RESUMO
Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 µM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.
RESUMO
The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compound library which showed significantly improved selectivity for the parasite's folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was observed for compound 5b. This work represents a step forward in the identification of effective antileishmania agents.
Assuntos
Leishmania/enzimologia , Oxirredutases/antagonistas & inibidores , Tripanossomicidas/síntese química , Linhagem Celular , Sinergismo Farmacológico , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Ácido Fólico/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Leishmania/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Leishmania major/enzimologia , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , Simulação de Acoplamento Molecular , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Pirimetamina/farmacologia , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/farmacologiaRESUMO
A characteristic, well-studied feature of the pathogenic protists belonging to the family Trypanosomatidae is the compartmentalisation of the major part of the glycolytic pathway in peroxisome-like organelles, hence designated glycosomes. Such organelles containing glycolytic enzymes appear to be present in all members of the Kinetoplastea studied, and have recently also been detected in a representative of the Diplonemida, but they are absent from the Euglenida. Glycosomes therefore probably originated in a free-living, common ancestor of the Kinetoplastea and Diplonemida. The initial sequestering of glycolytic enzymes inside peroxisomes may have been the result of a minor mistargeting of proteins, as generally observed in eukaryotic cells, followed by preservation and its further expansion due to the selective advantage of this specific form of metabolic compartmentalisation. This selective advantage may have been a largely increased metabolic flexibility, allowing the organisms to adapt more readily and efficiently to different environmental conditions. Further evolution of glycosomes involved, in different taxonomic lineages, the acquisition of additional enzymes and pathways - often participating in core metabolic processes - as well as the loss of others. The acquisitions may have been promoted by the sharing of cofactors and crucial metabolites between different pathways, thus coupling different redox processes and catabolic and anabolic pathways within the organelle. A notable loss from the Trypanosomatidae concerned a major part of the typical peroxisomal H(2)O(2)-linked metabolism. We propose that the compartmentalisation of major parts of the enzyme repertoire involved in energy, carbohydrate and lipid metabolism has contributed to the multiple development of parasitism, and its elaboration to complicated life cycles involving consecutive different hosts, in the protists of the Kinetoplastea clade.
Assuntos
Glicólise , Kinetoplastida/metabolismo , Microcorpos/metabolismo , Evolução Biológica , Euglênidos/metabolismo , Euglenozoários/metabolismo , Kinetoplastida/genética , Redes e Vias MetabólicasRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The aim of the study was to evaluate the in vitro antitrypanosomal and antileishmanial activity of crude extracts of 10 plant species traditionally used in Benin to treat parasitic infections. MATERIALS AND METHODS: For each species, dichloromethane, methanol and aqueous extracts were tested. Their antitrypanosomal and antileishmanial activities were evaluated in vitro on Trypanosoma brucei brucei (strain 427) (Tbb) and on promastigotes of Leishmania mexicana mexicana (MHOM/BZ/84/BEL46) (Lmm). RESULTS: The best growth inhibition was observed with the dichloromethane extracts of aerial parts of Acanthospermum hispidum DC. (Asteraceae) (IC(50)=14.5 µg/ml on Tbb and 11.1 µg/ml on Lmm), twigs of Keetia leucantha (K. Krause) Bridson (syn. Plectronia leucantha Krause) (IC(50)=5.8 µg/ml on Tbb), aerial parts of Byrsocarpus coccineus Schumach. & Thonn (syn. Rourea coccinea (Schumach. & Thonn.) Hook.f.) (IC(50)=14.7 µg/ml on Tbb) and aerial parts of Carpolobia lutea G.Don. (IC(50)=18.3 µg/ml on Tbb). All these extracts had a low cytotoxicity. It is not the case for the methanolic and water extracts of roots of Anchomanes difformis (Blume) Engl. (IC(50)=14.7 and 13.8 µg/ml on Tbb) which were toxic at the same concentration range on WI38, human cells. A bio-guided fractionation of the most active extract of Keetia leucantha allowed to identify oleanolic acid and ursolic acid as responsible for the observed activities. CONCLUSION: Our study gives some justification for antiparasitic activity of some investigated plants.
Assuntos
Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Medicinas Tradicionais Africanas , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Trypanosoma brucei brucei/efeitos dos fármacos , Antiprotozoários/isolamento & purificação , Benin , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Humanos , Leishmania/crescimento & desenvolvimento , Testes de Sensibilidade Parasitária , Extratos Vegetais/isolamento & purificação , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Glycolysis and glyconeogenesis play crucial roles in the ATP supply and synthesis of glycoconjugates, important for the viability and virulence, respectively, of the human-pathogenic stages of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. These pathways are, therefore, candidate targets for antiparasite drugs. The glycolytic/gluconeogenic enzyme enolase is generally highly conserved, with similar overall fold and identical catalytic residues in all organisms. Nonetheless, potentially important differences exist between the trypanosomatid and host enzymes, with three unique, reactive residues close to the active site of the former that might be exploited for the development of new drugs. In addition, enolase is found both in the secretome and in association with the surface of Leishmania spp. where it probably functions as plasminogen receptor, playing a role in the parasite's invasiveness and virulence, a function possibly also present in the other trypanosomatids. This location and possible function of enolase offer additional perspectives for both drug discovery and vaccination.
RESUMO
Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) which is fatal if left untreated. This disease occurs in 36 African countries, south of the Sahara, where 60 million people are at risk of acquiring infection. The current chemotherapy relies on only four drugs, three of which were developed more than 60 years ago. These drugs have many limitations, ranging from oral inabsorption, acute toxicities, short duration of action and the emergence of trypanosomal resistance. Despite decades of use of most of the current trypanocides, little is known about their mode of action. That being said, African trypanosomes continue to be among the most extensively studied parasitic protists to date. Many of their intriguing biological features have been well documented and can be viewed as attractive targets for antitrypanosomal chemotherapy. A considerable number of natural products with diverse molecular structures have revealed antiparasitic potency in the laboratory and represent interesting lead compounds for the development of new and urgently needed antiparasitics. The major validated drug targets in T. brucei are discussed with particular emphasis on those known to be attacked by natural compounds.
Assuntos
Produtos Biológicos/farmacologia , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , África/epidemiologia , Animais , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Metabolismo dos Carboidratos/efeitos dos fármacos , DNA Topoisomerases/efeitos dos fármacos , DNA de Cinetoplasto/efeitos dos fármacos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Resistência a Medicamentos , Farnesiltranstransferase/efeitos dos fármacos , Humanos , Oxirredutases/efeitos dos fármacos , Proteínas de Protozoários/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Tripanossomicidas/química , Tripanossomicidas/isolamento & purificação , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/epidemiologia , Tubulina (Proteína)/efeitos dos fármacosRESUMO
Folate analogue inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new molecules with specificity for PTR1, we carried out a virtual screening of the Available Chemicals Directory (ACD) database to select compounds that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like molecules with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compounds both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compounds showed efficacy only in combination. In toxicity tests against human fibroblasts, several compounds showed low toxicity. One compound, 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies, was active in combination and is suitable for early preclinical evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.
Assuntos
Fármacos do Sistema Nervoso Central/química , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Tripanossomicidas/química , Benzotiazóis/síntese química , Benzotiazóis/química , Benzotiazóis/farmacologia , Fármacos do Sistema Nervoso Central/síntese química , Fármacos do Sistema Nervoso Central/farmacologia , Desenho de Fármacos , Sinergismo Farmacológico , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Leishmania/efeitos dos fármacos , Leishmania/enzimologia , Oxirredutases/química , Testes de Sensibilidade Parasitária , Pirimetamina/análogos & derivados , Pirimetamina/síntese química , Pirimetamina/química , Pirimetamina/farmacologia , Riluzol/análogos & derivados , Riluzol/síntese química , Riluzol/química , Riluzol/farmacologia , Bibliotecas de Moléculas Pequenas , Tetra-Hidrofolato Desidrogenase/química , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologiaRESUMO
Several 5-O-alkyl- and 5-C-alkyl-mannitol bis-phosphates were synthesized and comparatively assayed as inhibitors of fructose bis-phosphate aldolases (Fbas) from rabbit muscle (taken as surrogate model of the human enzyme) and from Trypanosoma brucei. A limited selectivity was found in several instances. Crystallographic studies confirm that the 5-O-methyl derivative binds competitively with substrate and the 5-O-methyl moiety penetrating deeper into a shallow hydrophobic pocket at the active site. This observation can lead to the preparation of selective competitive or irreversible inhibitors of the parasite Fba.
RESUMO
In continuation of our study on medicinal plants of Cameroon, stem barks of Polyalthia suaveolens were phytochemically studied. This investigation yielded a new indolosesquiterpene alkaloid, named polysin (1) and four hitherto known alkaloids (2-5). Polysin (1) appeared as a competitive reversible inhibitor (K(i)=10 microM) of phosphofructo kinase (PFK) of Trypanosoma brucei with respect to fructose-6-phosphate (K(i)/K(M)=0.05) and could be used in the design of new trypanocidal drugs. The other isolated compounds (2-5) also exhibited interesting inhibitory effects on selected glycolytic enzymes (PFK, glyceraldehyde-3-phosphate dehydrogenase and aldolase).
Assuntos
Alcaloides/farmacologia , Polyalthia/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Alcaloides/química , Alcaloides/isolamento & purificação , Camarões , Inibidores Enzimáticos/farmacologia , Glicólise/efeitos dos fármacos , Fosfofrutoquinases/antagonistas & inibidores , Fitoterapia , Plantas Medicinais/química , Sesquiterpenos , Tripanossomicidas/química , Tripanossomicidas/isolamento & purificação , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/metabolismoRESUMO
Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6 degrees rigid body rocking motion of the A- and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.
Assuntos
Leishmania mexicana/enzimologia , Modelos Químicos , Modelos Moleculares , Proteínas de Protozoários/química , Piruvato Quinase/química , Regulação Alostérica/fisiologia , Animais , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Piruvato Quinase/metabolismoRESUMO
Aldolase (ALD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Trypanosoma brucei are considered to be promising targets for chemotherapeutic treatment of African sleeping sickness, because glycolysis is the single source of ATP for the parasite when living in the human bloodstream. Moreover, these enzymes appeared to possess distinct kinetic and structural properties that have already been exploited for the discovery of effective and selective inhibitors with trypanocidal activity. Here we present an experimental, quantitative assessment of the importance of these enzymes for the glycolytic pathway. This was achieved by decreasing the concentrations of ALD and GAPDH by RNA interference. The effects of these knockdowns on parasite growth, levels of various enzymes and transcripts, enzyme activities and glucose consumption were studied. A partial depletion of ALD and GAPDH was already sufficient to rapidly kill the trypanosomes. An effect was also observed on the activity of some other glycolytic enzymes.
Assuntos
Frutose-Bifosfato Aldolase/genética , Variação Genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/parasitologia , Animais , Linhagem Celular , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Frutose-Bifosfato Aldolase/metabolismo , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Humanos , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/metabolismoRESUMO
Polycarpol, sitosterol and sitosterol-3-O-beta-D-glucoside isolated for the first time from Piptostigma preussi (Annonaceae) occur regularly in some Annonaceae such as Piptostigma genus. Polycarpol exhibits interesting antitrypanosomal activity with an ED(50) value of 5.11 microM on Trypanosoma brucei cells. Moreover, it inhibits T. brucei glycolytic enzymes GAPDH and PFK with IC(50) values of 650 and 180 microM respectively.
Assuntos
Annonaceae/química , Extratos Vegetais/farmacologia , Hidrocarbonetos Policíclicos Aromáticos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Glucosídeos/isolamento & purificação , Casca de Planta , Extratos Vegetais/química , Caules de Planta , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , Sitosteroides/isolamento & purificação , Tripanossomicidas/isolamento & purificação , Trypanosoma brucei brucei/enzimologiaRESUMO
We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening (4 A) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.
Assuntos
Leishmania mexicana/enzimologia , Estrutura Quaternária de Proteína , Piruvato Quinase/química , Sulfatos/química , Trifosfato de Adenosina/química , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Piruvato Quinase/genética , Piruvato Quinase/metabolismoRESUMO
The glycolytic pathway has been considered a potential drug target against the parasitic protozoan species of Trypanosoma and Leishmania. We report the design and the synthesis of inhibitors targeted against Trypanosoma brucei phosphofructokinase (PFK) and Leishmania mexicana pyruvate kinase (PyK). Stepwise library synthesis and inhibitor design from a rational starting point identified furanose sugar amino amides as a novel class of inhibitors for both enzymes with IC(50) values of 23microM and 26microM against PFK and PyK, respectively. Trypanocidal activity also showed potency in the low micromolar range and confirms these inhibitors as promising candidates for the development towards the design of anti-trypanosomal drugs.
Assuntos
Inibidores Enzimáticos/farmacologia , Chumbo/química , Leishmania mexicana/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicólise , Concentração Inibidora 50 , Leishmania mexicana/enzimologia , Leishmania mexicana/metabolismo , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Testes de Sensibilidade Parasitária , Fosfofrutoquinases/antagonistas & inibidores , Piruvato Quinase/antagonistas & inibidores , Estereoisomerismo , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/metabolismoRESUMO
Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.
Assuntos
Microcorpos/metabolismo , Proteínas de Protozoários/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Estágios do Ciclo de Vida , Microcorpos/ultraestrutura , Dados de Sequência Molecular , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Ligação Proteica , Transporte Proteico , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Análise de Sequência de Proteína , Homologia de Sequência , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/ultraestruturaRESUMO
Protozoan Kinetoplastida, including the pathogenic trypanosomatids of the genera Trypanosoma and Leishmania, compartmentalize several important metabolic systems in their peroxisomes which are designated glycosomes. The enzymatic content of these organelles may vary considerably during the life-cycle of most trypanosomatid parasites which often are transmitted between their mammalian hosts by insects. The glycosomes of the Trypanosoma brucei form living in the mammalian bloodstream display the highest level of specialization; 90% of their protein content is made up of glycolytic enzymes. The compartmentation of glycolysis in these organelles appears essential for the regulation of this process and enables the cells to overcome short periods of anaerobiosis. Glycosomes of all other trypanosomatid forms studied contain an extended glycolytic pathway catalyzing the aerobic fermentation of glucose to succinate. In addition, these organelles contain enzymes for several other processes such as the pentose-phosphate pathway, beta-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines, ether-lipids and squalenes. The enzymatic content of glycosomes is rapidly changed during differentiation of mammalian bloodstream-form trypanosomes to the forms living in the insect midgut. Autophagy appears to play an important role in trypanosomatid differentiation, and several lines of evidence indicate that it is then also involved in the degradation of old glycosomes, while a population of new organelles containing different enzymes is synthesized. The compartmentation of environment-sensitive parts of the metabolic network within glycosomes would, through this way of organelle renewal, enable the parasites to adapt rapidly and efficiently to the new conditions.
Assuntos
Leishmania/metabolismo , Microcorpos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/metabolismo , Animais , Compartimento Celular , Diferenciação Celular , Glicólise , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
Peroxisomes, glyoxysomes and glycosomes are related organelles found in different organisms. The morphology and enzymic content of the different members of this organelle family differ considerably, and may also be highly dependent on the cell's environmental conditions or life cycle. However, all peroxisome-like organelles have in common a number of characteristic enzymes or enzyme systems, notably enzymes dealing with reactive oxygen species. All organelles of the family follow essentially the same route of biogenesis, but with species-specific differences. Sets of proteins called peroxins are involved in different aspects of the formation and proliferation of peroxisomes such as import of proteins in the organellar matrix, insertion of proteins in the membrane, etc. In different eukaryotic lineages these functions are carried out by often--but not always--homologous yet poorly conserved peroxins. The process of biogenesis and the nature of the proteins involved suggest that all members of the peroxisome family evolved from a single organelle in an ancestral eukaryotic cell. This original peroxisome was possibly derived from a cellular membrane system such as the endoplasmic reticulum. Most of the organism-specific functions of the extant organelles have been acquired later in evolution.
Assuntos
Glioxissomos/metabolismo , Membranas Intracelulares/metabolismo , Microcorpos/metabolismo , Peroxissomos/metabolismo , Transporte Proteico , Animais , Evolução Molecular , Humanos , Proteínas/metabolismoRESUMO
A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.
Assuntos
Sangue/parasitologia , Glicólise , Trypanosoma brucei brucei/metabolismo , Animais , Antiporters/genética , Proteínas de Bactérias/genética , Linhagem Celular , Escherichia coli/genética , Genes Reporter , Cinética , Modelos Biológicos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA de Protozoário/genética , RNA Interferente Pequeno , Transfecção , Trypanosoma brucei brucei/isolamento & purificaçãoRESUMO
Kinetoplastida are protozoan organisms that probably diverged early in evolution from other eukaryotes. They are characterized by a number of unique features with respect to their energy and carbohydrate metabolism. These organisms possess peculiar peroxisomes, called glycosomes, which play a central role in this metabolism; the organelles harbour enzymes of several catabolic and anabolic routes, including major parts of the glycolytic and pentosephosphate pathways. The kinetoplastid mitochondrion is also unusual with regard to both its structural and functional properties.In this review, we describe the unique compartmentation of metabolism in Kinetoplastida and the metabolic properties resulting from this compartmentation. We discuss the evidence for our recently proposed hypothesis that a common ancestor of Kinetoplastida and Euglenida acquired a photosynthetic alga as an endosymbiont, contrary to the earlier notion that this event occurred at a later stage of evolution, in the Euglenida lineage alone. The endosymbiont was subsequently lost from the kinetoplastid lineage but, during that process, some of its pathways of energy and carbohydrate metabolism were sequestered in the kinetoplastid peroxisomes, which consequently became glycosomes. The evolution of the kinetoplastid glycosomes and the possible selective advantages of these organelles for Kinetoplastida are discussed. We propose that the possession of glycosomes provided metabolic flexibility that has been important for the organisms to adapt easily to changing environmental conditions. It is likely that metabolic flexibility has been an important selective advantage for many kinetoplastid species during their evolution into the highly successful parasites today found in many divergent taxonomic groups.Also addressed is the evolution of the kinetoplastid mitochondrion, from a supposedly pluripotent organelle, attributed to a single endosymbiotic event that resulted in all mitochondria and hydrogenosomes of extant eukaryotes. Furthermore, indications are presented that Kinetoplastida may have acquired other enzymes of energy and carbohydrate metabolism by various lateral gene transfer events different from those that involved the algal- and alpha-proteobacterial-like endosymbionts responsible for the respective formation of the glycosomes and mitochondria.
RESUMO
We report here the first crystal structure of a stable isosteric analogue of 1,3-bisphospho-d-glyceric acid (1,3-BPGA) bound to the catalytic domain of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) in which the two phosphoryl moieties interact with Arg249. This complex possibly illustrates a step of the catalytic process by which Arg249 may induce compression of the product formed, allowing its expulsion from the active site. Structural modifications were introduced into this isosteric analogue and the respective inhibitory effects of the resulting diphosphorylated compounds on T. cruzi and Trypanosoma brucei gGAPDHs were investigated by enzymatic inhibition studies, fluorescence spectroscopy, site-directed mutagenesis, and molecular modelling. Despite the high homology between the two trypanomastid gGAPDHs (> 95%), we have identified specific interactions that could be used to design selective irreversible inhibitors against T. cruzi gGAPDH.