Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum
2007; Elsevier BV; Volume: 282; Issue: 49 Linguagem: Inglês
10.1074/jbc.m703643200
ISSN1083-351X
Autores Tópico(s)Trypanosoma species research and implications
ResumoDuring the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria. During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria. With an estimated 2 million deaths annually to its name (1Snow R.W. Guerra C.A. Noor A.M. Myint H.Y. Hay S.I. Nature. 2005; 434: 214-217Crossref PubMed Scopus (2130) Google Scholar), the human malaria parasite Plasmodium falciparum continues to present an enormous public health challenge. Clinical manifestations of infection appear as the parasite replicates within host erythrocytes. During this replication cycle, the parasite endocytoses and catabolizes large amounts (up to 75%) of host hemoglobin to constituent amino acids (2Krugliak M. Zhang J. Ginsburg H. Mol. Biochem. Parasitol. 2002; 119: 249-256Crossref PubMed Scopus (164) Google Scholar, 3Loria P. Miller S. Foley M. Tilley L. Biochem. J. 1999; 339: 363-370Crossref PubMed Scopus (222) Google Scholar). This process makes available large quantities of free amino acids for parasite protein synthesis (4Sherman I.W. Tanigoshi L. Int. J. Biochem. 1970; 1: 635-637Crossref Scopus (38) Google Scholar) and may also modulate the osmotic environment of the host cell (5Lew V.L. Tiffert T. Ginsburg H. Blood. 2003; 101: 4189-4194Crossref PubMed Scopus (197) Google Scholar) and/or create space inside the red cell for parasite growth (6Allen R.J. Kirk K. Trends Parasitol. 2004; 20: 7-10Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Amino acids derived from hemoglobin catabolism are also important for the uptake of isoleucine from the extracellular environment (7Martin R.E. Kirk K. Blood. 2007; 109: 2217-2224Crossref PubMed Scopus (89) Google Scholar). As hemoglobin catabolism is essential for parasite replication and is currently a major focus of anti-malarial drug development efforts, a comprehensive understanding of the biochemistry of this pathway is an urgent priority. Hemoglobin catabolism occurs in an acidic, degradative organelle termed the food vacuole (FV) 2The abbreviations used are: FV, food vacuole; AMC, amidomethylcoumarin; AP, aminopeptidase; APP, aminopeptidase P; PfDAP, P. falciparum aspartyl aminopeptidase; DPAP1, dipeptidyl aminopeptidase 1; E-64, N-(trans-epoxysuccinyl)-l-leucine 4-guanidinobutylamide; HA, hemagglutinin; PfLAP, P. falciparum leucyl aminopeptidase; LDH, lactate dehydrogenase; βNA, β-naphthylamine; PBS, phosphate-buffered saline; PfA-M1, P. falciparum aminopeptidase N; PM II, plasmepsin II; YFP, yellow fluorescent protein; AA, amino acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) propane-1,3-diol; PBS, phosphate-buffered saline; FMK, fluoromethyl ketone; MOPS, 4-morpholinepropanesulfonic acid; GFP, green fluorescent protein. 2The abbreviations used are: FV, food vacuole; AMC, amidomethylcoumarin; AP, aminopeptidase; APP, aminopeptidase P; PfDAP, P. falciparum aspartyl aminopeptidase; DPAP1, dipeptidyl aminopeptidase 1; E-64, N-(trans-epoxysuccinyl)-l-leucine 4-guanidinobutylamide; HA, hemagglutinin; PfLAP, P. falciparum leucyl aminopeptidase; LDH, lactate dehydrogenase; βNA, β-naphthylamine; PBS, phosphate-buffered saline; PfA-M1, P. falciparum aminopeptidase N; PM II, plasmepsin II; YFP, yellow fluorescent protein; AA, amino acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) propane-1,3-diol; PBS, phosphate-buffered saline; FMK, fluoromethyl ketone; MOPS, 4-morpholinepropanesulfonic acid; GFP, green fluorescent protein. or digestive vacuole. In the lumen of the FV, endopeptidases of diverse catalytic mechanism and specificity (plasmepsin (PM) I, II, and IV, histo-aspartic protease, falcipain-2, -2′, and -3, and falcilysin) contribute to the hydrolysis of the α- and β-globin chains to oligopeptides (8Goldberg D.E. Curr. Top. Microbiol. Immunol. 2005; 295: 275-291Crossref PubMed Scopus (172) Google Scholar, 9Rosenthal P.J. Curr. Opin. Hematol. 2002; 9: 140-145Crossref PubMed Scopus (103) Google Scholar). These oligopeptides are further hydrolyzed to dipeptides by the FV exopeptidase dipeptidyl aminopeptidase 1 (10Klemba M. Gluzman I. Goldberg D.E. J. Biol. Chem. 2004; 279: 43000-43007Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The fate of these di- and oligopeptides is unclear. A widely cited model holds that the terminal stages of hemoglobin degradation occur in the cytosol, with peptides exported from the FV for hydrolysis by cytosolic aminopeptidases (11Gavigan C.S. Dalton J.P. Bell A. Mol. Biochem. Parasitol. 2001; 117: 37-48Crossref PubMed Scopus (86) Google Scholar, 12Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biochem. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar). This model is based largely on the apparent lack of aminopeptidase (AP) activity in extracts of enriched FVs (12Kolakovich K.A. Gluzman I.Y. Duffin K.L. Goldberg D.E. Mol. Biochem. Parasitol. 1997; 87: 123-135Crossref PubMed Scopus (111) Google Scholar), on the localization of two P. falciparum APs to the cytosol (13Allary M. Schrevel J. Florent I. Parasitology. 2002; 125: 1-10Crossref PubMed Scopus (81) Google Scholar, 14Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Stack C.M. Dalton J.P. J. Biol. Chem. 2006; 281: 1741-1745Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and on the apparent absence of carboxypeptidase homologs from the annotated parasite genome sequence (16Wu Y. Wang X. Liu X. Wang Y. Genome Res. 2003; 13: 601-616Crossref PubMed Scopus (191) Google Scholar). Support for this model is weakened, however, by the absence of experimental evidence for peptide export from the FV, and by the presence of uncharacterized AP homologs in the P. falciparum genome sequence that could play a role in vacuolar peptide catabolism. To test this model, we have examined the roles of four P. falciparum AP homologs. These four enzymes were selected because, to the best of our knowledge at the time of this study, they constituted the entire repertoire of AP activities encoded by the P. falciparum genome, excluding the housekeeping methionine aminopeptidases. To gain insight into the functions of these four APs, we have determined the subcellular distribution of each. We have attempted to inactivate each AP by disrupting its coding sequence to assess their importance for intraerythrocytic growth and to determine their utility as potential drug targets. To further probe the roles of APs in peptide catabolism and parasite growth, the effect of exogenous amino acids on the toxicity of the AP inhibitor bestatin was examined. Generation of Constructs—The green fluorescent protein allele gfpmut2 (17Cormack B.P. Valdivia R.H. Falkow S. Gene (Amst.). 1996; 173: 33-38Crossref PubMed Scopus (2499) Google Scholar) was converted to the yellow fluorescent protein (YFP) allele Citrine (18Griesbeck O. Baird G.S. Campbell R.E. Zacharias D.A. Tsien R.Y. J. Biol. Chem. 2001; 276: 29188-29194Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar) by introduction of the mutations A65G, Q69M, and T203Y using the QuikChange mutagenesis kit (Stratagene). The mutation A206K was also introduced to eliminate the weak tendency of GFP to dimerize (19Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1763) Google Scholar). The Citrine sequence, preceded by the tobacco etch virus protease cleavage site (ENLYFQS) and followed by the Softag1 epitope tag GSLAELLNAGLGGS (20Thompson N.E. Arthur T.M. Burgess R.R. Anal. Biochem. 2003; 323: 171-179Crossref PubMed Scopus (24) Google Scholar), was introduced into the AvrII/NotI sites of the plasmid pPM2GT (21Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (109) Google Scholar) to produce pPM2CIT2. To create plasmids for the generation of chromosomal AP-YFP fusions, 3′-1-kb fragments of PfAPP, PfDAP, PfA-M1, and PfLAP coding sequence up to but not including the stop codon were PCR-amplified from P. falciparum 3D7 genomic DNA and cloned into the XhoI/AvrII sites of pPM2CIT2. HA tag constructs were produced by excising the AvrII/NotI YFP fragment from the AP-YFP plasmids and cloning in-frame a sequence encoding GGGYPYDVPDYA (HA tag underlined) into the same sites. For gene disruption constructs, a kilobase of sequence from the 5′ end of each AP coding sequence was cloned into the XhoI/AvrII sites of pPM2CIT2. Sequences of primers used for PCR amplification of AP sequences are provided in supplemental Table S1. Parasite Culture and Transfection—P. falciparum clone 3D7 was grown in human O+ erythrocytes in RPMI medium supplemented as described previously (21Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (109) Google Scholar). RPMI containing 148 μm isoleucine as the only amino acid (I medium) was prepared as described (22Liu J. Istvan E.S. Gluzman I.Y. Gross J. Goldberg D.E. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8840-8845Crossref PubMed Scopus (271) Google Scholar). Cultures were synchronized by sorbitol treatment (23Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2802) Google Scholar). Parasites were transfected with 75-100 μg of plasmid DNA using low voltage electroporation conditions (24Fidock D.A. Wellems T.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10931-10936Crossref PubMed Scopus (412) Google Scholar), and 10 nm WR99210 was added after 48 h. Resistant parasites appearing after 18-24 days were subjected to drug cycling whereby WR99210 was removed from the media for 21 days and then applied until resistant parasites reappeared. After two drug cycles, populations of parasites expressing PfDAP-YFP, PfAPP-YFP, and PfLAP-YFP were obtained. All parasites in each population appeared to possess a chromosomal AP-YFP chimera, as nonfluorescent parasites were no longer observed. In contrast, after two drug cycles only a low percentage of parasites expressed PfA-M1-YFP, as determined microscopically; this necessitated the isolation of clonal fluorescent parasite lines by limiting dilution. Clone F11 was used for this study. Parasites stably transfected with gene disruption episomes were cycled off/on drug two (PfDAP) or three times (PfAPP, PfLAP, and PfA-M1). Clonal parasite lines containing a disrupted PfDAP gene were obtained by limiting dilution; clone C2 was used for this study. Southern Blot Analysis of Transfected Parasites—To detect targeted integration of the episome in parasite lines that were expressing YFP or HA fusions or were transfected with gene disruption constructs, genomic DNA was isolated from saponin-treated parasites using the QiaAmp DNA blood mini kit (Qiagen), digested with one or two restriction enzymes (enzymes used for YFP fusion and gene disruption lines are indicated in supplemental Figs. S1 and S8, respectively), and resolved on a 0.6% agarose gel. After transfer to a Nytran+ membrane (GE Biosciences) and blocking, AP loci were detected using probes complementary to the episomal AP targeting sequences. Probe labeling and detection were carried out using the AlkPhos direct labeling kit (GE Biosciences). Fluorescence Microscopy—For live imaging of parasites expressing AP-YFP fusions, cultures were mounted under a coverslip after addition of the vital nuclear stain Hoechst 33342 (5 μm). Images were collected on a Zeiss AxioImager equipped with an MRm Axiocam digital camera using a 100×/1.4NA objective lens. For HA tag localization, parasites were fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde, permeabilized, and blocked, as described previously (25Tonkin C.J. van Dooren G.G. Spurck T.P. Struck N.S. Good R.T. Handman E. Cowman A.F. McFadden G.I. Mol. Biochem. Parasitol. 2004; 137: 13-21Crossref PubMed Scopus (345) Google Scholar), and then incubated with an affinity-purified rabbit anti-HA antibody (Invitrogen) followed by an Alexa 594-conjugated anti-rabbit secondary antibody (Invitrogen). Cells were then allowed to settle onto polyethyleneimine-coated coverslips and were mounted with ProLong Gold containing the nuclear stain 4′,6-diamidino-2-phenylindole (Invitrogen). Images were converted to TIF files, and contrast was adjusted using Adobe Photoshop CS2. Trophozoite and FV Isolation and Preparation of Extracts—Trophozoites were isolated from infected erythrocyte cultures by treating with 1 mg/ml saponin in Dulbecco's phosphate-buffered saline (PBS) for 10 min on ice. Parasites were recovered by centrifugation at 1940 × g for 10 min at 4 °C, washed once with cold PBS, and then stored at -80 °C. Extracts used in Fig. 5C were prepared by resuspending parasite pellets in 500 μl of 25 mm sodium MOPS, pH 7.0, 100 mm NaCl and sonicating three times for 10 s. Soluble extracts were generated by centrifuging the crude lysate at 100,000 × g for 1 h at 4 °C. For normalization of enzyme rate data, the parasitemia of infected erythrocyte cultures was determined from a Giemsa-stained thin smear and used to calculate the number of parasites in the extract. To prepare extracts for immunoblotting, synchronized trophozoite and schizont parasites were isolated by saponin treatment as described in the above paragraph and stored at -80 °C. Frozen parasite pellets were resuspended in PBS containing the following protease inhibitors: 10 μm pepstatin, 10 μm N-(trans-epoxysuccinyl)-l-leucine 4-guanidinobutylamide (E-64), 0.5 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, and 1 mm sodium EDTA. Inclusion of these inhibitors was required to prevent artifactual proteolysis of AP-YFP fusions during sample preparation. Reducing SDS-PAGE sample buffer was added, and the sample was mixed and immediately placed in a boiling water bath for 3 min. Insoluble material was removed by centrifugation at 12,000 × g for 2 min. Food vacuoles were prepared from a parasite line expressing PM II-GFP (21Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (109) Google Scholar) as described previously (26Saliba K.J. Folb P.I. Smith P.J. Biochem. Pharmacol. 1998; 56: 313-320Crossref PubMed Scopus (91) Google Scholar). Briefly, ∼5 × 109 saponin-treated trophozoites were washed twice with cold PBS and resuspended in 500 μl of cold water that had been adjusted to pH 4.5 with HCl. Resuspended parasites were triturated four times through a 27-gauge needle. After centrifugation at 18,000 × g for 2 min, parasites were resuspended in cold uptake buffer (25 mm HEPES, 25 mm NaHCO3, 5 mm sodium phosphate, 100 mm KCl, 10 mm NaCl, 2 mm MgSO4, pH 7.4). DNase I was added (5 μl of a 5 mg/ml solution), and the preparation was incubated for 5 min at 37 °C. After centrifugation at 18,000 × g for 2 min, the pellet was resuspended in 100 μl of cold uptake buffer, added to 1.3 ml of 42% Percoll, 0.25 m sucrose, 1 mm MgCl2, pH 7.4, triturated twice as described above, and centrifuged at 18,000 × g for 10 min at 4 °C. The material at the bottom of the tube was washed once with uptake buffer, resuspended in 100 μl of uptake buffer, and then subjected to a second Percoll enrichment step. The second enrichment, which was carried out exactly as the first, reduced contamination with unlysed trophozoites. The presence of intact, GFP-containing FVs was confirmed microscopically. Isolated vacuoles were stored at -80 °C. Extracts of FVs and trophozoites (for Fig. 3) were prepared by suspension in 25 mm sodium MOPS, pH 7.0, 100 mm NaCl, 0.1% Triton X-100, and insoluble material was removed by centrifugation at 100,000 × g for 30 min at 4 °C. Immunoblotting—Parasite extracts were resolved on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with affinity-purified anti-GFP antibody 6556 (Abcam, 1:10,000 dilution) and a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Biosciences). Chemiluminescent signal was developed with the ECL kit (GE Biosciences). Blots were stripped and reprobed with rabbit anti-BiP (1:10,000 (27Kumar N. Koski G. Harada M. Aikawa M. Zheng H. Mol. Biochem. Parasitol. 1991; 48: 47-58Crossref PubMed Scopus (142) Google Scholar)) to assess relative loading levels. Enzyme Assays—Peptidase assays were carried out in 96-well plates with fluorogenic substrates as follows: (i) DPAP1 with 200 μm Pro-Arg-amidomethylcoumarin (PR-AMC; Bachem) as described previously (10Klemba M. Gluzman I. Goldberg D.E. J. Biol. Chem. 2004; 279: 43000-43007Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar); (ii) PfA-M1 with 200 μm Ala-AMC (Sigma) in 50 mm Tris-HCl, pH 7.5 (Ala-AMC is efficiently hydrolyzed by PfA-M1 but not PfLAP (15Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar)); (iii) PfAPP with 200 μm lysine(Nϵ-2-aminobenzoyl)-Pro-Pro-4-nitroanilide (Bachem (28Stockel-Maschek A. Stiebitz B. Koelsch R. Neubert K. Anal. Biochem. 2003; 322: 60-67Crossref PubMed Scopus (23) Google Scholar)) in 50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1 mm MnCl2, 10 μm bestatin; (iv) PfDAP in a coupled assay (29Wilk S. Wilk E. Magnusson R.P. J. Biol. Chem. 1998; 273: 15961-15970Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) containing 100 μm Asp-Ala-Pro-β-naphthylamide and 0.2 units/100 μl of rabbit kidney dipeptidyl peptidase IV in 50 mm Tris-HCl, pH 7.5, 10 μm bestatin. When enzyme extracts were prepared with Triton X-100 (Fig. 3), 0.1% Triton X-100 was included in all assays. Lactate dehydrogenase (LDH) activity was detected spectrophotometrically at 650 nm in a lactate oxidation assay (30Makler M.T. Hinrichs D.J. Am. J. Trop. Med. Hyg. 1993; 48: 205-210Crossref PubMed Scopus (469) Google Scholar) containing 100 mm Tris-HCl, pH 9.2, 100 mm lactate, 1 mm 3-acetylpyridine adenine dinucleotide, 2 mm phenazine ethosulfate, 1 mg/ml nitro blue tetrazolium, and 0.2% Triton X-100. Enzyme rates were calculated by linear regression using Kaleidagraph 4.0. Enrichment of DPAP1 and Ala-Pro-Ala-βNA Assays—Approximately 108 trophozoite-stage parasites were suspended in 50 mm BisTris-HCl, pH 6.0, and lysed by sonication. The extract was clarified by centrifugation at 100,000 × g for 1 h at 4 °C. Supernatant was loaded onto a MonoQ column equilibrated in 50 mm BisTris-HCl, pH 6.0, and proteins were eluted with a linear gradient from 0 to 1 m NaCl. Fractions containing DPAP1 were identified using the substrate PR-AMC as described under "Enzyme Assays." Assays for Ala-Pro-Ala-βNA hydrolysis contained either clarified FV lysate, prepared as described above, or 5 μl of a MonoQ fraction enriched in DPAP1 supplemented with 0, 0.3, 1, or 3 μg of purified recombinant PfAPP 3K. Bompiani and M. Klemba, manuscript in preparation. in 50 mm sodium MOPS, pH 5.5, 2 mm dithiothreitol, 30 mm NaCl, and 500 μm Ala-Pro-Ala-βNA (Bachem). Assays of FV extract also contained 0.1% Triton X-100. When inhibitors were included in the assay, concentrations were as follows: 1 μm E-64, 10 μm bestatin, 1 μm pepstatin, or 1 μm Pro-Arg-fluoromethyl ketone (PR-FMK). All inhibitors were obtained from commercial sources except PR-FMK, which was custom-synthesized (Enzyme Systems Products). Bestatin Treatment—P. falciparum 3D7 parasites were cultured for at least three generations in I medium. Aliquots of a synchronous ring-stage parasite culture were centrifuged at 860 × g for 3 min, and infected cell pellets were resuspended in either fresh I medium or in regular RPMI medium containing all 20 amino acids. Bestatin was added to 200-μl aliquots of cultures in 96-well flat-bottom culture dishes to give a concentration range of 0.03-30 μm. A separate aliquot of culture was resuspended in RPMI medium lacking all amino acids to induce amino acid starvation. All assays were carried out in triplicate. Parasite cultures were incubated for 65 h and then fixed with 0.1% glutaraldehyde in PBS. After permeabilization of cells with 0.25% Triton X-100 in PBS and staining of DNA with 400 nm YOYO-1 (Invitrogen) in PBS, parasite fluorescence was determined on a Coulter Epics XL MCL flow cytometer equipped with a 488 nm laser. Flow cytometry histogram overlays, parasitemias, and mean fluorescence intensities were generated using FlowJo version 8.3 (Treestar). Aminopeptidases in the P. falciparum Genome—The four APs studied here were identified through molecular biological (31Florent I. Derhy Z. Allary M. Monsigny M. Mayer R. Schrevel J. Mol. Biochem. Parasitol. 1998; 97: 149-160Crossref PubMed Scopus (65) Google Scholar) and bioinformatic (16Wu Y. Wang X. Liu X. Wang Y. Genome Res. 2003; 13: 601-616Crossref PubMed Scopus (191) Google Scholar) approaches. The designated names and nearest homologs of these sequences are listed in Table 1. One of them, aminopeptidase N (PfA-M1), has broad specificity at the substrate P1 position (13Allary M. Schrevel J. Florent I. Parasitology. 2002; 125: 1-10Crossref PubMed Scopus (81) Google Scholar), whereas that of leucyl aminopeptidase (PfLAP) is restricted to hydrophobic P1 residues (15Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). (The convention of Schechter and Berger (32Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4738) Google Scholar) is used to designate the positions of residues in the substrate and the corresponding binding sites in the peptidase.) Aminopeptidase P (PfAPP) and aspartyl aminopeptidase (PfDAP) have not yet been characterized but are predicted by homology to have highly restricted P1′ and P1 specificities, respectively (Table 1). Thus, each P. falciparum AP appears to have a distinct specificity range, and together they represent the apparent sum total of AP activities that could play a role in peptide catabolism. Four methionine APs encoded in the genome (16Wu Y. Wang X. Liu X. Wang Y. Genome Res. 2003; 13: 601-616Crossref PubMed Scopus (191) Google Scholar) were not considered likely to participate directly in oligopeptide catabolism, as they function in the co-translational removal of methionine from the N termini of newly synthesized proteins (33Bradshaw R.A. Brickey W.W. Walker K.W. Trends Biochem. Sci. 1998; 23: 263-267Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). These methionine APs were therefore not analyzed in this study.TABLE 1P. falciparum aminopeptidases examined in this studyNameGene IDNearest homologSpecificitySignal peptideLocalizationPfAPPPF14_0517Aminopeptidase PX-ProYesFV, CytPfA-M1MAL13P1.56Aminopeptidase NBroad P1YesFV, NucPfLAPPF14_0439Leucyl aminopeptidaseHydrophobic P1NoCytPfDAPPF11570cAspartyl aminopeptidaseAsp/Glu-XNoCyt Open table in a new tab Aminopeptidase Localization—Stable parasite lines expressing each AP in Table 1 as a fusion with the yellow fluorescent protein (YFP) allele Citrine were generated. As depicted in Fig. 1A, each chromosomal AP gene was modified to encode an AP-YFP fusion by homologous recombination with a transfected episome (see also supplemental Fig. S1). Transcription of each single copy AP-YFP chimera was controlled by the respective endogenous AP promoter; therefore, physiologically relevant patterns of AP expression were expected to be maintained, as observed previously with this approach (21Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (109) Google Scholar). All four AP-YFP parasite lines exhibited fluorescence during the asexual erythrocytic cycle (Fig. 1B and supplemental Figs. S2-5). Two APs were observed in the parasite's food vacuole, the site of hemoglobin catabolism: PfAPP (Fig. 1B and supplemental Fig. S2) and PfA-M1 (Fig. 1B and supplemental Fig. S3). Interestingly, both of these APs also accumulated in a second compartment as follows: the cytosol (PfAPP) and the nucleus (PfA-M1). The two other APs (PfDAP and PfLAP) appeared in the cytosol throughout the erythrocytic cycle (Fig. 1B and supplemental Figs. S4 and S5). Both PfDAP and PfLAP were clearly excluded from the FV, and PfDAP also appeared to be excluded from the nucleus. In addition to its cytosolic location, PfLAP appeared in punctate structures in mature schizonts (Fig. 1B). The cellular location of these fluorescent spots is not clear; they did not co-localize with markers for rhoptries, micronemes, the endoplasmic reticulum, the Golgi apparatus, the apicoplast, or the mitochondrion (supplemental Fig. S6). Localization assignments for all APs are summarized in Table 1. SDS-soluble protein extracts from each AP-YFP parasite line were subjected to immunoblotting with an anti-YFP antibody to assess the number and sizes of YFP-containing polypeptides (Fig. 2). Both PfLAP-YFP and PfDAP-YFP were present as intact fusions in their respective extracts. PfLAP-YFP exhibited a higher mobility than would be expected for the full-length fusion protein (Fig. 2). The apparent decrease in size of ∼15 kDa is consistent with loss of the N-terminal asparagine-rich region (15Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Interestingly, removal of this region was required for the expression of active recombinant PfLAP (15Stack C.M. Lowther J. Cunningham E. Donnelly S. Gardiner D.L. Trenholme K.R. Skinner-Adams T.S. Teuscher F. Grembecka J. Mucha A. Kafarski P. Lua L. Bell A. Dalton J.P. J. Biol. Chem. 2007; 282: 2069-2080Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar); our data suggest that the asparagine-rich region is absent from PfLAP in vivo. Intact fusion protein was also observed in extracts of PfAPP-YFP and PfA-M1-YFP parasites; however, a 25-kDa YFP species was present that presumably reflects cleavage of YFP from the fusions. This is not a surprising outcome considering that both of these AP-YFP fusions are targeted to the FV and exposed to high levels of aspartic and cysteine endopeptidase activities. Cleavage of GFP was previously observed with fusions to the resident FV peptidases PM II and d
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