Plasmodium Falciparum
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 4786-4790, June 1991 Biochemistry Chloroquine inhibits heme-dependent protein synthesis in Plasmodium falciparum (malaria paraite/cell-free system/translation/protein synthesis in situ/eukaryotic initiation factor 2a kinase) NAMITA SUROLIA* AND GOVINDARAJAN PADMANABAN*t#§ *Department of Biochemistry, tCentre for Genetic Engineering, and *Jawaharlal Nehru Centre for Advanced Scientific Research, Indian Institute of Science, Bangalore 560 012, India Communicated by Irving M. London, December 26, 1990 ABSTRACT A cell-free protein-synthesizing system has Heme is required for protein synthesis in the reticulocyte been reconstituted using the S-30 fraction or ribosomes and the lysate (10, 11) and a few nonerythroid systems as well (12). S-100 fraction from Plasmodiumfalckarum. Addition of heme We have investigated the possibility whether chloroquine can in vitro stimulates cell-free protein synthesis strikingly. Chlo- inhibit the translation process of the parasite by virtue of roquine inhibits the heme-dependent protein synthesis in the complex formation with heme, if the parasite protein syn- parasite lysate. The drug has also been found to inhibit parasite thesis were to be heme dependent. Earlier, the protein protein synthesis in situ at therapeutic concentrations soon synthesis rates obtained with Plasmodia infecting birds and after addition to parasite cultures. Ribosomes as well as the animals were low, and it was generally concluded that S-100 fraction isolated from such chloroquine-treated cultures chloroquine is not a potent inhibitor of protein synthesis (13, are defective in protein synthesis. Addition of hemin plus 14). In the present study we demonstrate that an early event glucose 6-phosphate or high concentrations of GTP, cAMP, in chloroquine action is an inhibition of heme-dependent and an active preparation of eIF-2 to the parasite cell-free protein synthesis in the parasite. system restores protein synthesis to a sigfant extent in chloroquine-treated cultures. Under conditions of inhibition of protein synthesis in situ by chloroquine in the culture, the MATERIALS AND METHODS parasite eukaryotic initiation factor 2a- (eIF-2a) is phospho- Maintenance of the Malarial Parasite. Plasmodium falci- rylated in the parasite lysate to a greater extent than that parum FCK 2 strain, a local isolate from Karnataka State, observed in the control culture. Addition of hemin in vitro India, was cultured in human O-positive washed erythrocytes suppresses this phosphorylation. eIF-2a kinase activity is pres- in vitro using standard techniques (15). ent in the parasite lysate and is not a contaminant derived from Synchronization of Parasites in Culture. Parasites were the human erythrocytes used to culture the parasite. The synchronized using 5% D-sorbitol (16) and the cultures were heme-chloroquine interactive effects can also be demonstrated pooled at late trophozoite stage with 12-15% parasitaemia. with purified eIF-2a kinase from rabbit reticulocyte lysate. It Parasites were released from the erythrocytes with 0.15% is proposed that chloroquine inhibits heme-dependent protein saponin (17). synthesis in the parasite and this is an early evefit mediating the Preparation of the Parasite S-30 Fraction, Isolation of Ri- growth-inhibitory effects of the drug. bosomes, and Constitution of the Cell-Free System. The free parasites were lysed with 4 vol ofice-cold lysing solution [100 It is rather surprising that the mechanism of action of mM KCI/7 mM Mg(OAc)2/380 mM sucrose/6.5 mM 2-mer- chloroquine, a widely used antimalarial drug, is not fully captoethanol/50 mM Tris HCl, pH 7.4/0.14% (vol/vol) Tri- understood. An early suggestion based on intercalation of ton X-100] as described for Plasmodium knowlesi (18). Leu- chloroquine with DNA and inhibition ofDNA synthesis (1, 2) peptin (15 gM) and placental ribonuclease inhibitor (20 units) has not found favor in view of the millimolar concentrations were added to the free parasites before adding lysing solu- required for the purpose, whereas nanomolar concentrations tion. The suspension was then centrifuged at 12,000 x g for in the culture are growth inhibitory. There has, however, 20 min at 40C. The supernatant was passed through a G-50 been a recent attempt to revive this theory (3, 4). Chloroquine spun column and used as the S-30 fraction. The 12,000 x g is known to accumulate in the acid vesicles ofthe parasite and supernatant was centrifuged twice at 10,000 x g for 10 min increase the pH and osmolality of the vesicles, leading to and the second 10,000 x g supernatant was centrifuged at swelling and membrane leakiness (5). However, it has also 105,000 x g for 60 min at 40C to sediment ribosomes. The been suggested that alkalinization ofthe vesicles per se is not ribosomal pellet was rinsed with 200 p.l of buffer A [0.25 M adequate to explain the toxicity of chloroquine, and substan- sucrose/25 mM KCI/5 mM Mg(OAc)2/50 mM Tris-HCI, pH tial alkalinization does not occur within the therapeutic drug 7.4] and resuspended in the same buffer. The postribosomal concentration range (6). It is known that intraerythrocytic supernatant was passed through a G-50 spun column and Plasmodium digests up to 75% of the host cell hemoglobin in used as the S-100 fraction. Cell-free protein synthesis was the acid food vacuoles, and the generated heme is present as carried out in 50 ILI containing 20 mM Hepes (pH 7.5), 1 mM an inert complex referred to as malaria pigment or hemozoin ATP, 0.1 mM GTP, 5 mM creatine phosphate, 45 units of (7). Chloroquine has been demonstrated to bind to ferripro- creatine phosphokinase per ml, 0.5 mM glucose 6- phosphate, toporphyrin IX with high affinity in vitro and in situ, and it has 7 mM Mg(OAc)2, 110 mM potassium acetate, 0.375 mM been suggested that the drug can sequester heme, interfering spermidine, 2.5 mM dithiothreitol, 2 pug ofwheat germ tRNA, with the formation ofthe inert pigment, and the complex can 40 p.M (each) of 19 amino acids except methionine (20 p.Ci, lyse the parasite membrane (7-9). However, there have been 800 Ci/mmol; 1 Ci = 37 GBq; Amersham), and the S-30 arguments against this theory (3, 6). Abbreviations: eIF-2, eukaryotic initiation factor 2; eIF-2a, a sub- The publication costs of this article were defrayed in part by page charge unit (38 kDa) of eIF-2; HRI, heme-regulated inhibitor. payment. This article must therefore be hereby marked "advertisement" §To whom reprint requests should be addressed at: Department of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Biochemistry, Indian Institute ofScience, Bangalore 560 012, India. 4786 Downloaded by guest on September 26, 2021 Biochemistry: Surolia and Padmanaban Proc. NatL. Acad. Sci. USA 88 (1991) 4787 fraction or S-100 plus ribosomes (10 Ag of rRNA). Hemin, denaturing buffer and analyzed by NaDodSO4/PAGE and when added, was at 15 ,uM concentration. Chloroquine autoradiography. phosphate and hemin solutions were prepared fresh and were preincubated together on ice for 5 min before the addition of RESULTS other components. Reactions were initiated with the addition of the S-30 fraction or ribosomes and incubated at 30'C, Chloroquine Inhibits Heme-Dependent Protein Synthesis in samples (5 p.1) were spotted onto Whatman 1MM filter discs, Rabbit Reticulocyte Lysate. The reticulocyte lysate used was and radioactivity was measured after washing with trichloro- stimulated 3-fold with 20 p.M hemin during a 30-min incuba- acetic acid (12). All assays were run in triplicate and zero time tion period. The response to hemin added 10 min after the values were subtracted. start of incubation was found to be decreased by 50%. Preparation of Reticulocyte Lysate and Cell-Free Protein Chloroquine at a concentration of 10 p.M blocked the heme- Synthesis. Reticulocyte lysate was prepared and cell-free mediated stimulation by 80% (Fig. 1). Higher concentrations protein synthesis with [35S]methionine was carried out in 25 of chloroquine did not lead to any significant increase in the A.l using standard protocols (10, 12). extent of inhibition obtained. Again, chloroquine at these Phosphorylation of Proteins in Reticulocyte and Parasite concentrations did not inhibit the basal protein synthesis Lysate and NaDodSO4/PAGE. Phosphorylation of the para- obtained in the absence of added hemin. site eukaryotic initiation factor 2a- (eIF-2a) was assessed in Heme Stimulates Protein Synthesis in Parasite Lysate and the parasite lysate prepared from in situ chloroquine-treated Chloroquine Inhibits This Process. Addition of heme (15 p.M) and control cultures. Reaction mixtures were the same as that to the parasite lysate (S-30) resulted ir. stimulation ofparasite for protein synthesis except that unlabeled ATP was replaced protein synthesis by 3-fold (Fig. 2). The effect of a range of with [y-32PJATP (25 p.Ci, 3000 Ci/mmol; Amersham) and heme concentrations was tested and 15 pM was found to be [35S]methionine was omitted. The protein synthesis mixture optimal. Chloroquine at 8 p.M was found to strikingly inhibit (50 1.l) was incubated at 300C for 5 min before [y32P]ATP the heme-dependent increase in protein synthesis. An in- addition. Samples (5 ,ul) were removed 2 min after 32p crease in chloroquine concentration to 16 p.M only marginally addition and mixed with equal volumes of NaDodSO4/ increases the extent of inhibition. At these chloroquine protein dissociation buffer. These samples were analyzed on concentrations, protein synthesis in the absence of added 10o polyacrylamide gels (0.1% NaDodSO4; bisacrylamide- heme is not inhibited by more than 15% (data not presented). :acrylamide ratio, 1:38.5), stained with Coomassie blue, and Chloroquine Inhibits Protein Synthesis in P.