RESUMO
The protozoan parasite Leishmania, responsible for leishmaniasis, is one of the few aerobic organisms that cannot synthesize the essential molecule heme. Therefore, it has developed specialized pathways to scavenge it from its host. In recent years, some proteins involved in the import of heme, such as LHR1 and LFLVCRB, have been identified, but relevant aspects regarding the process remain unknown. Here, we characterized the kinetics of the uptake of the heme analogue Zn(II) Mesoporphyrin IX (ZnMP) in Leishmania major promastigotes as a model of a parasite causing cutaneous leishmaniasis with special focus on the force that drives the process. We found that ZnMP uptake is an active, inducible, and pH-dependent process that does not require a plasma membrane proton gradient but requires the presence of the monovalent cations Na+ and/or K+. In addition, we demonstrated that this parasite can efflux this porphyrin against a concentration gradient. We also found that ZnMP uptake differs among different dermotropic or viscerotropic Leishmania species and does not correlate with LHR1 or LFLVCRB expression levels. Finally, we showed that these transporters have only partially overlapping functions. Altogether, these findings contribute to a deeper understanding of an important process in the biology of this parasite.
Assuntos
Leishmania major , Leishmaniose Cutânea , Porfirinas , Heme/metabolismo , Humanos , Leishmania major/metabolismo , Leishmaniose Cutânea/parasitologia , Metaloporfirinas , Porfirinas/metabolismo , PrótonsRESUMO
Leishmaniasis comprises a group of neglected diseases caused by the protozoan parasite Leishmania spp. As is the case for other trypanosomatid parasites, Leishmania is auxotrophic for heme and must scavenge this essential compound from its human host. In mammals, the SLC transporter FLVCR2 mediates heme import across the plasma membrane. Herein we identify and characterize Leishmania major FLVCRb (LmFLVCRb), the first member of the FLVCR family studied in a non-metazoan organism. This protein localizes to the plasma membrane of the parasite and is able to bind heme. LmFLVCRb levels in Leishmania, which are modulated by overexpression thereof or the abrogation of an LmFLVCRb allele, correlate with the ability of the parasite to take up porphyrins. Moreover, injection of LmFLVCRb cRNA to Xenopus laevis oocytes provides these cells with the ability to take up heme. This process is temperature dependent, requires monovalent ions and is inhibited at basic pH, characteristics shared by the uptake of heme by Leishmania parasites. Interestingly, LmFLVCRb is essential as CRISPR/Cas9-mediated knockout parasites were only obtained in the presence of an episomal copy of the gene. In addition, deletion of just one of the alleles of the LmFLVCRb gene markedly impairs parasite replication as intracellular amastigotes as well as its virulence in an in vivo model of cutaneous leishmaniasis. Collectively, these results show that Leishmania parasites can rescue heme through plasma membrane transporter LFLVCRb, which could constitute a novel target for therapeutic intervention against Leishmania and probably other trypanosomatid parasites in which FLVCR genes are also present.
Assuntos
Heme/metabolismo , Leishmania major/metabolismo , Leishmaniose/parasitologia , Macrófagos/parasitologia , Proteínas de Membrana Transportadoras/metabolismo , Porfirinas/metabolismo , Proteínas de Protozoários/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Leishmania major/patogenicidade , Leishmaniose/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Oócitos/metabolismo , Oócitos/parasitologia , Proteínas de Protozoários/genética , Receptores Virais/genética , Homologia de Sequência , Virulência , Xenopus laevisRESUMO
Heme is an essential molecule synthetized through a broadly conserved 8-step route that has been lost in trypanosomatid parasites. Interestingly, Leishmania reacquired by horizontal gene transfer from γ-proteobacteria the genes coding for the last 3 enzymes of the pathway. Here we show that intracellular amastigotes of Leishmania major can scavenge heme precursors from the host cell to fulfill their heme requirements, demonstrating the functionality of this partial pathway. To dissect its role throughout the L. major life cycle, the significance of L. major ferrochelatase (LmFeCH), the terminal enzyme of the route, was evaluated. LmFeCH expression in a heterologous system demonstrated its activity. Knockout promastigotes lacking lmfech were not able to use the ferrochelatase substrate protoporphyrin IX as a source of heme. In vivo infection of Phlebotomus perniciosus with knockout promastigotes shows that LmFeCH is not required for their development in the sandfly. In contrast, the replication of intracellular amastigotes was hampered in vitro by the deletion of lmfech. However, LmFeCH-/- parasites produced disease in a cutaneous leishmaniasis murine model in a similar way as control parasites. Therefore, although L. major can synthesize de novo heme from macrophage precursors, this activity is dispensable being an unsuited target for leishmaniasis treatment.-Orrego, L. M., Cabello-Donayre, M., Vargas, P., Martínez-García, M., Sánchez, C., Pineda-Molina, E., Jiménez, M., Molina, R., Pérez-Victoria, J. M. Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major.
Assuntos
Ferroquelatase/metabolismo , Heme/biossíntese , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/metabolismo , Proteínas de Protozoários/metabolismo , Psychodidae/metabolismo , Virulência , Sequência de Aminoácidos , Animais , Coproporfirinogênio Oxidase/metabolismo , Feminino , Ferroquelatase/química , Ferroquelatase/genética , Leishmaniose Cutânea/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Conformação Proteica , Protoporfirinogênio Oxidase/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Psychodidae/parasitologia , Homologia de SequênciaRESUMO
Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.
Assuntos
Heme/metabolismo , Hemoglobinas/metabolismo , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Citosol/metabolismo , Endocitose/fisiologia , Leishmania/metabolismo , Leishmaniose/sangue , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/parasitologiaRESUMO
The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca(2+), and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei.
Assuntos
Aminoquinolinas/farmacologia , Antimaláricos/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Administração Oral , Aminoquinolinas/metabolismo , Antimaláricos/metabolismo , Humanos , Lisossomos/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestruturaRESUMO
Protozoan parasites are responsible for dramatic, neglected diseases. The automatic determination of intracellular parasite burden from fluorescence microscopy images is a challenging problem. Recent advances in deep learning are transforming this process, however, high-performance algorithms have not been developed. The limitations in image acquisition, especially for intracellular parasites, make this process complex. For this reason, traditional image-processing methods are not easily transferred between different datasets and segmentation-based strategies do not have a high performance. Here, we propose a novel method FiCRoN, based on fully convolutional regression networks (FCRNs), as a promising new tool for estimating intracellular parasite burden. This estimation requires three values, intracellular parasites, infected cells and uninfected cells. FiCRoN solves this problem as multi-task learning: counting by regression at two scales, a smaller one for intracellular parasites and a larger one for host cells. It does not use segmentation or detection, resulting in a higher generalization of counting tasks and, therefore, a decrease in error propagation. Linear regression reveals an excellent correlation coefficient between manual and automatic methods. FiCRoN is an innovative freedom-respecting image analysis software based on deep learning, designed to provide a fast and accurate quantification of parasite burden, also potentially useful as a single-cell counter.
Assuntos
Aprendizado Profundo , Parasitos , Humanos , Animais , Algoritmos , Software , Microscopia de Fluorescência , Processamento de Imagem Assistida por Computador/métodosRESUMO
OBJECTIVES: New drugs are required to treat neglected diseases caused by trypanosomatid parasites such as Leishmania, Trypanosoma brucei and Trypanosoma cruzi. An Achilles' heel of these parasites is their heme auxotrophy; they have an absolute dependence on scavenging this molecule from the host, and trypanosomatid HRG heme transporters (TrypHRG) play an important role in this process. As these proteins are essential for the parasites and have low similarity with their human orthologue, they have been proposed as attractive therapeutic targets. Here, we have developed two yeast-based assays that allow an inexpensive high-throughput screening of TrypHRG inhibitors within a cellular context. METHODS: We first assessed that Leishmania major, Leishmania donovani and T. brucei HRG proteins were heterologously expressed in the digestive vacuole membrane of a mutant heme auxotrophic yeast strain. Here, TrypHRG imports hemoglobinderived heme into the cytosol, allowing mutant yeast to grow in the presence of low hemoglobin concentrations and promoting the activity of hemeproteins such as catalase, which was used as a reporter of cytosolic heme levels. RESULTS: In the presence of a TrypHRG inhibitor, both catalase activity (test 1) and yeast growth (test 2) were diminished, being easily monitored. The assays were then tested on a pilot scale for HTS purposes using a collection of repurposing drugs and food antioxidants. Some of the TrypHRG inhibitors identified in yeast presented strong trypanocidal and leishmanicidal activity in the submicromolar range, proving the potential of this approach. CONCLUSIONS: Cumulatively, it was shown that the inhibition bioassays developed were robust and applicable to large-scale HTS.
Assuntos
Leishmania , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Catalase , Bioensaio , HemeRESUMO
Three families of non-reducing trisaccharide fatty acid monoesters bearing C10 to C18 acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to µM. The new compounds are capable of dissolving phospholipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed ß-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.
Assuntos
Detergentes/química , Ácidos Graxos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Trissacarídeos/química , Bioquímica , Lipossomos , Micelas , SolubilidadeRESUMO
The characterization of LABCG5, a new intracellular ATP-binding cassette protein in Leishmania donovani, is described. Unlike other ABCG half-transporters, LABCG5 is not involved in either drug resistance or phospholipid efflux. However, we provide evidence suggesting that this protein is involved in intracellular haem trafficking. Thus, downregulation of LABCG5 function produced upon overexpression of an inactive version of the protein caused a dramatic growth arrest unless a haemin supplement was added or the mutated gene was eliminated. Supplementation with haemoglobin, an upstream metabolite normally sufficient to meet parasite haem requirements, was unable to rescue the growth defect phenotype. Haemoglobin endocytosis was not hampered in dominant-negative parasites and neither was haem uptake, a process that we show here to be dependent on a specific transporter. In contrast, LABCG5 function was required for the correct intracellular trafficking of haemoglobin-bound porphyrins to the mitochondria, not affecting the routing of free haem. Finally, LABCG5 binds haem through hydrophobic and electrostatic interactions. Altogether, these data suggest that LABCG5 is involved in the salvage of the haem released after the breakdown of internalized haemoglobin. As Leishmania is auxotrophic for haem, the pharmacological targeting of this route could represent a novel approach to control fatal visceral leishmaniasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Heme/metabolismo , Leishmania donovani/metabolismo , Leishmaniose Visceral/parasitologia , Proteínas de Protozoários/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Biológico , Linhagem Celular , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/metabolismo , Transporte Proteico , Proteínas de Protozoários/genéticaRESUMO
Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania.
Assuntos
Trifosfato de Adenosina/metabolismo , Aminoquinolinas/farmacologia , Antiprotozoários/farmacologia , Leishmania major/efeitos dos fármacos , Leishmania major/metabolismo , Resistência a Medicamentos , Glicólise , Piruvato Quinase/metabolismoRESUMO
Although oral miltefosine represented an important therapeutic advance in the treatment of leishmaniasis, the appearance of resistance remains a serious threat. LMDR1/LABCB4, a P-glycoprotein-like transporter included in the Leishmania ABC (ATP-binding cassette) family, was the first molecule shown to be involved in experimental miltefosine resistance. LMDR1 pumps drugs out of the parasite, thereby decreasing their intracellular accumulation. Sitamaquine, another promising oral drug for leishmaniasis, is currently in phase 2b clinical trials. The physicochemical features of this drug suggested to us that it could be considered for use as an LMDR1 inhibitor. Indeed, we report herein that nonleishmanicidal concentrations of sitamaquine reverse miltefosine resistance in a multidrug resistance Leishmania tropica line that overexpresses LMDR1. This reversal effect is due to modulation of the LMDR1-mediated efflux of miltefosine. In addition, sitamaquine is not a substrate of LMDR1, as this transporter does not affect sitamaquine accumulation or sensitivity in the parasite. Likewise, we show that ketoconazole, another oral leishmanicidal drug known to interact with ABC transporters, is also able to reverse LMDR1-mediated miltefosine resistance, although with a lower efficiency than sitamaquine. Molecular docking on a three-dimensional homology model of LMDR1 showed different preferential binding sites for each substrate-inhibitor pair, thus explaining this different behavior. Finally, we show that sitamaquine is also able to modulate the antimony resistance mediated by MRPA/LABCC3, another ABC transporter involved in experimental and clinical antimony resistance in this parasite. Taken together, these data suggest that the combination of sitamaquine with miltefosine or antimony could avoid the appearance of resistance mediated by these membrane transporters in Leishmania.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Aminoquinolinas/farmacologia , Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania tropica/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminoquinolinas/metabolismo , Aminoquinolinas/uso terapêutico , Antimônio/uso terapêutico , Antiprotozoários/metabolismo , Farmacorresistência Bacteriana , Cetoconazol/metabolismo , Cetoconazol/farmacologia , Leishmania tropica/metabolismo , Leishmaniose/tratamento farmacológico , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Ligação ProteicaRESUMO
Species of the protozoan Leishmania are causative agents of human leishmaniasis, a disease that results in significant death, disability, and disfigurement around the world. The parasite is transmitted to a mammalian host by a sand fly vector where it develops as an intracellular parasite within macrophages. This process requires the acquisition of nutritional iron and heme from the host as Leishmania lacks the capacity for de novo heme synthesis and does not contain cytosolic iron-storage proteins. Proteins involved in Leishmania iron and heme transport and metabolism have been identified and shown to be crucial for the parasite's growth and replication within the host. Consequently, a detailed understanding of how these parasites harness host pathways for survival may lay the foundation for promising new therapeutic intervention against leishmaniasis.
Assuntos
Heme/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Ferro/metabolismo , Leishmania/metabolismo , Leishmaniose/parasitologia , Animais , HumanosRESUMO
The multidrug transporter ABCG2, a membrane protein with six transmembrane segments, can be overexpressed with the baculovirus/insect cell system. However, ABCG2 is produced as two species with different migration behavior via SDS-PAGE. Evidences suggest that this is due to the accumulation of an immature ABCG2 species, since: (i) the upper species, with higher apparent molecular weight, was favored by treatments reducing the rate of protein synthesis; (ii) the lower species was accumulated in presence of an endoplasmic reticulum stress inducer, and could be converted into the upper species during electrophoresis with 9 M urea; (iii) each species was differently solubilized by detergents: the upper species was partially solubilized by non-ionic and zwitterionic detergents, whereas the lower one required stronger surfactants; (iv) membrane ATPase activity from infected insect cells was essentially associated to the upper species. Altogether, these results suggest that although the insect cell/baculovirus system is not ideally adapted to overexpress human ABCG2, it is able to produce appreciable amounts of purified protein and the addition of agents reducing the rate of protein synthesis improves the homogeneity, making it a suitable heterologous expression system.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Clonagem Molecular/métodos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/isolamento & purificação , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Animais , Baculoviridae/genética , Linhagem Celular , Cicloeximida/farmacologia , Endopeptidase K/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Insetos/citologia , Proteínas de Neoplasias/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
The parasitic protozoan Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds. Here, we explore circular bacteriocin AS-48's ability to kill clinically relevant bloodstream forms of T. brucei gambiense, T. brucei rhodesiense and T. brucei brucei. AS-48 exhibited excellent anti-trypanosomal activity in vitro (EC50â¯=â¯1-3â¯nM) against the three T. brucei subspecies, but it was innocuous to human cells at 104-fold higher concentrations. In contrast to its antibacterial action, AS-48 does not kill the parasite through plasma membrane permeabilization but by targeting intracellular compartments. This was evidenced by the fact that vital dye internalization-prohibiting concentrations of AS-48 could kill the parasite at 37⯰C but not at 4⯰C. Furthermore, AS-48 interacted with the surface of the parasite, at least in part via VSG, its uptake was temperature-dependent and clathrin-depleted cells were less permissive to the action of AS-48. The bacteriocin also caused the appearance of myelin-like structures and double-membrane autophagic vacuoles. These changes in the parasite's ultrastructure were confirmed by fluorescence microscopy as AS-48 induced the production of EGFP-ATG8.2-labeled autophagosomes. Collectively, these results indicate AS-48 kills the parasite through a mechanism involving clathrin-mediated endocytosis of VSG-bound AS-48 and the induction of autophagic-like cell death. As AS-48 has greater in vitro activity than the drugs currently used to treat T. brucei infection and does not present any signs of toxicity in mammalian cells, it could be an attractive lead compound for the treatment of sleeping sickness and nagana.
Assuntos
Autofagia/efeitos dos fármacos , Bacteriocinas/farmacologia , Morte Celular/efeitos dos fármacos , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Endocitose/efeitos dos fármacos , Humanos , Microscopia de Fluorescência , Temperatura , Trypanosoma brucei brucei/ultraestrutura , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Tripanossomíase Africana/sangue , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologiaRESUMO
Overexpression of breast cancer resistance protein ABCG2 confers multidrug resistance in cancer cells. The GF120918-sensitive drug efflux activity of human wild-type (R482) ABCG2-transfected cells was used for rational screening of inhibitory flavonoids and establishment of structure-activity relationships. Flavones were found more efficient than flavonols, isoflavones, and flavanones. Differentially substituted flavone derivatives indicated positive OH effects at position 5, in contrast to positions 3 and 7. A methoxy at position 7 was slightly positive in tectochrysin, whereas a strong positive effect was produced by prenylation at position 6. The potency of 6-prenylchrysin was comparable with that of GF120918 (IC50 = 0.3 micromol/L). Both 6-prenylchrysin and tectochrysin seemed specific for ABCG2 because no interaction was detected with either P-glycoprotein or MRP1. The ABCG2 resistance profile in vitro is altered by mutation at amino acid 482. The R482T mutation limited the effect of prenylation on ABCG2 inhibition. Whereas GF120918 strongly inhibited the ATPase activity of wild-type ABCG2, neither 6-prenylchrysin nor tectochrysin altered the activity. In contrast, all three inhibitors stimulated the ATPase activity of mutant ABCG2. 6-Prenylchrysin at 0.5 micromol/L efficiently sensitized the growth of wild-type ABCG2-transfected cells to mitoxantrone, whereas higher concentrations were required for the mutant ones. In contrast, 1 micromol/L tectochrysin was sufficient to fully sensitize mutant ABCG2-transfected cells, whereas higher concentrations were required for the wild-type ones. Both flavones exhibited a lower intrinsic cytotoxicity than GF120918 and were apparently not transported by ABCG2. 6-Prenylchrysin and tectochrysin therefore constitute new and promising inhibitors for the reversal of ABCG2-mediated drug transport.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Flavonoides/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Acridinas/farmacologia , Adenosina Trifosfatases/metabolismo , Benzimidazóis/farmacocinética , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Flavonoides/química , Humanos , Mitoxantrona/farmacocinética , Mitoxantrona/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Rodaminas/farmacocinética , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/farmacologia , TransfecçãoRESUMO
BACKGROUND: Mitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host. METHODS: LmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software. RESULTS: LmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the generation of cytosolic ISC. Indeed, docking analyzes performed with a LmABCB3 structural model using trypanothione, the main thiol in this parasite, as a ligand showed how both, LmABCB3 and yeast ATM1, contain a similar thiol-binding pocket. Additionally, we show solid evidence suggesting that LmABCB3 is an essential gene as dominant negative inhibition of LmABCB3 is lethal for the parasite. Moreover, the abrogation of only one allele of the gene did not impede promastigote growth in axenic culture but prevented the replication of intracellular amastigotes and the virulence of the parasites in a mouse model of cutaneous leishmaniasis. CONCLUSIONS: Altogether our results present the previously undescribed LmABCB3 as an unusual mitochondrial ABC transporter essential for Leishmania survival through its role in the generation of heme and cytosolic ISC. Hence, LmABCB3 could represent a novel target to combat leishmaniasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Leishmania major/genética , Leishmaniose/parasitologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Heme/metabolismo , Humanos , Ferro/metabolismo , Leishmania major/metabolismo , Leishmania major/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Moleculares , Transporte Proteico , Enxofre/metabolismo , VirulênciaRESUMO
The hydrophobic estradiol-derivative RU49953 inhibits the energy-dependent interaction of yeast multidrug-transporter Pdr5p with its fluorescent drug-substrate rhodamine 6G. The potent inhibition is competitive towards drug binding (Ki=23+/-6 nM), whereas nucleoside-triphosphate hydrolysis is two-orders-of-magnitude less sensitive. RU49953 constitutes the most efficient inhibitor of drug binding to a yeast multidrug ABC exporter reported so far.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Mifepristona/análogos & derivados , Mifepristona/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Ligação Competitiva/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/metabolismo , Mifepristona/química , Rodaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidoresRESUMO
The hemolytic activity and solubilizing capacity of two families of non-reducing trisaccharide fatty acid monoesters have been studied to assess their usefulness as surfactants for pharmaceutical applications. The carbohydrate-based surfactants investigated included homologous series of raffinose and melezitose monoesters bearing C10 to C18 acyl chains prepared by lipase-catalyzed synthesis in organic media. The hemolytic activity was determined in vitro using a static method based on the addition of the surfactants to an erythrocyte suspension and subsequent spectrophotometric determination of the released hemoglobin. The effect of the carbohydrate head group, the acyl chain length and the regioisomeric purity was investigated. In all cases, the carbohydrate monoester surfactants decreased their hemolytic activity (with respect to their critical micelle concentration) when increasing the length of the acyl chain. A very similar behaviour was observed either the carbohydrate head-group (raffinose and melezitose) or regardless of the regioisomeric purity. Interestingly, decanoyl (C10) and lauroyl (C12) monoesters were just marginally hemolytic at their critical micelle concentrations while the longer palmitoyl (C16) and (C18) stearoyl monoesters become hemolytic at concentrations much higher than their respective cmc. The palmitoyl and stearoyl monoesters also displayed higher solubilization capacity than the shorter acyl chain monoesters in a solubilization assay of a hydrophobic dye as a model drug mimic. These results suggest that raffinose and melezitose monoesters with long-chain fatty acids (C16 to C18) are promising surfactants for pharmaceutical applications and could be an alternative to the use of current commercial nonionic polyoxyethylene-based surfactants in parenteral formulations.
Assuntos
Hemólise/efeitos dos fármacos , Rafinose/farmacologia , Tensoativos/química , Trissacarídeos/farmacologia , Carboidratos/química , Química Farmacêutica/métodos , Corantes/química , Ácidos Graxos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipase/metabolismo , Micelas , Rafinose/química , Trissacarídeos/químicaRESUMO
Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2(K/M)) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2(K/M) expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2(K/M) are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2(K/M) did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Leishmania/metabolismo , Leishmania/patogenicidade , Fosfatidilserinas/metabolismo , Proteínas de Protozoários/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Feminino , Leishmania/genética , Leishmania major/genética , Leishmania major/metabolismo , Leishmania major/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genéticaRESUMO
The multidrug resistance phenotype of cancer cells has been often related to overexpression of plasma membrane ATP-binding cassette transporters, which are able to efflux many types of drug by using the energy of ATP hydrolysis. ABCG2 is a half-transporter recently involved. Its purification would help to understand the mechanism of both transport and its inhibition. Biophysical, structural, and functional studies are consuming great amounts of homogeneous purified proteins and require efficient overexpression systems. Heterologous overexpression of human membrane proteins is actually a challenge because these proteins are toxic for the host, and both translation and chaperone systems of the host are not well adapted to the biosynthesis of human proteins. Overexpression of ABCG2 has been assayed in both bacterial and insect cell/baculovirus systems. Although it was highly overexpressed in bacterial system, neither transport nor ATPase activity was found within inverted membrane vesicles. By contrast, insect cells/baculovirus system produces a low amount of protein, a part of which is active.