 15.
Article in English | MEDLINE | ID: mdl-3197724

ABSTRACT

Using a radioimmunoassay for rabbit-serum albumin, platelet-activating-factor-induced serum-albumin release by platelets was monitored under non-aggregating conditions. The four main results from this study are as follows. The EC50 of the release was of the same order of magnitude as the aggregation EC50 in the presence of calcium. The release took place within 2 min and was inhibited by BN 52021, which is a very specific inhibitor of the platelet-activating-factor-aggregating effect. Serum-albumin release was much greater than serotonin release.


Subject(s)
Blood Platelets/metabolism , Calcium/pharmacology , Platelet Activating Factor/pharmacology , Serum Albumin/metabolism , Animals , Blood Platelets/drug effects , Kinetics , Rabbits , Radioimmunoassay , Serotonin/blood
8.
Biochemistry ; 26(11): 2972-8, 1987 Jun 02.
Article in English | MEDLINE | ID: mdl-3038172

ABSTRACT

Spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were added to human platelet suspensions. Due to the partial water solubility of these spin-labeled lipids which possess a relatively short beta-chain (C5), they incorporate rapidly in membranes. The orientation of the spin-labels within the platelet plasma membrane was assessed by following the spontaneous reduction at 37 and 4 degrees C due to endogenous reducing agents present in the cytosol. The rate of spontaneous reduction showed unambiguously that the labels incorporated initially in the outer leaflet of the plasma membrane and that the rate of outside-inside translocation of the aminophospholipids was faster than that of the choline derivatives. For example, at 37 degrees C, the half-time for the transverse diffusion of a phosphatidylcholine analogue was found to be of the order of 40 min, while it was less than 7 min for the phosphatidylserine analogue. At low temperatures, a fraction of the labels gave rise to a strongly immobilized ESR component. This fraction, which corresponded to 20-30% of the initial spin-label concentration, was found resistant to chemical reduction from the inner side of the membrane and also to externally added reducing agents such as ascorbate. Presumably these immobilized lipids are trapped in a gel phase formed in the outer leaflet at 4 degrees C. Cell aging, which depletes the cells of ATP, resulted in the progressive inhibition of the fast transport of the aminophospholipids from the outer to inner leaflet. Treatment of the cells with iodoacetamide completely blocked the transverse diffusion of the spin-labels.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Blood Platelets/metabolism , Phospholipids/blood , Spin Labels/metabolism , Adenosine Triphosphate/blood , Biological Transport , Blood Platelets/cytology , Blood Platelets/ultrastructure , Electron Spin Resonance Spectroscopy , Humans , Iodoacetamide/pharmacology , Kinetics , Microscopy, Electron
9.
Biochim Biophys Acta ; 860(2): 194-200, 1986 Aug 21.
Article in English | MEDLINE | ID: mdl-3017416

ABSTRACT

The interactions between a spin-labeled analogue of PAF-acether (designated as (0,2)PAF) and different human blood components (platelets, erythrocytes, and serum) have been studied. The rate of spin probe reduction by cytosol provided information about the internalization processes when the hydrolysis rate was also available. Although erythrocyte reactivity is lower than that of platelets, erythrocytes, because of their greater numbers, removed (0,2)PAF from whole blood faster than platelets. Lastly, erythrocytes may be more efficient traps for (0,2)PAF than serum acetylhydrolase. Criteria for extending these results to genuine PAF-acether are also discussed.


Subject(s)
Blood Platelets/metabolism , Erythrocytes/metabolism , Platelet Activating Factor/metabolism , Spin Labels , Electron Spin Resonance Spectroscopy , Free Radicals , Humans , Hydrolysis , Nitrogen Oxides , Oxidation-Reduction , Plasma/metabolism
10.
Chem Phys Lipids ; 37(3): 215-26, 1985.
Article in English | MEDLINE | ID: mdl-4042252

ABSTRACT

The multistep synthesis of a photoreactive, radioactive and aggregating analogue of platelet-activating factor (PAF)-acether is described. The photoreactive and radioactive moiety was added at the last step; the specific radioactivity was higher than 1000 Ci/mmol. The concentration of this new analogue which causes 50% of aggregation of platelets were of the same order of magnitude as for synthetic snPAF-acether, so as for two other analogues having a bulky group at the omega end of the fatty ether chain. The photoreactivity was proved by the covalent binding of the analogue to protein (BSA) after 10-min irradiation times at 300 nm. The binding was largely prevented by prior (not by later) addition of a high concentration of lyso phosphatidyl choline.


Subject(s)
Platelet Activating Factor/analysis , Acetylation , Chemical Phenomena , Chemistry , Humans , Iodine Radioisotopes , Isotope Labeling , Magnetic Resonance Spectroscopy
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