42 results on '"Klemba M"'
Search Results
2. A bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases
- Author
-
McGowan, S., primary, Klemba, M., additional, and Greebaum, D.C., additional
- Published
- 2011
- Full Text
- View/download PDF
3. Metabolism of host lysophosphatidylcholine in Plasmodium falciparum -infected erythrocytes.
- Author
-
Liu J, Fike KR, Dapper C, and Klemba M
- Subjects
- Animals, Humans, Plasmodium falciparum, Lysophosphatidylcholines metabolism, Lysophospholipase genetics, Lysophospholipase metabolism, Erythrocytes metabolism, Fatty Acids metabolism, Lipase metabolism, Protozoan Proteins metabolism, Malaria, Falciparum parasitology, Parasites metabolism
- Abstract
The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum -infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy., Competing Interests: Competing interests statement:The authors declare no competing interest.
- Published
- 2024
- Full Text
- View/download PDF
4. Metabolism of host lysophosphatidylcholine in Plasmodium falciparum -infected erythrocytes.
- Author
-
Liu J, Dapper C, and Klemba M
- Abstract
The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using a novel assay for LPC hydrolysis in P. falciparum -infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, are the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ , loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically-relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.
- Published
- 2023
- Full Text
- View/download PDF
5. Leveraging a Fluorescent Fatty Acid Probe to Discover Cell-Permeable Inhibitors of Plasmodium falciparum Glycerolipid Biosynthesis.
- Author
-
Dapper C, Liu J, and Klemba M
- Subjects
- Animals, Humans, Fatty Acids metabolism, Fluorescent Dyes metabolism, Phospholipids metabolism, Mammals, Plasmodium falciparum metabolism, Antimalarials pharmacology
- Abstract
A sensitive and quantitative fluorescence-based approach is presented for characterizing fatty acid acquisition and lipid biosynthesis by asexually replicating, intraerythrocytic Plasmodium falciparum. We show that a BODIPY-containing, green-fluorescent fatty acid analog is efficiently and rapidly incorporated into parasite neutral lipids and phospholipids. Prelabeling with a red-fluorescent ceramide analog permits normalization and enables reliable quantitation of glycerolipid labeling. Inhibition of lipid labeling by competition with natural fatty acids and by acyl-coenzyme A synthetase and diacylglycerol acyltransferase inhibitors demonstrates that the fluorescent fatty acid probe is acquired, activated, and transferred to lipids through physiologically-relevant pathways. To assess its utility in discovering small molecules that block parasite lipid biosynthesis, the lipid labeling assay was used to screen a panel of mammalian lipase inhibitors and a selection of compounds from the "Malaria Box" anti-malarial collection. Several compounds were identified that inhibited the incorporation of the fluorescent fatty acid probe into lipids in cultured parasites at low micromolar concentrations. Two contrasting profiles of suppression of neutral lipid and phospholipid synthesis were observed, which implies the inhibition of distinct pathways. IMPORTANCE The human malaria parasite Plasmodium falciparum relies on fatty acid scavenging to supply this essential precursor of lipid synthesis during its asexual replication cycle in human erythrocytes. This dependence on host fatty acids represents a potential vulnerability that can be exploited to develop new anti-malarial therapies. The quantitative experimental approach described here provides a platform for simultaneously interrogating multiple facets of lipid metabolism- fatty acid uptake, fatty acyl-CoA synthesis, and neutral lipid and phospholipid biosynthesis- and of identifying cell-permeable inhibitors that are active in situ .
- Published
- 2022
- Full Text
- View/download PDF
6. In vitro and in vivo evaluation of the antimalarial MMV665831 and structural analogs.
- Author
-
Ding S, Fike KR, Klemba M, and Carlier PR
- Subjects
- Animals, Antimalarials chemistry, Antimalarials therapeutic use, Disease Models, Animal, Malaria drug therapy, Malaria parasitology, Mannich Bases pharmacology, Mannich Bases therapeutic use, Mice, Plasmodium berghei pathogenicity, Antimalarials pharmacology, Mannich Bases chemistry, Plasmodium falciparum drug effects
- Abstract
Antimalarial candidates possessing novel mechanisms of action are needed to control drug resistant Plasmodium falciparum. We were drawn to Malaria Box compound 1 (MMV665831) by virtue of its excellent in vitro potency, and twelve analogs were prepared to probe its structure-activity relationship. Modulation of the diethyl amino group was fruitful, producing compound 25, which was twice as potent as 1 against cultured parasites. Efforts were made to modify the phenolic Mannich base functionality of 1, to prevent formation of a reactive quinone methide. Homologated analog 28 had reduced potency relative to 1, but still inhibited growth with EC
50 ≤ 200 nM. Thus, the antimalarial activity of 1 does not derive from quinone methide formation. Chemical stability studies on dimethyl analog 2 showed remarkable hydrolytic stability of both the phenolic Mannich base and ethyl ester moieties, and 1 was evaluated for in vivo efficacy in P. berghei-infected mice (40 mg/kg, oral). Unfortunately, no reduction in parasitemia was seen relative to control. These results are discussed in the context of measured plasma and hepatocyte stabilities, with reference to structurally-related, orally-efficacious antimalarials., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
7. Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparum.
- Author
-
Elahi R, Ray WK, Dapper C, Dalal S, Helm RF, and Klemba M
- Subjects
- Biotin analogs & derivatives, Humans, Hydrolases antagonists & inhibitors, Life Cycle Stages, Lipolysis, Plasmodium falciparum growth & development, Proteomics, Serine metabolism, Erythrocytes parasitology, Hydrolases metabolism, Plasmodium falciparum enzymology, Protozoan Proteins metabolism
- Abstract
Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/β hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.
- Published
- 2019
- Full Text
- View/download PDF
8. Internalization of Erythrocyte Acylpeptide Hydrolase Is Required for Asexual Replication of Plasmodium falciparum.
- Author
-
Elahi R, Dapper C, and Klemba M
- Subjects
- Cells, Cultured, Humans, Malaria, Falciparum parasitology, Plasmodium falciparum growth & development, Protozoan Proteins metabolism, Erythrocytes enzymology, Erythrocytes parasitology, Host-Parasite Interactions, Peptide Hydrolases metabolism, Plasmodium falciparum physiology, Reproduction, Asexual
- Abstract
The human malaria parasite Plasmodium falciparum causes disease as it replicates within the host's erythrocytes. We have found that an erythrocyte serine hydrolase, acylpeptide hydrolase (APEH), accumulates within developing asexual parasites. Internalization of APEH was associated with a proteolytic event that reduced the size of the catalytic polypeptide from 80 to 55 kDa. A triazole urea APEH inhibitor, termed AA74-1, was employed to characterize the role of parasite-internalized APEH. In cell lysates, AA74-1 was a potent and highly selective inhibitor of both host erythrocyte and parasite-internalized APEH. When added to cultures of ring-stage parasites, AA74-1 was a poor inhibitor of replication over one asexual replication cycle; however, its potency increased dramatically after a second cycle. This enhancement of potency was not abrogated by the addition of exogenous isopentenyl pyrophosphate, the sole essential product of apicoplast metabolism. High-potency inhibition of parasite growth could be effected by adding AA74-1 to schizont-stage parasites, which resulted in parasite death at the early trophozoite stage of the ensuing replication cycle. Analysis of APEH inhibition in intact cultured cells revealed that host erythrocyte APEH, but not the parasite-internalized APEH pool, was inhibited by exogenous AA74-1. Our data support a model for the mode of parasiticidal activity of AA74-1 whereby sustained inactivation of host erythrocyte APEH is required prior to merozoite invasion and during parasite asexual development. Together, these findings provide evidence for an essential catalytic role for parasite-internalized APEH. IMPORTANCE Nearly half a million deaths were attributed to malaria in 2017. Protozoan parasites of the genus Plasmodium cause disease in humans while replicating asexually within the host's erythrocytes, with P. falciparum responsible for most of the mortality. Understanding how Plasmodium spp. have adapted to their unique host erythrocyte environment is important for developing malaria control strategies. Here, we demonstrate that P. falciparum coopts a host erythrocyte serine hydrolase termed acylpeptide hydrolase. By showing that the parasite requires acylpeptide hydrolase activity for replication, we expand our knowledge of host cell factors that contribute to robust parasite growth., (Copyright © 2019 Elahi et al.)
- Published
- 2019
- Full Text
- View/download PDF
9. Evidence for Regulation of Hemoglobin Metabolism and Intracellular Ionic Flux by the Plasmodium falciparum Chloroquine Resistance Transporter.
- Author
-
Lee AH, Dhingra SK, Lewis IA, Singh MK, Siriwardana A, Dalal S, Rubiano K, Klein MS, Baska KS, Krishna S, Klemba M, Roepe PD, Llinás M, Garcia CRS, and Fidock DA
- Subjects
- Amodiaquine pharmacology, Antimalarials pharmacology, Artemisinins pharmacology, Calcium metabolism, Cells, Cultured, Erythrocytes metabolism, Erythrocytes parasitology, Gene Expression, Humans, Ion Transport drug effects, Ionophores pharmacology, Membrane Transport Proteins metabolism, Monensin pharmacology, Mutation, Nigericin pharmacology, Plasmodium falciparum genetics, Plasmodium falciparum metabolism, Protozoan Proteins metabolism, Pyrrolidinones pharmacology, Chloroquine pharmacology, Drug Resistance, Multiple genetics, Erythrocytes drug effects, Hemoglobins metabolism, Host-Parasite Interactions, Membrane Transport Proteins genetics, Plasmodium falciparum drug effects, Protozoan Proteins genetics
- Abstract
Plasmodium falciparum multidrug resistance constitutes a major obstacle to the global malaria elimination campaign. Specific mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate resistance to the 4-aminoquinoline drug chloroquine and impact parasite susceptibility to several partner agents used in current artemisinin-based combination therapies, including amodiaquine. By examining gene-edited parasites, we report that the ability of the wide-spread Dd2 PfCRT isoform to mediate chloroquine and amodiaquine resistance is substantially reduced by the addition of the PfCRT L272F mutation, which arose under blasticidin selection. We also provide evidence that L272F confers a significant fitness cost to asexual blood stage parasites. Studies with amino acid-restricted media identify this mutant as a methionine auxotroph. Metabolomic analysis also reveals an accumulation of short, hemoglobin-derived peptides in the Dd2 + L272F and Dd2 isoforms, compared with parasites expressing wild-type PfCRT. Physiologic studies with the ionophores monensin and nigericin support an impact of PfCRT isoforms on Ca
2+ release, with substantially reduced Ca2+ levels observed in Dd2 + L272F parasites. Our data reveal a central role for PfCRT in regulating hemoglobin catabolism, amino acid availability, and ionic balance in P. falciparum, in addition to its role in determining parasite susceptibility to heme-binding 4-aminoquinoline drugs.- Published
- 2018
- Full Text
- View/download PDF
10. Two cap residues in the S1 subsite of a Plasmodium falciparum M1-family aminopeptidase promote broad specificity and enhance catalysis.
- Author
-
Rosati M, Dalal S, and Klemba M
- Subjects
- Amino Acid Substitution, Aminopeptidases genetics, Catalysis, Hydrolysis, Mutation, Plasmodium falciparum genetics, Protozoan Proteins genetics, Substrate Specificity, Aminopeptidases metabolism, Plasmodium falciparum enzymology, Protozoan Proteins metabolism
- Abstract
The aminopeptidase PfA-M1 is a key contributor to peptide catabolism in the human malaria parasite Plasmodium falciparum. PfA-M1 substrate specificity is shaped by the cylindrical S1 subsite, which accommodates the sidechain of the substrate P1 residue. At the top of the S1 subsite are two "cap" residues, E572 and M1034, that are positioned to influence S1 subsite specificity. In this study, we have mutated the cap residues, individually and together, and have evaluated the effects on PfA-M1 specificity and catalytic efficiency. When the P1 residue was too small to engage the cap residues, the mutations had no effect on catalysis. Hydrolysis of dipeptide substrates with a basic P1 residue was significantly impaired in the E572A mutant, most likely due to the loss of a stabilizing salt bridge between E572 and the P1 sidechain. With M1034A, a substantial reduction in catalytic efficiency was observed when the P1 sidechain was large and non-polar. The double E572A/M1034A exhibited significant decreases in catalytic efficiency for most substrates. This effect was not reversed with the polar substitutions E572N/M1034Q, which replaced the PfA-M1 cap residues with those of Escherichia coli aminopeptidase N. Both E572 and M1034 contributed to the binding of the competitive aminopeptidase inhibitor bestatin., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
11. Comparative Proteomics and Functional Analysis Reveal a Role of Plasmodium falciparum Osmiophilic Bodies in Malaria Parasite Transmission.
- Author
-
Suárez-Cortés P, Sharma V, Bertuccini L, Costa G, Bannerman NL, Sannella AR, Williamson K, Klemba M, Levashina EA, Lasonder E, and Alano P
- Subjects
- Animals, Anopheles parasitology, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases metabolism, Female, Germ Cells metabolism, Mutation, Protozoan Proteins genetics, Subtilisins metabolism, Organelles metabolism, Plasmodium falciparum physiology, Proteomics methods, Protozoan Proteins analysis
- Abstract
An essential step in the transmission of the malaria parasite to the Anopheles vector is the transformation of the mature gametocytes into gametes in the mosquito gut, where they egress from the erythrocytes and mate to produce a zygote, which matures into a motile ookinete. Osmiophilic bodies are electron dense secretory organelles of the female gametocytes which discharge their contents during gamete formation, suggestive of a role in gamete egress. Only one protein with no functional annotation, Pfg377, is described to specifically reside in osmiophilic bodies in Plasmodium falciparum Importantly, Pfg377 defective gametocytes lack osmiophilic bodies and fail to infect mosquitoes, as confirmed here with newly produced pfg377 disrupted parasites. The unique feature of Pfg377 defective gametocytes of lacking osmiophilic bodies was here exploited to perform comparative, label free, global and affinity proteomics analyses of mutant and wild type gametocytes to identify components of these organelles. Subcellular localization studies with fluorescent reporter gene fusions and specific antibodies revealed an osmiophilic body localization for four out of five candidate gene products analyzed: the proteases PfSUB2 (subtilisin 2) and PfDPAP2 (Dipeptidyl aminopeptidase 2), the ortholog of the osmiophilic body component of the rodent malaria gametocytes PbGEST and a previously nonannotated 13 kDa protein. These results establish that osmiophilic bodies and their components are dispensable or marginally contribute (PfDPAP2) to gamete egress. Instead, this work reveals a previously unsuspected role of these organelles in P. falciparum development in the mosquito vector., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
12. Parallel inhibition of amino acid efflux and growth of erythrocytic Plasmodium falciparum by mefloquine and non-piperidine analogs: Implication for the mechanism of antimalarial action.
- Author
-
Ghavami M, Dapper CH, Dalal S, Holzschneider K, Klemba M, and Carlier PR
- Subjects
- Animals, Inhibitory Concentration 50, Plasmodium falciparum growth & development, Plasmodium falciparum metabolism, Antimalarials pharmacology, Mefloquine pharmacology, Plasmodium falciparum drug effects
- Abstract
Despite the troubling psychiatric side-effects it causes in some patients, mefloquine (MQ) has been used for malaria prophylaxis and therapy, due to its activity against all Plasmodium species, its ease of dosing, and its relative safety in children and pregnant women. Yet at present there is no consensus on the mechanism of antimalarial action of MQ. Two leading hypotheses for the mechanism of MQ are inhibition of heme crystallization and inhibition of host cell hemoglobin endocytosis. In this report we show that MQ is a potent and rapid inhibitor of amino acid efflux from intact parasitized erythrocytes, which is a measure of the in vivo rate of host hemoglobin endocytosis and catabolism. To further explore the mechanism of action of MQ, we have compared the effects of MQ and 18 non-piperidine analogs on amino acid efflux and parasite growth. Among these closely related compounds, an excellent correlation over nearly 4 log units is seen for 50% inhibition concentration (IC50) values for parasite growth and leucine efflux. These data and other observations are consistent with the hypothesis that the antimalarial action of these compounds derives from inhibition of hemoglobin endocytosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
13. Integrated quantitative phase and birefringence microscopy for imaging malaria-infected red blood cells.
- Author
-
Li C, Chen S, Klemba M, and Zhu Y
- Subjects
- Birefringence, Hemeproteins chemistry, Humans, Plasmodium falciparum, Erythrocytes parasitology, Image Processing, Computer-Assisted methods, Malaria, Falciparum diagnostic imaging, Microscopy methods
- Abstract
A dual-modality birefringence/phase imaging system is presented. The system features a crystal retarder that provides polarization mixing and generates two interferometric carrier waves in a single signal spectrum. The retardation and orientation of sample birefringence can then be measured simultaneously based on spectral multiplexing interferometry. Further, with the addition of a Nomarski prism, the same setup can be used for quantitative differential interference contrast (DIC) imaging. Sample phase can then be obtained with two-dimensional integration. In addition, birefringence-induced phase error can be corrected using the birefringence data. This dual-modality approach is analyzed theoretically with Jones calculus and validated experimentally with malaria-infected red blood cells. The system generates not only corrected DIC and phase images, but a birefringence map that highlights the distribution of hemozoin crystals.
- Published
- 2016
- Full Text
- View/download PDF
14. Amino acid efflux by asexual blood-stage Plasmodium falciparum and its utility in interrogating the kinetics of hemoglobin endocytosis and catabolism in vivo.
- Author
-
Dalal S and Klemba M
- Subjects
- Androstadienes metabolism, Artemisinins metabolism, Enzyme Inhibitors metabolism, Metabolic Flux Analysis standards, Metabolism, Plasmodium falciparum drug effects, Reference Standards, Valine analogs & derivatives, Valine metabolism, Wortmannin, Amino Acids metabolism, Biological Transport, Active, Endocytosis, Hemoglobins metabolism, Metabolic Flux Analysis methods, Plasmodium falciparum metabolism
- Abstract
The endocytosis and catabolism of large quantities of host cell hemoglobin is a hallmark of the intraerythrocytic asexual stage of the human malaria parasite Plasmodium falciparum. It is known that the parasite's production of amino acids from hemoglobin far exceeds its metabolic needs. Here, we show that P. falciparum effluxes large quantities of certain non-polar (Ala, Leu, Val, Pro, Phe, Gly) and polar (Ser, Thr, His) amino acids to the external medium. That these amino acids originate from hemoglobin catabolism is indicated by the strong correlation between individual amino acid efflux rates and their abundances in hemoglobin, and the ability of the food vacuole falcipain inhibitor E-64d to greatly suppress efflux rates. We then developed a rapid, sensitive and precise method for quantifying flux through the hemoglobin endocytic-catabolic pathway that is based on leucine efflux. Optimization of the method involved the generation of a novel amino acid-restricted RPMI formulation as well as the validation of D-norvaline as an internal standard. The utility of this method was demonstrated by characterizing the effects of the phosphatidylinositol-3-kinase inhibitors wortmannin and dihydroartemisinin on the kinetics of Leu efflux. Both compounds rapidly inhibited Leu efflux, which is consistent with a role for phosphtidylinositol-3-phosphate production in the delivery of hemoglobin to the food vacuole; however, wortmannin inhibition was transient, which was likely due to the instability of this compound in culture medium. The simplicity, convenience and non-invasive nature of the Leu efflux assay described here makes it ideal for characterizing the in vivo kinetics of hemoglobin endocytosis and catabolism, for inhibitor target validation studies, and for medium-throughput screens to identify novel inhibitors of cytostomal endocytosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
15. Isoprenoid precursor biosynthesis is the essential metabolic role of the apicoplast during gametocytogenesis in Plasmodium falciparum.
- Author
-
Wiley JD, Merino EF, Krai PM, McLean KJ, Tripathi AK, Vega-Rodríguez J, Jacobs-Lorena M, Klemba M, and Cassera MB
- Subjects
- Animals, Gametogenesis, Organophosphorus Compounds, Plasmodium falciparum growth & development, Apicoplasts metabolism, Hemiterpenes biosynthesis, Life Cycle Stages, Plasmodium falciparum metabolism
- Abstract
The malaria parasite harbors a relict plastid called the apicoplast and its discovery opened a new avenue for drug discovery and development due to its unusual, nonmammalian metabolism. The apicoplast is essential during the asexual intraerythrocytic and hepatic stages of the parasite, and there is strong evidence supporting its essential metabolic role during the mosquito stages of the parasite. Supply of the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is the essential metabolic function of the apicoplast during the asexual intraerythrocytic stages. However, the metabolic role of the apicoplast during gametocyte development, the malaria stages transmitted to the mosquito, remains unknown. In this study, we showed that production of IPP for isoprenoid biosynthesis is the essential metabolic function of the apicoplast during gametocytogenesis, by obtaining normal gametocytes lacking the apicoplast when supplemented with IPP. When IPP supplementation was removed early in gametocytogenesis, developmental defects were observed, supporting the essential role of isoprenoids for normal gametocytogenesis. Furthermore, mosquitoes infected with gametocytes lacking the apicoplast developed fewer and smaller oocysts that failed to produce sporozoites. This finding further supports the essential role of the apicoplast in establishing a successful infection in the mosquito vector. Our study supports isoprenoid biosynthesis as a valid drug target for development of malaria transmission-blocking inhibitors., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
- Full Text
- View/download PDF
16. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.
- Author
-
Poreba M, Mihelic M, Krai P, Rajkovic J, Krezel A, Pawelczak M, Klemba M, Turk D, Turk B, Latajka R, and Drag M
- Subjects
- Amino Acids metabolism, Animals, Cathepsin C metabolism, Cattle, Dipeptides chemical synthesis, Dipeptides metabolism, Humans, Kinetics, Molecular Structure, Plasmodium falciparum chemistry, Protozoan Proteins metabolism, Substrate Specificity, Amino Acids chemistry, Cathepsin C chemistry, Dipeptides chemistry, Plasmodium falciparum enzymology, Protozoan Proteins chemistry
- Abstract
Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.
- Published
- 2014
- Full Text
- View/download PDF
17. Evidence for a Golgi-to-endosome protein sorting pathway in Plasmodium falciparum.
- Author
-
Krai P, Dalal S, and Klemba M
- Subjects
- Adaptor Proteins, Vesicular Transport metabolism, Amino Acid Sequence, Blotting, Southern, Brefeldin A, Endosomes metabolism, Fluorescent Antibody Technique, Golgi Apparatus metabolism, Immunoblotting, Molecular Sequence Data, Organelles physiology, Sequence Alignment, rab GTP-Binding Proteins genetics, rab GTP-Binding Proteins metabolism, rab7 GTP-Binding Proteins, Endosomes physiology, Golgi Apparatus physiology, Plasmodium falciparum physiology, Protein Transport physiology
- Abstract
During the asexual intraerythrocytic stage, the malaria parasite Plasmodium falciparum must traffic newly-synthesized proteins to a broad array of destinations within and beyond the parasite's plasma membrane. In this study, we have localized two well-conserved protein components of eukaryotic endosomes, the retromer complex and the small GTPase Rab7, to define a previously-undescribed endosomal compartment in P. falciparum. Retromer and Rab7 co-localized to a small number of punctate structures within parasites. These structures, which we refer to as endosomes, lie in close proximity to the Golgi apparatus and, like the Golgi apparatus, are inherited by daughter merozoites. However, the endosome is clearly distinct from the Golgi apparatus as neither retromer nor Rab7 redistributed to the endoplasmic reticulum upon brefeldin A treatment. Nascent rhoptries (specialized secretory organelles required for invasion) developed adjacent to endosomes, an observation that suggests a role for the endosome in rhoptry biogenesis. A P. falciparum homolog of the sortilin family of protein sorting receptors (PfSortilin) was localized to the Golgi apparatus. Together, these results elaborate a putative Golgi-to-endosome protein sorting pathway in asexual blood stage parasites and suggest that one role of retromer is to mediate the retrograde transport of PfSortilin from the endosome to the Golgi apparatus.
- Published
- 2014
- Full Text
- View/download PDF
18. A naturally variable residue in the S1 subsite of M1 family aminopeptidases modulates catalytic properties and promotes functional specialization.
- Author
-
Dalal S, Ragheb DRT, Schubot FD, and Klemba M
- Subjects
- Amino Acid Substitution, Catalysis, Dipeptides chemistry, Dipeptides genetics, Escherichia coli genetics, Humans, Hydrolysis, Plasmodium falciparum genetics, CD13 Antigens chemistry, CD13 Antigens genetics, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Evolution, Molecular, Mutation, Missense, Plasmodium falciparum enzymology, Protozoan Proteins chemistry, Protozoan Proteins genetics
- Abstract
M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.
- Published
- 2013
- Full Text
- View/download PDF
19. Characterization of a glycerophosphodiesterase with an unusual tripartite distribution and an important role in the asexual blood stages of Plasmodium falciparum.
- Author
-
Denloye T, Dalal S, and Klemba M
- Subjects
- Cations, Divalent metabolism, Coenzymes metabolism, Cytosol enzymology, Gene Knockout Techniques, Genes, Essential, Glycerophospholipids metabolism, Hydrolysis, Kinetics, Magnesium metabolism, Phosphoric Diester Hydrolases genetics, Plasmodium falciparum genetics, Vacuoles enzymology, Virulence Factors genetics, Erythrocytes parasitology, Phosphoric Diester Hydrolases metabolism, Plasmodium falciparum enzymology, Plasmodium falciparum physiology, Virulence Factors metabolism
- Abstract
Catabolism of glycerophospholipids during the rapid growth of the asexual intraerythrocytic malaria parasite may contribute to membrane recycling and the acquisition of lipid biosynthetic precursors from the host. To better understand the scope of lipid catabolism in Plasmodium falciparum, we have characterized a malarial homolog of bacterial glycerophosphodiesterases. These enzymes catalyze the hydrolysis of glycerophosphodiesterases that are generated by phospholipase-catalyzed removal of the two acyl groups from glycerophospholipids. The P. falciparum glycerophosphodiesterase (PfGDPD) exhibits an unusual tripartite distribution during the asexual blood stage with pools of enzyme in the parasitophorous vacuole, food vacuole and cytosol. Efforts to disrupt the chromosomal PfGDPD coding sequence were unsuccessful, which implies that the enzyme is important for efficient parasite growth. Tagging of the endogenous pool of PfGDPD with a conditional aggregation domain partially perturbed the distribution of the enzyme in the parasitophorous vacuole but had no discernable effect on growth in culture. Kinetic characterization of the hydrolysis of glycerophosphocholine by recombinant PfGDPD, an Mg(2+)-dependent enzyme, yielded steady-state parameters that were comparable to those of a homologous bacterial glycerophosphodiesterase. Together, these results suggest a physiological role for PfGDPD in glycerophospholipid catabolism in multiple subcellular compartments. Possibilities for what this role might be are discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
20. Engagement of the S1, S1' and S2' subsites drives efficient catalysis of peptide bond hydrolysis by the M1-family aminopeptidase from Plasmodium falciparum.
- Author
-
Dalal S, Ragheb DR, and Klemba M
- Subjects
- Amino Acid Motifs, Catalytic Domain, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Oligopeptides chemistry, Substrate Specificity, Zinc chemistry, Aminopeptidases chemistry, Plasmodium falciparum enzymology, Proteolysis, Protozoan Proteins chemistry
- Abstract
The M1-family aminopeptidase PfA-M1 catalyzes the last step in the catabolism of human hemoglobin to amino acids in the Plasmodium falciparum food vacuole. In this study, the structural features of the substrate that promote efficient PfA-M1-catalyzed peptide bond hydrolysis were analyzed. X-Ala and Ala-X dipeptide substrates were employed to characterize the specificities of the enzyme's S1 and S1' subsites. Both subsites exhibited a preference for basic and hydrophobic sidechains over polar and acidic sidechains. The relative specificity of the S1 subsite was similar over the pH range 5.5-7.5. Substrate P1 and P1' residues affected both K(m) and k(cat), revealing that sidechain-subsite interactions not only drive the formation of the Michaelis complex but also influence the rates of ensuing chemical steps. Only a small fraction of the available binding energy was exploited in interactions between substrate sidechains and the S1 and S1' subsites, which indicates a modest level of complementarity. There was no correlation between S1 and S1' specificities and amino acid abundance in hemoglobin. Interactions between PfA-M1 and the backbone atoms of the P1' and P2' residues as well as the P2' sidechain further contributed to the catalytic efficiency of substrate hydrolysis. By demonstrating the engagement of multiple, broad-specificity subsites in PfA-M1, these studies provide insight into how this enzyme is able to efficiently generate amino acids from highly sequence-diverse di- and oligopeptides in the food vacuole., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
21. Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases.
- Author
-
Harbut MB, Velmourougane G, Dalal S, Reiss G, Whisstock JC, Onder O, Brisson D, McGowan S, Klemba M, and Greenbaum DC
- Subjects
- Amino Acid Sequence, Aminopeptidases metabolism, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Hemoglobins metabolism, Leucine chemistry, Leucine pharmacology, Leucyl Aminopeptidase antagonists & inhibitors, Models, Molecular, Molecular Sequence Data, Peptide Library, Peptides chemistry, Peptides metabolism, Plasmodium falciparum drug effects, Plasmodium falciparum enzymology, Protein Array Analysis, Protein Processing, Post-Translational drug effects, Substrate Specificity drug effects, Aminopeptidases antagonists & inhibitors, Leucine analogs & derivatives, Malaria parasitology, Molecular Probe Techniques, Molecular Probes metabolism, Multigene Family
- Abstract
Malaria causes worldwide morbidity and mortality, and while chemotherapy remains an excellent means of malaria control, drug-resistant parasites necessitate the discovery of new antimalarials. Peptidases are a promising class of drug targets and perform several important roles during the Plasmodium falciparum erythrocytic life cycle. Herein, we report a multidisciplinary effort combining activity-based protein profiling, biochemical, and peptidomic approaches to functionally analyze two genetically essential P. falciparum metallo-aminopeptidases (MAPs), PfA-M1 and Pf-LAP. Through the synthesis of a suite of activity-based probes (ABPs) based on the general MAP inhibitor scaffold, bestatin, we generated specific ABPs for these two enzymes. Specific inhibition of PfA-M1 caused swelling of the parasite digestive vacuole and prevented proteolysis of hemoglobin (Hb)-derived oligopeptides, likely starving the parasite resulting in death. In contrast, inhibition of Pf-LAP was lethal to parasites early in the life cycle, prior to the onset of Hb degradation suggesting that Pf-LAP has an essential role outside of Hb digestion.
- Published
- 2011
- Full Text
- View/download PDF
22. Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism.
- Author
-
Ragheb D, Dalal S, Bompiani KM, Ray WK, and Klemba M
- Subjects
- Amino Acid Sequence, Aminopeptidases chemistry, Aminopeptidases isolation & purification, Animals, Base Sequence, Blotting, Western, DNA Primers, Erythrocytes parasitology, Humans, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Aminopeptidases metabolism, Hemoglobins metabolism, Plasmodium falciparum enzymology, Vacuoles metabolism
- Abstract
Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68- and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steady-state kinetic parameters for both aminoacyl-β-naphthylamide and unmodified dipeptide substrates over the pH range 5.0-8.5 reveals that k(cat) is relatively insensitive to pH, whereas K(m) increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0-5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.
- Published
- 2011
- Full Text
- View/download PDF
23. Synthesis of new (-)-bestatin-based inhibitor libraries reveals a novel binding mode in the S1 pocket of the essential malaria M1 metalloaminopeptidase.
- Author
-
Velmourougane G, Harbut MB, Dalal S, McGowan S, Oellig CA, Meinhardt N, Whisstock JC, Klemba M, and Greenbaum DC
- Subjects
- Antimalarials chemistry, Antimalarials pharmacology, Binding Sites, CD13 Antigens chemistry, Crystallography, X-Ray, Leucine chemical synthesis, Leucine chemistry, Leucine pharmacology, Models, Molecular, Molecular Structure, Plasmodium falciparum enzymology, Protein Binding, Small Molecule Libraries, Stereoisomerism, Structure-Activity Relationship, Antimalarials chemical synthesis, CD13 Antigens antagonists & inhibitors, Leucine analogs & derivatives, Plasmodium falciparum drug effects
- Abstract
The malarial PfA-M1 metallo-aminopeptidase is considered a putative drug target. The natural product dipeptide mimetic, bestatin, is a potent inhibitor of PfA-M1. Herein we present a new, efficient, and high-yielding protocol for the synthesis of bestatin derivatives from natural and unnatural N-Boc-d-amino acids. A diverse library of bestatin derivatives was synthesized with variants at the side chain of either the α-hydroxy-β-amino acid (P1) or the adjacent natural α-amino acid (P1'). Surprisingly, we found that extended aromatic side chains at the P1 position resulted in potent inhibition against PfA-M1. To understand these data, we determined the X-ray cocrystal structures of PfA-M1 with two derivatives having either a Tyr(OMe) 15 or Tyr(OBzl) 16 at the P1 position and observed substantial inhibitor-induced rearrangement of the primary loop within the PfA-M1 pocket that interacts with the P1 side chain. Our data provide important insights for the rational design of more potent and selective inhibitors of this enzyme that may eventually lead to new therapies for malaria.
- Published
- 2011
- Full Text
- View/download PDF
24. Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.
- Author
-
Wang F, Krai P, Deu E, Bibb B, Lauritzen C, Pedersen J, Bogyo M, and Klemba M
- Subjects
- Amino Acids metabolism, Cathepsin C antagonists & inhibitors, Cathepsin C isolation & purification, Chlorides metabolism, Enzyme Activators metabolism, Fluorescent Dyes metabolism, Hemoglobins metabolism, Humans, Hydrogen-Ion Concentration, Kinetics, Protease Inhibitors metabolism, Protein Multimerization, Protozoan Proteins antagonists & inhibitors, Protozoan Proteins isolation & purification, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Substrate Specificity, Cathepsin C metabolism, Plasmodium falciparum enzymology, Protozoan Proteins metabolism
- Abstract
Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
25. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.
- Author
-
Deu E, Yang Z, Wang F, Klemba M, and Bogyo M
- Subjects
- Animals, Cathepsin C antagonists & inhibitors, Cathepsin C metabolism, Humans, Liver cytology, Plasmodium falciparum cytology, Protease Inhibitors pharmacology, Rats, Small Molecule Libraries pharmacology, Substrate Specificity, Cell Extracts, Drug Evaluation, Preclinical methods, High-Throughput Screening Assays methods, Molecular Probes metabolism
- Abstract
Background: High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities., Methodology and Principal Findings: Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates., Conclusions: We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.
- Published
- 2010
- Full Text
- View/download PDF
26. Evidence for catalytic roles for Plasmodium falciparum aminopeptidase P in the food vacuole and cytosol.
- Author
-
Ragheb D, Bompiani K, Dalal S, and Klemba M
- Subjects
- Animals, Apoenzymes chemistry, Catalysis, Cryoelectron Microscopy methods, Cytosol metabolism, Humans, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Microscopy, Fluorescence methods, Models, Biological, Protein Structure, Quaternary, Aminopeptidases physiology, Cytosol enzymology, Plasmodium falciparum enzymology, Vacuoles enzymology
- Abstract
The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the food vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37 degrees C in the pH range 5.0-7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.
- Published
- 2009
- Full Text
- View/download PDF
27. On the location of the aminopeptidase N homolog PfA-M1 in Plasmodium falciparum.
- Author
-
Klemba M
- Subjects
- Animals, CD13 Antigens metabolism, Cytosol enzymology, Plasmodium falciparum enzymology, Protozoan Proteins metabolism
- Published
- 2009
- Full Text
- View/download PDF
28. Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum.
- Author
-
Dalal S and Klemba M
- Subjects
- Amino Acids metabolism, Amino Acids pharmacology, Aminopeptidases antagonists & inhibitors, Animals, Cell Proliferation drug effects, Cytoplasm enzymology, Humans, Hydrolysis drug effects, Leucine analogs & derivatives, Leucine pharmacology, Malaria, Falciparum drug therapy, Protease Inhibitors pharmacology, Protozoan Proteins antagonists & inhibitors, Aminopeptidases metabolism, Hemoglobins metabolism, Malaria, Falciparum enzymology, Plasmodium falciparum enzymology, Protozoan Proteins metabolism, Vacuoles enzymology
- Abstract
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.
- Published
- 2007
- Full Text
- View/download PDF
29. Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum.
- Author
-
Klonis N, Tan O, Jackson K, Goldberg D, Klemba M, and Tilley L
- Subjects
- Animals, Endocytosis genetics, Genes, Reporter, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Hemoglobins chemistry, Hemoglobins genetics, Hydrogen-Ion Concentration, Plasmodium falciparum genetics, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Transfection, Vacuoles genetics, Vacuoles physiology, Endocytosis physiology, Hemoglobins metabolism, Plasmodium falciparum chemistry, Plasmodium falciparum physiology, Vacuoles chemistry
- Abstract
The DV (digestive vacuole) of the malaria parasite, Plasmodium falciparum, is the site of Hb (haemoglobin) digestion and haem detoxification and, as a consequence, the site of action of CQ (chloroquine) and related antimalarials. However, the precise pH of the DV and the endocytic vesicles that feed it has proved difficult to ascertain. We have developed new methods using EGFP [enhanced GFP (green fluorescent protein)] to measure the pH of intracellular compartments. We have generated a series of transfectants in CQ-sensitive and -resistant parasite strains expressing GFP chimaeras of the DV haemoglobinase, plasmepsin II. Using a quantitative flow cytometric assay, the DV pH was determined to be 5.4-5.5. No differences were detected between CQ-sensitive and -resistant strains. We have also developed a method that relies on the pH dependence of GFP photobleaching kinetics to estimate the pH of the DV compartment. This method gives a pH estimate consistent with the intensity-based measurement. Accumulation of the pH-sensitive probe, LysoSensor Blue, in the DV confirms the acidity of this compartment and shows that the cytostomal vesicles are not measurably acidic, indicating that they are unlikely to be the site of Hb digestion or the site of CQ accumulation. We show that a GFP probe located outside the DV reports a pH value close to neutral. The transfectants and methods that we have developed represent useful tools for investigating the pH of GFP-containing compartments and should be of general use in other systems.
- Published
- 2007
- Full Text
- View/download PDF
30. A role for falcilysin in transit peptide degradation in the Plasmodium falciparum apicoplast.
- Author
-
Ponpuak M, Klemba M, Park M, Gluzman IY, Lamppa GK, and Goldberg DE
- Subjects
- Acyl Carrier Protein metabolism, Animals, Artificial Gene Fusion, Computational Biology, Genes, Protozoan, Genes, Reporter, Green Fluorescent Proteins analysis, Green Fluorescent Proteins genetics, Metalloendopeptidases genetics, Microscopy, Confocal, Microscopy, Fluorescence, Microscopy, Immunoelectron, Phylogeny, Plasmodium falciparum genetics, Plasmodium falciparum ultrastructure, Protozoan Proteins genetics, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins genetics, Sequence Deletion, Sequence Homology, Amino Acid, Metalloendopeptidases metabolism, Peptides metabolism, Plasmodium falciparum enzymology, Plastids metabolism, Protozoan Proteins metabolism
- Abstract
Falcilysin (FLN) is a zinc metalloprotease thought to degrade globin peptides in the acidic vacuole of the human malaria parasite Plasmodium falciparum. The enzyme has been found to have acidic or neutral pH optima on different peptides and to have additional distribution outside the food vacuole. These data suggested that FLN has an additional function in the parasite. To further probe the functions of FLN, we created a transgenic parasite clone expressing a chromosomally encoded FLN-GFP fusion. Unexpectedly, FLN was found in the apicoplast, an essential chloroplast-like organelle. Nuclear encoded apicoplast proteins are targeted to the organelle by a bipartite N-terminal sequence comprised of a signal sequence followed by a positively charged transit peptide domain. Free transit peptides are thought to be toxic to the plastid and need to be rapidly degraded after proteolytic release from proproteins. We hypothesized that FLN may participate in transit peptide degradation in the apicoplast based on its preference for basic residues at neutral pH and on phylogenetic comparison with other M16 family metalloproteases. In vitro cleavage by FLN of the transit peptide from the apicoplast-resident acyl carrier protein supports this idea. The importance of FLN for parasite development is suggested by our inability to truncate the chromosomal FLN open reading frame. Our work indicates that FLN is an attractive target for antimalarial development.
- Published
- 2007
- Full Text
- View/download PDF
31. Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum.
- Author
-
Klemba M and Goldberg DE
- Subjects
- Amino Acid Sequence, Animals, Aspartic Acid Endopeptidases biosynthesis, Aspartic Acid Endopeptidases chemistry, Aspartic Acid Endopeptidases genetics, Cell Fractionation, Cell Membrane enzymology, Endoplasmic Reticulum chemistry, Gene Expression Regulation, Golgi Apparatus enzymology, Molecular Sequence Data, Pepstatins metabolism, Plasmodium falciparum growth & development, Protein Binding, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Aspartic Acid Endopeptidases metabolism, Endoplasmic Reticulum enzymology, Plasmodium falciparum enzymology
- Abstract
Aspartic proteases participate in a wide variety of cellular processes in eukaryotic organisms. The genome of the human malaria parasite Plasmodium falciparum encodes 10 aspartic protease homologs. Functions have been assigned to four of these: plasmepsins I, II, IV and histo-aspartic protease are key players in the catabolism of hemoglobin in the food vacuole. The functions of the other six remain obscure. To better understand the roles of aspartic proteases in blood stage growth and asexual reproduction of P. falciparum, we have characterized the biosynthesis, cellular location and pepstatin-binding properties of plasmepsin V (PM V). PM V is expressed over the course of asexual intraerythrocytic development. The amount of PM V in the parasite is lowest in the ring stage and increases steadily through schizogony. The proregion of this aspartic protease homolog exhibits remarkable interspecies diversity and appears not to be removed following biosynthesis. In intraerythrocytic parasites, PM V is located in the endoplasmic reticulum but not in ERD2-associated Golgi structures. Fractionation and solubilization experiments demonstrate that PM V is an integral membrane protein, a result that is consistent with the presence of a C-terminal putative transmembrane domain in the PM V sequence. In contrast to the food vacuole plasmepsins, detergent-solubilized PM V does not bind the aspartic protease inhibitor pepstatin. Together, these results strongly suggest that the role of PM V in P. falciparum is distinct from those of previously characterized plasmepsins.
- Published
- 2005
- Full Text
- View/download PDF
32. A Plasmodium falciparum dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation.
- Author
-
Klemba M, Gluzman I, and Goldberg DE
- Subjects
- Amino Acid Sequence, Animals, Blotting, Southern, Cathepsin C chemistry, Cathepsin C metabolism, Cell Division, Chromosomes metabolism, Cloning, Molecular, Erythrocytes metabolism, Humans, Hydrolysis, Immunoblotting, Immunoprecipitation, Microscopy, Fluorescence, Microscopy, Immunoelectron, Models, Biological, Models, Genetic, Molecular Sequence Data, Peptides chemistry, Proteins chemistry, Recombinant Fusion Proteins chemistry, Sequence Homology, Amino Acid, Subcellular Fractions, Time Factors, Cathepsin C physiology, Hemoglobins metabolism, Plasmodium falciparum metabolism, Vacuoles metabolism
- Abstract
Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum requires the catabolism of large amounts of host cell hemoglobin. Endoproteolytic digestion of hemoglobin to short oligopeptides occurs in an acidic organelle called the food vacuole. How amino acids are generated from these peptides is not well understood. To gain insight into this process, we have studied a plasmodial ortholog of the lysosomal exopeptidase cathepsin C. The plasmodial enzyme dipeptidyl aminopeptidase 1 (DPAP1) was enriched from parasite extract by two different approaches and was shown to possess hydrolytic activity against fluorogenic dipeptide substrates. To localize DPAP1 we created a transgenic parasite line expressing a chromosomally encoded DPAP1-green fluorescent protein fusion. Green fluorescent protein fluorescence was observed in the food vacuole of live transgenic parasites, and anti-DPAP1 antibody labeled the food vacuole in parasite cryosections. Together these data implicate DPAP1 in the generation of dipeptides from hemoglobin-derived oligopeptides. To assess the significance of DPAP1, we attempted to ablate DPAP1 activity from blood stage parasites by truncating the chromosomal DPAP1-coding sequence. The inability to disrupt the coding sequence indicates that DPAP1 is important for asexual proliferation. The proenzyme form of DPAP1 was found to accumulate in the parasitophorous vacuole of mature parasites. This observation suggests a trafficking route for DPAP1 through the parasitophorous vacuole to the food vacuole.
- Published
- 2004
- Full Text
- View/download PDF
33. Plasmodium falciparum cysteine protease falcipain-1 is not essential in erythrocytic stage malaria parasites.
- Author
-
Sijwali PS, Kato K, Seydel KB, Gut J, Lehman J, Klemba M, Goldberg DE, Miller LH, and Rosenthal PJ
- Subjects
- Animals, Base Sequence, Cysteine Endopeptidases genetics, Cysteine Proteinase Inhibitors pharmacology, DNA, Protozoan genetics, Erythrocytes parasitology, Gene Targeting, Genes, Protozoan, Humans, In Vitro Techniques, Malaria, Falciparum drug therapy, Malaria, Falciparum parasitology, Plasmodium falciparum genetics, Plasmodium falciparum growth & development, Cysteine Endopeptidases physiology, Plasmodium falciparum enzymology
- Abstract
Among potential new targets for antimalarial chemotherapy are Plasmodium falciparum cysteine proteases, known as falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases that may have additional functions. The function of falcipain-1 remains uncertain. To better characterize the role of falcipain-1 in erythrocytic parasites, we disrupted the falcipain-1 gene and characterized recombinant parasites. Disruption of the falcipain-1 gene was confirmed with Southern blots, and loss of expression of falcipain-1 was confirmed with immunoblots and by loss of labeling with a specific protease inhibitor. Compared with wild-type parasites, falcipain-1 knockout parasites developed normally, with the same morphology, multiplication rate, and invasion efficiency, and without significant differences in sensitivity to cysteine protease inhibitors. In wild-type and knockout parasites, cysteine protease inhibitors blocked hemoglobin hydrolysis in trophozoites, with a subsequent block in rupture of erythrocytes by mature schizonts, but they did not inhibit erythrocyte invasion by merozoites. Our results indicate that although falcipain-1 is expressed by erythrocytic parasites, it is not essential for normal development during this stage or for erythrocyte invasion.
- Published
- 2004
- Full Text
- View/download PDF
34. Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum.
- Author
-
Klemba M, Beatty W, Gluzman I, and Goldberg DE
- Subjects
- Animals, Animals, Genetically Modified genetics, Animals, Genetically Modified metabolism, Aspartic Acid Endopeptidases antagonists & inhibitors, Aspartic Acid Endopeptidases genetics, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum ultrastructure, Enzyme Inhibitors pharmacology, Enzyme Precursors antagonists & inhibitors, Enzyme Precursors genetics, Enzyme Precursors metabolism, Green Fluorescent Proteins, Hemoglobins metabolism, Immunohistochemistry, Intracellular Membranes metabolism, Intracellular Membranes ultrastructure, Luminescent Proteins genetics, Luminescent Proteins metabolism, Microscopy, Electron, Models, Biological, Nuclear Envelope metabolism, Nuclear Envelope ultrastructure, Plasmodium falciparum genetics, Plasmodium falciparum ultrastructure, Protein Transport genetics, Protozoan Proteins, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Vacuoles genetics, Vacuoles ultrastructure, Aspartic Acid Endopeptidases metabolism, Plasmodium falciparum metabolism, Vacuoles metabolism
- Abstract
A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.
- Published
- 2004
- Full Text
- View/download PDF
35. Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
- Author
-
Banerjee R, Liu J, Beatty W, Pelosof L, Klemba M, and Goldberg DE
- Subjects
- Amino Acid Sequence, Animals, Aspartic Acid Endopeptidases genetics, Erythrocytes parasitology, Hemoglobins metabolism, Humans, Plasmodium falciparum genetics, Plasmodium falciparum pathogenicity, Protozoan Proteins, Recombinant Proteins genetics, Recombinant Proteins metabolism, Vacuoles enzymology, Aspartic Acid Endopeptidases metabolism, Plasmodium falciparum enzymology
- Abstract
Hemoglobin degradation is a metabolic process that is central to the growth and maturation of the malaria parasite Plasmodium falciparum. Two aspartic proteases that initiate degradation, plasmepsins (PMs) I and II, have been identified and extensively characterized. Eight additional PM genes are present in the P. falciparum genome. To better understand the enzymology of hemoglobin degradation, it is necessary to determine which of these genes are expressed when hemoglobin degradation is occurring, which encode active enzymes, and which gene products are found in the food vacuole where catabolism takes place. Our genome-wide analysis reveals that PM I, II, and IV and histo-aspartic protease encode hemoglobin-degrading food vacuole proteases. Despite having a histidine in place of one of the catalytic aspartic acids conserved in other aspartic proteases, histo-aspartic protease is an active hydrolase.
- Published
- 2002
- Full Text
- View/download PDF
36. Biological roles of proteases in parasitic protozoa.
- Author
-
Klemba M and Goldberg DE
- Subjects
- Animals, Enzyme Inhibitors metabolism, Eukaryota cytology, Eukaryota physiology, Host-Parasite Interactions, Humans, Endopeptidases metabolism, Eukaryota enzymology, Eukaryota pathogenicity, Protozoan Proteins metabolism
- Abstract
Proteases from a variety of protozoan parasites have been characterized at the molecular and cellular levels, and the many roles that proteases play in these organisms are coming into focus. Central roles have been proposed for proteases in diverse processes such as host cell invasion and egress, encystation, excystation, catabolism of host proteins, differentiation, cell cycle progression, cytoadherence, and both stimulation and evasion of host immune responses. Detailed structural and functional characterization of parasite proteases has led to novel insights into the workings of these fascinating catalytic machines. The possibility of developing selective inhibitors of key proteases of pathogenic parasites into novel chemotherapeutic strategies is being vigorously explored.
- Published
- 2002
- Full Text
- View/download PDF
37. Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets.
- Author
-
Coombs GH, Goldberg DE, Klemba M, Berry C, Kay J, and Mottram JC
- Subjects
- Amino Acid Sequence, Animals, Antiprotozoal Agents therapeutic use, Aspartic Acid Endopeptidases chemistry, Aspartic Acid Endopeptidases genetics, Aspartic Acid Endopeptidases metabolism, Eukaryota drug effects, Eukaryota enzymology, Eukaryota genetics, Humans, Molecular Sequence Data, Plasmodium falciparum drug effects, Plasmodium falciparum genetics, Polymerase Chain Reaction, Protozoan Infections drug therapy, Sequence Alignment, Sequence Homology, Antiprotozoal Agents pharmacology, Aspartic Acid Endopeptidases drug effects, Malaria, Falciparum drug therapy, Plasmodium falciparum enzymology
- Abstract
All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.
- Published
- 2001
- Full Text
- View/download PDF
38. Chromosomal integration of tcb chlorocatechol degradation pathway genes as a means of expanding the growth substrate range of bacteria to include haloaromatics.
- Author
-
Klemba M, Jakobs B, Wittich RM, and Pieper D
- Subjects
- Blotting, Southern, Chlorobenzoates metabolism, Cloning, Molecular, Genes, Bacterial, Multigene Family, Phenotype, Plasmids genetics, Polymerase Chain Reaction, Pseudomonas genetics, Pseudomonas metabolism, Pseudomonas putida metabolism, Catechols metabolism, Conjugation, Genetic, Hydrocarbons, Chlorinated metabolism, Pseudomonas putida genetics, Pseudomonas putida growth & development
- Abstract
The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.
- Published
- 2000
- Full Text
- View/download PDF
39. Characterization of metal binding by a designed protein: single ligand substitutions at a tetrahedral Cys2His2 site.
- Author
-
Klemba M and Regan L
- Subjects
- Binding Sites, Cadmium chemistry, Carrier Proteins chemistry, Carrier Proteins genetics, Cobalt chemistry, Metalloproteins chemistry, Metalloproteins genetics, Models, Molecular, Mutagenesis, Insertional, Protein Conformation, Protein Engineering, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Zinc chemistry, Carrier Proteins metabolism, Metalloproteins metabolism, Metals metabolism
- Abstract
The tetrahedral Cys2His2 Zn(II)-binding site in the de novo designed protein Z alpha 4 [Regan, L., & Clarke, N. D. (1990) Biochemistry 29, 10878] has been studied by independently mutating each of the metal-binding ligands to alanine. The contribution of each ligand to the geometry and affinity of metal binding has been characterized using Co(II), Zn(II), and Cd(II). The results indicate that all four ligands contribute to high-affinity metal binding in Z alpha 4. Two of the four metal-site mutants retain the tetrahedral Zn(II)-binding geometry of Z alpha 4, with one water molecule presumed to bind in the vacant ligand position. These mutants provide the first examples of a demonstrated de novo tetrahedral three-coordinate site designed into a protein and as such are a first step toward the design of catalytic rather than structural Zn(II) sites. One of the metal-site mutants binds Zn(II) with either tetrahedral four-coordinate or five-coordinate geometry, while the last ligand-to-alanine substitution abolishes tetrahedral binding. The importance of ligand type for metal-binding in Z alpha 4 was investigated by characterizing two ligand-swap mutants in which a cysteine residue was replaced with a histidine. In both cases, tetrahedral metal binding was lost. Collectively, these results affirm the strategy used to design Z alpha 4 by showing that all designed liganding residues are participating in metal binding, and by suggesting that the tetrahedral geometry of the binding site is perturbed when the designed side chain ligands are replaced with alternate ligands.
- Published
- 1995
- Full Text
- View/download PDF
40. Novel metal-binding proteins by design.
- Author
-
Klemba M, Gardner KH, Marino S, Clarke ND, and Regan L
- Subjects
- Amino Acid Sequence, Binding Sites, Cadmium metabolism, Circular Dichroism, Cobalt metabolism, Cysteine metabolism, Histidine metabolism, Immunoglobulin G metabolism, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Protein Conformation, Thermodynamics, Zinc metabolism, Metalloproteins chemistry
- Abstract
We describe the successful design of a tetrahedral His3Cys Zn(II)-binding site in a small protein of known structure: the B1 domain of Streptococcal protein G. The B1 variants containing the novel metal-binding site were characterized using a combination of optical absorption, circular dichroism and NMR spectroscopies. The results indicate that the designed proteins bind Zn(II) with high affinity and tetrahedral coordination geometry, and that the overall secondary and tertiary structure of the B1 domain is maintained.
- Published
- 1995
- Full Text
- View/download PDF
41. Purification of human leukotriene C4 synthase from dimethylsulfoxide-differentiated U937 cells.
- Author
-
Nicholson DW, Klemba MW, Rasper DM, Metters KM, Zamboni RJ, and Ford-Hutchinson AW
- Subjects
- Cell Differentiation drug effects, Chromatography, Affinity, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Humans, Kinetics, Macromolecular Substances, Molecular Weight, Tumor Cells, Cultured, Dimethyl Sulfoxide pharmacology, Glutathione Transferase isolation & purification, Glutathione Transferase metabolism
- Abstract
Human leukotriene C4 (LTC4) synthase was purified > 10000-fold from dimethylsulfoxide-differentiated U937 cells. Steps included: (a) solubilization of membrane-bound LTC4 synthase from microsomal membranes by the anionic detergent taurocholate; (b) successive anion-exchange chromatography steps in the presence of taurocholate plus Triton X-100 (primary anion exchange) then taurocholate plus n-octyl glucoside (secondary anion exchange); and (c) LTC2-affinity chromatography on a matrix that was constructed by first biotinylating synthetic LTC2 then immobilizing the biotinylated LTC2 on streptavidin agarose. The purification of human LTC4 synthase was enabled by the finding that LTC4 synthase activity in preparations enriched > 500-fold was absolutely dependent on the presence in LTC4 synthase incubation mixtures of divalent cations (specifically Mg2+) and phospholipids (specifically phosphatidylcholine), and that reduced glutathione, which was required at 2-4 mM for stabilization of LTC4 synthase, irreversibly inactivated the enzyme when present at > or = 5 mM during freeze/thaw cycles. The > 10000-fold purified LTC4 synthase preparation was comprised of three polypeptides having molecular masses of 37.1, 24.5 and 18.0 kDa. An 18-kDa polypeptide in both microsomal membranes and in the LTC2-affinity purified fraction was specifically labelled by a radioiodinated LTC4 photoaffinity probe (azido 125I-LTC4). The Km values in the LTC2-affinity purified preparation for reduced glutathione and LTA4 were 1.83 mM and 19.6 microM (respectively), closely resembling the Km values in isolated human blood monocytes. The Vmax of LTC2-affinity purified LTC4 synthase was 2-4 mumol LTC4 formed .min-1 x mg-1.
- Published
- 1992
- Full Text
- View/download PDF
42. Human leukotriene C4 synthase expression in dimethyl sulfoxide-differentiated U937 cells.
- Author
-
Nicholson DW, Ali A, Klemba MW, Munday NA, Zamboni RJ, and Ford-Hutchinson AW
- Subjects
- Cell Differentiation drug effects, Cell Division, Cell Fractionation, Cell Line, Chromatography, High Pressure Liquid, Glutathione Transferase isolation & purification, Humans, Kinetics, SRS-A biosynthesis, SRS-A isolation & purification, Subcellular Fractions enzymology, Tumor Cells, Cultured, Cell Differentiation physiology, Dimethyl Sulfoxide pharmacology, Glutathione Transferase metabolism, Granulocytes enzymology
- Abstract
Leukotriene C4 (LTC4) synthase was highly expressed in the human U937 monoblast leukemia cell line when differentiated into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide. The specific activity of LTC4 synthase in differentiated cells (399.0 +/- 84.1 pmol of LTC4 formed.min-1.mg-1) was markedly higher (10-fold; p less than 0.001) than in undifferentiated U937 cells (39.9 +/- 16.7 pmol of LTC4 formed.min-1.mg-1) or freshly isolated blood monocytes (21.5 +/- 4.8 pmol of LTC4 formed.min-1.mg-1). The increase in LTC4 synthase activity following dimethyl sulfoxide-induced differentiation was substantially higher than the increase observed for other proteins involved in leukotriene biosynthesis. LTC4 synthase activity was unaffected in U937 cells differentiated by growth in the presence of phorbol 12-myristate 13-acetate. The HL-60 myeloblast leukemia cell line expressed higher LTC4 synthase levels when differentiated into either neutrophil-like or macrophage-like cells by growth in the presence of dimethyl sulfoxide or phorbol 12-myristate 13-acetate (respectively), but reached a specific activity comparable only to undifferentiated U937 cells. Human LTC4 synthase was found to be a unique membrane-bound enzymatic activity completely distinct from alpha, mu, pi, theta, and microsomal glutathione S-transferases, as determined by differential detergent solubilization, chromatographic separation, substrate specificity, and Western blot analysis. An 18-kDa polypeptide was specifically labeled in membranes from dimethyl sulfoxide-differentiated U937 cells using azido 125I-LTC4, a photoaffinity probe based on the product of the LTC4 synthase-catalyzed reaction. Photolabeling of the 18-kDa polypeptide was specifically competed for by LTC4 (greater than 50% at 0.1 microM) but not by 100,000-fold higher concentrations of reduced glutathione (10 mM). Elevation of both the level of the specifically photolabeled 18-kDa polypeptide and of LTC4 synthase specific activity occurred concomitantly with dimethyl sulfoxide differentiation of U937 cells. We conclude that differentiation of U937 cells into monocyte/macrophage-like cells by growth in the presence of dimethyl sulfoxide results in high levels of expression of LTC4 synthase activity. Human LTC4 synthase is a unique enzyme with a high degree of specificity for LTA4 and may therefore be dedicated exclusively to the formation of LTC4 in vivo. An 18-kDa membrane polypeptide, specifically labeled by a photoaffinity derivative of LTC4, is a candidate for being either LTC4 synthase or a subunit thereof.
- Published
- 1992
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.