RESUMO
ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.
Assuntos
Parasitos , Trypanosoma brucei brucei , Animais , Trypanosoma brucei brucei/metabolismo , Glicólise , Parasitos/metabolismo , Trifosfato de Adenosina/metabolismo , Modelos Teóricos , Proteínas de Protozoários/metabolismoRESUMO
Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 µM, and for ATP 291 ± 15 and 38 ± 2.2 µM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Fosfoglicerato Quinase/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Trypanosomiases are a group of tropical diseases that have devastating health and socio-economic effects worldwide. In humans, these diseases are caused by the pathogenic kinetoplastids Trypanosoma brucei, causing African trypanosomiasis or sleeping sickness, and Trypanosoma cruzi, causing American trypanosomiasis or Chagas disease. Currently, these diseases lack effective treatment. This is attributed to the high toxicity and limited trypanocidal activity of registered drugs, as well as resistance development and difficulties in their administration. All this has prompted the search for new compounds that can serve as the basis for the development of treatment of these diseases. Antimicrobial peptides (AMPs) are small peptides synthesized by both prokaryotes and (unicellular and multicellular) eukaryotes, where they fulfill functions related to competition strategy with other organisms and immune defense. These AMPs can bind and induce perturbation in cell membranes, leading to permeation of molecules, alteration of morphology, disruption of cellular homeostasis, and activation of cell death. These peptides have activity against various pathogenic microorganisms, including parasitic protists. Therefore, they are being considered for new therapeutic strategies to treat some parasitic diseases. In this review, we analyze AMPs as therapeutic alternatives for the treatment of trypanosomiases, emphasizing their possible application as possible candidates for the development of future natural anti-trypanosome drugs.
Assuntos
Doença de Chagas , Tripanossomicidas , Tripanossomíase Africana , Tripanossomíase , Animais , Humanos , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêutico , Tripanossomicidas/química , Peptídeos Antimicrobianos , Tripanossomíase/tratamento farmacológico , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Doença de Chagas/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/uso terapêuticoRESUMO
Previously, we reported the development of novel small molecules that are potent inhibitors of the glycolytic enzyme phosphofructokinase (PFK) of Trypanosoma brucei and related protists responsible for serious diseases in humans and domestic animals. Cultured bloodstream-form trypanosomes, which are fully reliant on glycolysis for their ATP production, are rapidly killed at submicromolar concentrations of these compounds, which have no effect on the activity of human PFKs and human cells. Single-day oral dosing cures stage 1 human trypanosomiasis in an animal model. Here we analyze changes in the metabolome of cultured trypanosomes during the first hour after addition of a selected PFK inhibitor, CTCB405. The ATP level of T. brucei drops quickly followed by a partial increase. Already within the first five minutes after dosing, an increase is observed in the amount of fructose 6-phosphate, the metabolite just upstream of the PFK reaction, while intracellular levels of the downstream glycolytic metabolites phosphoenolpyruvate and pyruvate show an increase and decrease, respectively. Intriguingly, a decrease in the level of O-acetylcarnitine and an increase in the amount of L-carnitine were observed. Likely explanations for these metabolomic changes are provided based on existing knowledge of the trypanosome's compartmentalized metabolic network and kinetic properties of its enzymes. Other major changes in the metabolome concerned glycerophospholipids, however, there was no consistent pattern of increase or decrease upon treatment. CTCB405 treatment caused less prominent changes in the metabolome of bloodstream-form Trypanosoma congolense, a ruminant parasite. This agrees with the fact that it has a more elaborate glucose catabolic network with a considerably lower glucose consumption rate than bloodstream-form T. brucei.
Assuntos
Fosfofrutoquinases , Trypanosoma , Animais , Humanos , Metaboloma , Metabolômica , Trifosfato de AdenosinaRESUMO
The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.
Assuntos
Glicólise/efeitos dos fármacos , Fosfofrutoquinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Trypanosoma/enzimologia , Tripanossomíase Africana/metabolismo , Tripanossomíase Africana/parasitologia , Doença Aguda , Regulação Alostérica/efeitos dos fármacos , Animais , Células Hep G2 , Humanos , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Camundongos , Parasitos/efeitos dos fármacos , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Relação Estrutura-Atividade , Trypanosoma/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológicoRESUMO
6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152â µM; K0.5F6P 147â µM), PFK-P the lowest affinities (K0.5ATP 276â µM; K0.5F6P 1333â µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160â µM; K0.5F6P 1360â µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfofrutoquinases , Transcrição Gênica , Regulação Alostérica , Frutosefosfatos/química , Frutosefosfatos/genética , Frutosefosfatos/metabolismo , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/genética , Especificidade de Órgãos , Fosfofrutoquinases/biossíntese , Fosfofrutoquinases/química , Fosfofrutoquinases/genética , FosforilaçãoRESUMO
Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK - to a much larger extent than Trypanosoma PFK - is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10-fold, compared to only a 1.3- and 1.4-fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme's 'reaching arms' and 'embracing arms' that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T-to-R equilibria for the two families, with the low-melting Leishmania PFK favouring the flexible inactive T-state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP-dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.
Assuntos
Monofosfato de Adenosina/metabolismo , Glicólise , Guanosina Trifosfato/metabolismo , Leishmania/enzimologia , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Trypanosoma brucei brucei/enzimologia , Monofosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Evolução Biológica , Domínio Catalítico , Cristalografia por Raios X , Gluconeogênese , Guanosina Trifosfato/química , Humanos , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.
Assuntos
Antioxidantes/metabolismo , Glutamato-Cisteína Ligase/genética , Glutationa/análogos & derivados , Proteínas de Protozoários/genética , Espermidina/análogos & derivados , Tiorredoxinas/genética , Trypanosoma cruzi/efeitos dos fármacos , Amida Sintases/genética , Amida Sintases/metabolismo , Butionina Sulfoximina/farmacologia , Linhagem Celular , Combinação de Medicamentos , Resistência a Medicamentos/genética , Fibroblastos/parasitologia , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/metabolismo , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Humanos , Peróxido de Hidrogênio/farmacologia , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Nitroimidazóis/farmacologia , Oxirredução , Estresse Oxidativo , Peroxidases/genética , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Espermidina/antagonistas & inibidores , Espermidina/biossíntese , Tiorredoxinas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genéticaRESUMO
Entamoeba histolytica has neither Krebs cycle nor oxidative phosphorylation activities; therefore, glycolysis is the main pathway for ATP supply and provision of carbon skeleton precursors for the synthesis of macromolecules. Glucose is metabolized through fermentative glycolysis, producing ethanol as its main end-product as well as some acetate. Amoebal glycolysis markedly differs from the typical Embden-Meyerhof-Parnas pathway present in human cells: (i) by the use of inorganic pyrophosphate, instead of ATP, as the high-energy phospho group donor; (ii) with one exception, the pathway enzymes can catalyze reversible reactions under physiological conditions; (iii) there is no allosteric regulation and sigmoidal kinetic behavior of key enzymes; and (iv) the presence of some glycolytic and fermentation enzymes similar to those of anaerobic bacteria. These peculiarities bring about alternative mechanisms of control and regulation of the PPi-dependent fermentative glycolysis in the parasite in comparison to the ATP-dependent and allosterically regulated glycolysis in many other eukaryotic cells. In this review, the current knowledge of the carbohydrate metabolism enzymes in E. histolytica is analyzed. Thermodynamics and stoichiometric analyses indicate 2 to 3.5 ATP yield per glucose metabolized, instead of the often presumed 5 ATP/glucose ratio. PPi derived from anabolism seems insufficient for PPi-glycolysis; hence, alternative ways of PPi supply are also discussed. Furthermore, the underlying mechanisms of control and regulation of the E. histolytica carbohydrate metabolism, analyzed by applying integral and systemic approaches such as Metabolic Control Analysis and kinetic modeling, contribute to unveiling alternative and promising drug targets.
Assuntos
Difosfatos/metabolismo , Entamoeba histolytica/metabolismo , Entamebíase/parasitologia , Glucose/metabolismo , Animais , Entamoeba histolytica/genética , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
In the search for therapeutic targets in the intermediary metabolism of trypanosomatids the gene essentiality criterion as determined by using knock-out and knock-down genetic strategies is commonly applied. As most of the evaluated enzymes/transporters have turned out to be essential for parasite survival, additional criteria and approaches are clearly required for suitable drug target prioritization. The fundamentals of Metabolic Control Analysis (MCA; an approach in the study of control and regulation of metabolism) and kinetic modeling of metabolic pathways (a bottom-up systems biology approach) allow quantification of the degree of control that each enzyme exerts on the pathway flux (flux control coefficient) and metabolic intermediate concentrations (concentration control coefficient). MCA studies have demonstrated that metabolic pathways usually have two or three enzymes with the highest control of flux; their inhibition has more negative effects on the pathway function than inhibition of enzymes exerting low flux control. Therefore, the enzymes with the highest pathway control are the most convenient targets for therapeutic intervention. In this review, the fundamentals of MCA as well as experimental strategies to determine the flux control coefficients and metabolic modeling are analyzed. MCA and kinetic modeling have been applied to trypanothione metabolism in Trypanosoma cruzi and the model predictions subsequently validated in vivo. The results showed that three out of ten enzyme reactions analyzed in the T. cruzi anti-oxidant metabolism were the most controlling enzymes. Hence, MCA and metabolic modeling allow a further step in target prioritization for drug development against trypanosomatids and other parasites.
Assuntos
Desenvolvimento de Medicamentos/métodos , Enzimas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Glutationa/análogos & derivados , Glutationa/metabolismo , Glicólise/fisiologia , Cinética , Modelos Biológicos , Espermidina/análogos & derivados , Espermidina/metabolismoRESUMO
The gluconeogenic enzyme fructose-1,6-bisphosphatase has been proposed as a potential drug target against Leishmania parasites that cause up to 20,000-30,000 deaths annually. A comparison of three crystal structures of Leishmania major fructose-1,6-bisphosphatase (LmFBPase) along with enzyme kinetic data show how AMP acts as an allosteric inhibitor and provides insight into its metal-dependent reaction mechanism. The crystal structure of the apoenzyme form of LmFBPase is a homotetramer in which the dimer of dimers adopts a planar conformation with disordered "dynamic loops". The structure of LmFBPase, complexed with manganese and its catalytic product phosphate, shows the dynamic loops locked into the active sites. A third crystal structure of LmFBPase complexed with its allosteric inhibitor AMP shows an inactive form of the tetramer, in which the dimer pairs are rotated by 18° relative to each other. The three structures suggest an allosteric mechanism in which AMP binding triggers a rearrangement of hydrogen bonds across the large and small interfaces. Retraction of the "effector loop" required for AMP binding releases the side chain of His23 from the dimer-dimer interface. This is coupled with a flip of the side chain of Arg48 which ties down the key catalytic dynamic loop in a disengaged conformation and also locks the tetramer in an inactive rotated T-state. The structure of the effector site of LmFBPase shows different structural features compared with human FBPases, thereby offering a potential and species-specific drug target.
Assuntos
Monofosfato de Adenosina/metabolismo , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/química , Leishmania major/enzimologia , Regulação Alostérica , Coenzimas , Cristalografia por Raios X , Inibidores Enzimáticos , Humanos , Cinética , Manganês/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização ProteicaRESUMO
BACKGROUND: In general, glycerol kinases (GKs) are transferases that catalyze phospho group transfer from ATP to glycerol, and the mechanism was suggested to be random bi-bi. The reverse reaction i.e. phospho transfer from glycerol 3-phosphate (G3P) to ADP is only physiologically feasible by the African trypanosome GK. In contrast to other GKs the mechanism of Trypanosoma brucei gambiense glycerol kinase (TbgGK) was shown to be in an ordered fashion, and proceeding via autophosphorylation. From the unique reaction mechanism of TbgGK, we envisaged its potential to possess phosphatase activity in addition to being a kinase. METHODS: Our hypothesis was tested by spectrophotometric and LC-MS/MS analyses using paranitrophenyl phosphate (pNPP) and TbgGK's natural substrate, G3P respectively. Furthermore, protein X-ray crystallography and site-directed mutagenesis were performed to examine pNPP binding, catalytic residues, and the possible reaction mechanism. RESULTS: In addition to its widely known and expected phosphotransferase (class II) activity, TbgGK can efficiently facilitate the hydrolytic cleavage of phosphoric anhydride bonds (a class III property). This phosphatase activity followed the classical Michaelis-Menten pattern and was competitively inhibited by ADP and G3P, suggesting a common catalytic site for both activities (phosphatase and kinase). The structure of the TGK-pNPP complex, and structure-guided mutagenesis implicated T276 to be important for the catalysis. Remarkably, we captured a crystallographic molecular snapshot of the phosphorylated T276 reaction intermediate. CONCLUSION: We conclude that TbgGK has both kinase and phosphatase activities. GENERAL SIGNIFICANCE: This is the first report on a bifunctional kinase/phosphatase enzyme among members of the sugar kinase family.
Assuntos
Glicerol Quinase/química , Monoéster Fosfórico Hidrolases/química , Conformação Proteica , Trypanosoma brucei gambiense/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cristalografia por Raios X , Glicerol/metabolismo , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Glicerofosfatos/metabolismo , Humanos , Nitrobenzenos/química , Monoéster Fosfórico Hidrolases/metabolismo , Especificidade por Substrato , Trypanosoma brucei gambiense/patogenicidadeRESUMO
The glycerol kinase (GK) of African human trypanosomes is compartmentalized in their glycosomes. Unlike the host GK, which under physiological conditions catalyzes only the forward reaction (ATP-dependent glycerol phosphorylation), trypanosome GK can additionally catalyze the reverse reaction. In fact, owing to this unique reverse catalysis, GK is potentially essential for the parasites survival in the human host, hence a promising drug target. The mechanism of its reverse catalysis was unknown; therefore, it was not clear if this ability was purely due to its localization in the organelles or whether structure-based catalytic differences also contribute. To investigate this lack of information, the X-ray crystal structure of this protein was determined up to 1.90 Å resolution, in its unligated form and in complex with three natural ligands. These data, in conjunction with results from structure-guided mutagenesis suggests that the trypanosome GK is possibly a transiently autophosphorylating threonine kinase, with the catalytic site formed by non-conserved residues. Our results provide a series of structural peculiarities of this enzyme, and gives unexpected insight into the reverse catalysis mechanism. Together, they provide an encouraging molecular framework for the development of trypanosome GK-specific inhibitors, which may lead to the design of new and safer trypanocidal drug(s).
Assuntos
Glicerol Quinase/química , Glicerol Quinase/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei gambiense/enzimologia , Difosfato de Adenosina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicerol , Glicerol Quinase/genética , Humanos , Modelos Moleculares , Mutagênese , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Trypanosoma brucei gambiense/química , Tripanossomíase Africana/parasitologiaRESUMO
Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.
Assuntos
Proteínas de Transporte/química , Coenzima A/química , Cisteína/química , Leishmania mexicana/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/química , Motivos de Aminoácidos , Biocatálise , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Domínio Catalítico , Coenzima A/metabolismo , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Leishmania mexicana/enzimologia , Leishmania mexicana/genética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genéticaRESUMO
All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in â¼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an â¼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/(RNAi)PGI double mutant when compared with the single mutants, and (iii) the (13)C enrichment of glycolytic and PPP intermediates from cells incubated with [U-(13)C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.
Assuntos
Glucose/metabolismo , Malato Desidrogenase/metabolismo , NADP/metabolismo , Via de Pentose Fosfato/fisiologia , Trypanosoma brucei brucei/metabolismo , Animais , Western Blotting , Células Cultivadas , Citosol/metabolismo , Desidroepiandrosterona/farmacologia , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Gluconeogênese/fisiologia , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Homeostase , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Malato Desidrogenase/genética , Espectrometria de Massas , Via de Pentose Fosfato/genética , Interferência de RNA , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Moscas Tsé-Tsé/parasitologiaRESUMO
PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Piruvato Quinase/antagonistas & inibidores , Animais , Arginina/metabolismo , Benzoatos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Sequência Conservada , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Leishmania mexicana/enzimologia , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Proteínas Recombinantes/metabolismo , Sacarina/análogos & derivados , Sacarina/farmacologia , Especificidade da Espécie , Relação Estrutura-Atividade , Suramina/farmacologiaRESUMO
The majority of the glycolytic enzymes in the African trypanosome are compartmentalised within peroxisome-like organelles, the glycosomes. Polypeptides harbouring peroxisomal targeting sequences (PTS type 1 or 2) are targeted to these organelles. This targeting is essential to parasite viability, as compartmentalisation of glycolytic enzymes prevents unregulated ATP-dependent phosphorylation of intermediate metabolites. Here, we report the surprising extra-glycosomal localisation of a PTS-2 bearing trypanosomal hexokinase, TbHK2. In bloodstream form parasites, the protein localises to both glycosomes and to the flagellum. Evidence for this includes fractionation and immunofluorescence studies using antisera generated against the authentic protein as well as detection of epitope-tagged recombinant versions of the protein. In the insect stage parasite, distribution is different, with the polypeptide localised to glycosomes and proximal to the basal bodies. The function of the extra-glycosomal protein remains unclear. While its association with the basal body suggests that it may have a role in locomotion in the insect stage parasite, no detectable defect in directional motility or velocity of cell movement were observed for TbHK2-deficient cells, suggesting that the protein may have a different function in the cell.
Assuntos
Hexoquinase/análise , Microcorpos/química , Microcorpos/enzimologia , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/enzimologia , Flagelos/química , Flagelos/enzimologia , Deleção de Genes , Hexoquinase/genética , Locomoção , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiologiaRESUMO
Ehrlich's pioneering chemotherapeutic experiments published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329-362) described the efficacy of a series of dye molecules including trypan blue and trypan red to eliminate trypanosome infections in mice. The molecular structures of the dyes provided a starting point for the synthesis of suramin, which was developed and used as a trypanocidal drug in 1916 and is still in clinical use. Despite the biological importance of these dye-like molecules, the mode of action on trypanosomes has remained elusive. Here we present crystal structures of suramin and three related dyes in complex with pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The phenyl sulfonate groups of all four molecules (suramin, Ponceau S, acid blue 80, and benzothiazole-2,5-disulfonic acid) bind in the position of ADP/ATP at the active sites of the pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. We show that suramin competitively inhibits PYKs from humans (muscle, tumor, and liver isoenzymes, K(i) = 1.1-17 µM), T. cruzi (K(i) = 108 µM), and L. mexicana (K(i) = 116 µM), all of which have similar active sites. Synergistic effects were observed when examining suramin inhibition in the presence of an allosteric effector molecule, whereby IC(50) values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition observed for suramin and into the mode of action of the dye-like molecules used in Ehrlich's original experiments.
Assuntos
Adenosina/metabolismo , Piruvato Quinase/antagonistas & inibidores , Suramina/farmacologia , Azul Tripano/farmacologia , Tripanossomicidas/farmacologia , Animais , Sítios de Ligação , Ligação Competitiva , Humanos , Concentração Inibidora 50 , Cinética , Leishmania mexicana/enzimologia , Camundongos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Azul Tripano/análogos & derivados , Trypanosoma cruzi/enzimologiaRESUMO
In kinetoplastid protists, glycolysis is compartmentalized in glycosomes, organelles belonging to the peroxisome family. The Trypanosoma brucei glycosomal enzyme triosephosphate isomerase (TPI) does not contain either of the two established peroxisome-targeting signals, but we identified a 22 amino acids long fragment, present at an internal position of the polypeptide, that has the capacity to route a reporter protein to glycosomes in transfected trypanosomes, as demonstrated by cell-fractionation experiments and corroborating immunofluorescence studies. This polypeptide-internal routing information seems to be unique for the sequence of the trypanosome enzyme: a reporter protein fused to a Saccharomyces cerevisiae peptide containing the sequence corresponding to the 22-residue fragment of the T. brucei enzyme, was not targeted to glycosomes. In yeasts, as in most other organisms, TPI is indeed exclusively present in the cytosol. These results suggest that it may be possible to develop new trypanocidal drugs by targeting specifically the glycosome import mechanism of TPI.
Assuntos
Microcorpos/metabolismo , Sinais Direcionadores de Proteínas , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismo , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Animais , Fracionamento Celular , Genes Reporter , Microscopia de Fluorescência , Modelos Moleculares , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/enzimologiaRESUMO
The crystal structure of the ATP-bound form of the tetrameric phosphofructokinase (PFK) from Trypanosoma brucei enables detailed comparisons to be made with the structures of the apoenzyme form of the same enzyme, as well as with those of bacterial ATP-dependent and PP(i)-dependent PFKs. The active site of T. brucei PFK (which is strictly ATP-dependent but belongs to the PP(i)-dependent family by sequence similarities) is a chimera of the two types of PFK. In particular, the active site of T. brucei PFK possesses amino acid residues and structural features characteristic of both types of PFK. Conformational changes upon ATP binding are observed that include the opening of the active site to accommodate the two substrates, MgATP and fructose 6-phosphate, and a dramatic ordering of the C-terminal helices, which act like reaching arms to hold the tetramer together. These conformational transitions are fundamentally different from those of other ATP-dependent PFKs. The substantial differences in structure and mechanism of T. brucei PFK compared with bacterial and mammalian PFKs give optimism for the discovery of species-specific drugs for the treatment of diseases caused by protist parasites of the trypanosomatid family.