RESUMEN
We describe a genetic syndrome due to PGM2L1 deficiency. PGM2 and PGM2L1 make hexose-bisphosphates, like glucose-1,6-bisphosphate, which are indispensable cofactors for sugar phosphomutases. These enzymes form the hexose-1-phosphates crucial for NDP-sugars synthesis and ensuing glycosylation reactions. While PGM2 has a wide tissue distribution, PGM2L1 is highly expressed in the brain, accounting for the elevated concentrations of glucose-1,6-bisphosphate found there. Four individuals (three females and one male aged between 2 and 7.5 years) with bi-allelic inactivating mutations of PGM2L1 were identified by exome sequencing. All four had severe developmental and speech delay, dysmorphic facial features, ear anomalies, high arched palate, strabismus, hypotonia, and keratosis pilaris. Early obesity and seizures were present in three individuals. Analysis of the children's fibroblasts showed that glucose-1,6-bisphosphate and other sugar bisphosphates were markedly reduced but still present at concentrations able to stimulate phosphomutases maximally. Hence, the concentrations of NDP-sugars and glycosylation of the heavily glycosylated protein LAMP2 were normal. Consistent with this, serum transferrin was normally glycosylated in affected individuals. PGM2L1 deficiency does not appear to be a glycosylation defect, but the clinical features observed in this neurodevelopmental disorder point toward an important but still unknown role of glucose-1,6-bisphosphate or other sugar bisphosphates in brain metabolism.
Asunto(s)
Glucosa-6-Fosfato/análogos & derivados , Mutación , Trastornos del Neurodesarrollo/patología , Fosfotransferasas/genética , Alelos , Niño , Preescolar , Femenino , Glucosa-6-Fosfato/biosíntesis , Glicosilación , Humanos , Masculino , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , LinajeRESUMEN
Neutropenia and neutrophil dysfunction in glycogen storage disease type 1b (GSD1b) and severe congenital neutropenia type 4 (SCN4), associated with deficiencies of the glucose-6-phosphate transporter (G6PT/SLC37A4) and the phosphatase G6PC3, respectively, are the result of the accumulation of 1,5-anhydroglucitol-6-phosphate in neutrophils. This is an inhibitor of hexokinase made from 1,5-anhydroglucitol (1,5-AG), an abundant polyol in blood. 1,5-AG is presumed to be reabsorbed in the kidney by a sodium-dependent-transporter of uncertain identity, possibly SGLT4/SLC5A9 or SGLT5/SLC5A10. Lowering blood 1,5-AG with an SGLT2-inhibitor greatly improved neutrophil counts and function in G6PC3-deficient and GSD1b patients. Yet, this effect is most likely mediated indirectly, through the inhibition of the renal 1,5-AG transporter by glucose, when its concentration rises in the renal tubule following inhibition of SGLT2. To identify the 1,5-AG transporter, both human and mouse SGLT4 and SGLT5 were expressed in HEK293T cells and transport measurements were performed with radiolabelled compounds. We found that SGLT5 is a better carrier for 1,5-AG than for mannose, while the opposite is true for human SGLT4. Heterozygous variants in SGLT5, associated with a low level of blood 1,5-AG in humans cause a 50-100% reduction in 1,5-AG transport activity tested in model cell lines, indicating that SGLT5 is the predominant kidney 1,5-AG transporter. These and other findings led to the conclusion that (1) SGLT5 is the main renal transporter of 1,5-AG; (2) frequent heterozygous mutations (allelic frequency > 1%) in SGLT5 lower blood 1,5-AG, favourably influencing neutropenia in G6PC3 or G6PT deficiency; (3) the effect of SGLT2-inhibitors on blood 1,5-AG level is largely indirect; (4) specific SGLT5-inhibitors would be more efficient to treat these neutropenias than SGLT2-inhibitors.
Asunto(s)
Neutropenia , Animales , Humanos , Ratones , Antiportadores , Células HEK293 , Riñón , Proteínas de Transporte de Membrana , Proteínas de Transporte de Monosacáridos/genética , Neutropenia/genética , Transportador 2 de Sodio-Glucosa/genéticaRESUMEN
Glycogen storage disease type Ib (GSD1b) and G6PC3-deficiency are rare autosomal recessive diseases caused by inactivating mutations in SLC37A4 (coding for G6PT) and G6PC3, respectively. Both diseases are characterized by neutropenia and neutrophil dysfunction due to the intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases. We recently showed that the use of SGLT2 inhibitor therapy to reduce tubular reabsorption of its precursor, 1,5-anhydroglucitol (1,5-AG), a glucose analog present in blood, successfully restored the neutropenia and neutrophil function in G6PC3-deficient and GSD1b patients. The intra-individual variability of response to the treatment and the need to adjust the dose during treatment, especially in pediatric populations, can only be efficiently optimized if the concentration of 1,5-AG in blood is monitored during treatment, together with the patients' clinical signs and symptoms. Monitoring the 1,5-AG levels would be greatly simplified if it could be performed on dry blood spots (DBS) which are easy to collect, store and transport. The challenge is to know if a suitable method can be developed to perform accurate and reproducible assays for 1,5-AG using DBS. Here, we describe and validate an assay that quantifies 1,5-AG in DBS using isotopic dilution quantitation by LC-MS/MS that should greatly facilitate patients' follow-up. 1,5-AG levels measured in plasma and DBS give comparable values. This assay was used to monitor the levels of 1,5-AG in DBS from 3 G6PC3-deficient and 6 GSD1b patients during treatment with SGLT2 inhibitors. We recommend this approach to verify the adequate therapeutical response and compliance to the treatment in G6PC3-deficient and GSD1b patients treated with SGLT2 inhibitors.
Asunto(s)
Desoxiglucosa , Enfermedad del Almacenamiento de Glucógeno Tipo I , Neutropenia , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Niño , Humanos , Cromatografía Liquida , Espectrometría de Masas en Tándem , Enfermedad del Almacenamiento de Glucógeno Tipo I/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/complicaciones , Neutropenia/genética , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Monoéster Fosfórico Hidrolasas , Proteínas de Transporte de Monosacáridos , AntiportadoresRESUMEN
Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.
Asunto(s)
Compuestos de Bencidrilo/uso terapéutico , Glucósidos/uso terapéutico , Enfermedad del Almacenamiento de Glucógeno Tipo I/complicaciones , Hexosafosfatos/sangre , Neutropenia/tratamiento farmacológico , Neutrófilos/patología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Compuestos de Bencidrilo/efectos adversos , Glucemia/análisis , Quimiotaxis de Leucocito/efectos de los fármacos , Preescolar , Reposicionamiento de Medicamentos , Resistencia a Medicamentos , Femenino , Glucósidos/efectos adversos , Enfermedad del Almacenamiento de Glucógeno Tipo I/sangre , Enfermedad del Almacenamiento de Glucógeno Tipo I/inmunología , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Granulocitos/química , Humanos , Recién Nacido , Proteína 2 de la Membrana Asociada a los Lisosomas/sangre , Masculino , Neutropenia/sangre , Uso Fuera de lo Indicado , Estallido Respiratorio/efectos de los fármacos , Inhibidores del Cotransportador de Sodio-Glucosa 2/efectos adversos , Adulto JovenRESUMEN
Neutropenia and neutrophil dysfunction found in deficiencies in G6PC3 and in the glucose-6-phosphate transporter (G6PT/SLC37A4) are due to accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), an inhibitor of hexokinase made from 1,5-anhydroglucitol (1,5-AG), an abundant polyol present in blood. Lowering blood 1,5-AG with an SGLT2 inhibitor greatly improved neutrophil counts and function in G6PC3-deficient mice and in patients with G6PT-deficiency. We evaluate this treatment in two G6PC3-deficient children. While neutropenia was severe in one child (PT1), which was dependent on granulocyte cololony-stimulating factor (GCSF), it was significantly milder in the other one (PT2), which had low blood 1,5-AG levels and only required GCSF during severe infections. Treatment with the SGLT2-inhibitor empagliflozin decreased 1,5-AG in blood and 1,5-AG6P in neutrophils and improved (PT1) or normalized (PT2) neutrophil counts, allowing to stop GCSF. On empagliflozin, both children remained infection-free (>1 year - PT2; >2 years - PT1) and no side effects were reported. Remarkably, sequencing of SGLT5, the gene encoding the putative renal transporter for 1,5-AG, disclosed a rare heterozygous missense mutation in PT2, replacing the extremely conserved Arg401 by a histidine. The higher urinary clearance of 1,5-AG explains the more benign neutropenia and the outstanding response to empagliflozin treatment found in this child. Our data shows that SGLT2 inhibitors are an excellent alternative to treat the neutropenia present in G6PC3-deficiency.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo I , Neutropenia , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Animales , Antiportadores/genética , Compuestos de Bencidrilo , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Glucósidos/uso terapéutico , Enfermedad del Almacenamiento de Glucógeno Tipo I/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Humanos , Ratones , Proteínas de Transporte de Monosacáridos/genética , Mutación , Neutropenia/tratamiento farmacológico , Neutropenia/genética , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Neutropenia represents an important problem in patients with genetic deficiency in either the glucose-6-phosphate transporter of the endoplasmic reticulum (G6PT/SLC37A4) or G6PC3, an endoplasmic reticulum phosphatase homologous to glucose-6-phosphatase. While affected granulocytes show reduced glucose utilization, the underlying mechanism is unknown and causal therapies are lacking. Using a combination of enzymological, cell-culture, and in vivo approaches, we demonstrate that G6PT and G6PC3 collaborate to destroy 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a close structural analog of glucose-6-phosphate and an inhibitor of low-KM hexokinases, which catalyze the first step in glycolysis in most tissues. We show that 1,5AG6P is made by phosphorylation of 1,5-anhydroglucitol, a compound normally present in human plasma, by side activities of ADP-glucokinase and low-KM hexokinases. Granulocytes from patients deficient in G6PC3 or G6PT accumulate 1,5AG6P to concentrations (â¼3 mM) that strongly inhibit hexokinase activity. In a model of G6PC3-deficient mouse neutrophils, physiological concentrations of 1,5-anhydroglucitol caused massive accumulation of 1,5AG6P, a decrease in glucose utilization, and cell death. Treating G6PC3-deficient mice with an inhibitor of the kidney glucose transporter SGLT2 to lower their blood level of 1,5-anhydroglucitol restored a normal neutrophil count, while administration of 1,5-anhydroglucitol had the opposite effect. In conclusion, we show that the neutropenia in patients with G6PC3 or G6PT mutations is a metabolite-repair deficiency, caused by a failure to eliminate the nonclassical metabolite 1,5AG6P.
Asunto(s)
Antiportadores/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Neutropenia/metabolismo , Fosforilación/fisiología , Animales , Muerte Celular/fisiología , Línea Celular , Retículo Endoplásmico/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Ratas WistarRESUMEN
Carnosine synthase is the ATP-dependent ligase responsible for carnosine (ß-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic ß-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈ 200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with ß-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed ß-alanyl-lysine, ß-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on ß-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from ß-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was â¼ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (ß-alanyl-lysine, ß-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.
Asunto(s)
Carnosina/análogos & derivados , Dipeptidasas , Dipéptidos , Animales , Carnosina/química , Carnosina/genética , Carnosina/metabolismo , Dipeptidasas/química , Dipeptidasas/genética , Dipeptidasas/metabolismo , Dipéptidos/química , Dipéptidos/genética , Dipéptidos/metabolismo , Células HEK293 , Humanos , Espectrometría de Masas , Ratones , Especificidad de Órganos/fisiología , Péptido Sintasas/química , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
PURPOSE: While the use of bone marrow concentrate (BMC) has been described in the treatment of rotator cuff tears, the impact of a rotator cuff injury on the mesenchymal stem cells (MSCs) content present in the human shoulder has not been determined, especially with regard to changes in the levels of MSCs at the tendon-bone interface. With the hypothesis that there was a decreased level of MSCs at the tendon-bone interface tuberosity in patients with rotator cuff tear, we assessed the level of MSCs in the tuberosity of the shoulder of patients undergoing a rotator cuff repair. METHODS: We analysed the data of 125 patients with symptomatic rotator cuff tears and of 75 control patients without rotator cuff injury. We recorded the following data: size of tear, number of torn tendons, aetiology of the tear, lag time between onset of shoulder symptoms/injury and repair, and also fatty infiltration of muscles. Mesenchymal stem cell content at the tendon-bone interface tuberosity was evaluated by bone marrow aspiration collected in the humeral tuberosities of patients at the beginning of surgery. RESULTS: A significant reduction in MSC content (from moderate, 30-50 %, to severe >70 %) at the tendon-bone interface tuberosity relative to the MSC content of the control was observed in all rotator cuff repair study patients. Severity of the decrease was statistically correlated to a number of factors, including the delay between onset of symptoms and surgery, number of involved tendons, fatty infiltration stage and increasing patient age. CONCLUSION: This study demonstrates that the level of MSCs present in the greater tuberosity of patients with a rotator cuff tear decreases as a function of a number of clinical factors, including lag time from tear onset to treatment, tear size, number of tears and stage of fatty infiltration, among others. This information may help the practices in using biologic augmentation of a rotator cuff repair.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Lesiones del Manguito de los Rotadores , Tejido Adiposo/patología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Manguito de los Rotadores/patología , Rotura , Articulación del Hombro/patología , Tendones , Cicatrización de Heridas/fisiologíaRESUMEN
Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ∆PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ∆PEX4 mutant.
Asunto(s)
Microcuerpos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Ubiquitinación , Animales , Línea Celular , Citosol/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Membranas Intracelulares/metabolismo , Estadios del Ciclo de Vida , Modelos Biológicos , Transporte de Proteínas , Proteínas Protozoarias/genética , Reproducibilidad de los Resultados , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/ultraestructura , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus Brucella. While most B. abortus, B. melitensis, and B. suis biovars grow slowly in complex media, they multiply intensely in livestock genitals and placenta indicating high metabolic capacities. Mutant analyses in vitro and in infection models emphasize that erythritol (abundant in placenta and genitals) is a preferred substrate of brucellae, and suggest hexoses, pentoses, and gluconeogenic substrates use in host cells. While Brucella sugar and erythritol catabolic pathways are known, growth on 3-4 carbon substrates persists in Fbp- and GlpX-deleted mutants, the canonical gluconeogenic fructose 1,6-bisphosphate (F1,6bP) bisphosphatases. Exploiting the prototrophic and fast-growing properties of B. suis biovar 5, we show that gluconeogenesis requires fructose-bisphosphate aldolase (Fba); the existence of a novel broad substrate bisphosphatase (Bbp) active on sedoheptulose 1,7-bisphosphate (S1,7bP), F1,6bP, and other phosphorylated substrates; that Brucella Fbp unexpectedly acts on S1,7bP and F1,6bP; and that, while active in B. abortus and B. melitensis, GlpX is disabled in B. suis biovar 5. Thus, two Fba-dependent reactions (dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate â F1,6bP; and dihydroxyacetone-phosphate + erythrose 4-phosphate â S1,7bP) can, respectively, yield fructose 6-phosphate and sedoheptulose 7-phosphate for classical gluconeogenesis and the Pentose Phosphate Shunt (PPS), the latter reaction opening a new gluconeogenic route. Since erythritol generates the PPS-intermediate erythrose 4-phosphate, and the Fba/Fbp-Bbp route predicts sedoheptulose 7-phosphate generation from erythrose 4-phosphate, we re-examined the erythritol connections with PPS. Growth on erythritol required transaldolase or the Fba/Fbp-Bbp pathway, strongly suggesting that Fba/Fbp-Bbp works as a PPS entry for both erythritol and gluconeogenic substrates in Brucella. We propose that, by increasing erythritol channeling into PPS through these peculiar routes, brucellae proliferate in livestock genitals and placenta in the high numbers that cause abortion and infertility, and make brucellosis highly contagious. These findings could be the basis for developing attenuated brucellosis vaccines safer in pregnant animals.
RESUMEN
Trypanosoma brucei contains peroxisome-like organelles designated glycosomes because they sequester the major part of the glycolytic pathway. Import of proteins into the peroxisomal matrix involves a protein complex associated with the peroxisomal membrane of which PEX13 is a component. Two very different PEX13 isoforms have recently been identified in T. brucei. A striking feature of one of the isoforms, TbPEX13.1, is the presence of a C-terminal type 1 peroxisomal-targeting signal (PTS1), the tripeptide TKL, conserved in its orthologues in all members of the Trypanosomatidae family so far studied, but absent from TbPEX13.2 and the PEX13s in all other organisms. Despite their differences, both TbPEX13s function as part of a docking complex for cytosolic receptors with bound matrix proteins to be imported. We further characterized TbPEX13.1's function in glycosomal matrix-protein import. It provides a frame to anchor another docking complex component, PEX14, to the glycosomal membrane or information to correctly position it within the membrane. To investigate the possible function of the C-terminal TKL, we determined the topology of the C-terminal half of TbPEX13.1 in the membrane and show that its SH3 domain, located immediately adjacent to the PTS1, is at the cytosolic face.
Asunto(s)
Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Trypanosoma brucei brucei/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/genética , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARNRESUMEN
Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy metabolism in these protozoan parasites. A 6-phosphofructo-2-kinase, responsible for the synthesis of this regulator, was highly purified from the bloodstream form of Trypanosoma brucei and kinetically characterized. By searching trypanosomatid genome databases, four genes encoding proteins homologous to the mammalian bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) were found for both T. brucei and the related parasite Leishmania major and four pairs in Trypanosoma cruzi. These genes were predicted to each encode a protein in which, at most, only a single domain would be active. Two of the T. brucei proteins showed most conservation in the PFK-2 domain, although one of them was predicted to be inactive due to substitution of residues responsible for ligating the catalytically essential divalent metal cation; the two other proteins were most conserved in the FBPase-2 domain. The two PFK-2-like proteins were expressed in Escherichia coli. Indeed, the first displayed PFK-2 activity with similar kinetic properties to that of the enzyme purified from T. brucei, whereas no activity was found for the second. Interestingly, several of the predicted trypanosomatid PFK-2/FBPase-2 proteins have long N-terminal extensions. The N-terminal domains of the two polypeptides with most similarity to mammalian PFK-2s contain a series of tandem repeat ankyrin motifs. In other proteins such motifs are known to mediate protein-protein interactions. Phylogenetic analysis suggests that the four different PFK-2/FBPase-2 isoenzymes found in Trypanosoma and Leishmania evolved from a single ancestral bifunctional enzyme within the trypanosomatid lineage. A possible explanation for the evolution of multiple monofunctional enzymes and for the presence of the ankyrin-motif repeats in the PFK-2 isoenzymes is presented.
Asunto(s)
Bases de Datos de Proteínas , Evolución Molecular , Modelos Biológicos , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/metabolismo , Trypanosomatina/enzimología , Secuencia de Aminoácidos , Animales , Ancirinas/química , Ancirinas/metabolismo , Expresión Génica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/aislamiento & purificación , Filogenia , Alineación de SecuenciaRESUMEN
Trypanosoma brucei is the causative agent of African sleeping sickness. Current work for the development of new drugs against this pathology includes evaluation of enzymes of the pentose phosphate pathway (PPP), which first requires a clear understanding of their function and mechanism of action. In this context, we focused on T. brucei 6-phosphogluconolactonase (Tb6PGL), which converts delta-6-phosphogluconolactone into 6-phosphogluconic acid in the second step of the PPP. We have determined the crystal structure of Tb6PGL in complex with two ligands, 6-phosphogluconic acid and citrate, at 2.2 A and 2.0 A resolution, respectively. We have performed molecular dynamics (MD) simulations on Tb6PGL in its empty form and in complex with delta-6-phosphogluconolactone, its natural ligand. Analysis of the structural data and MD simulations allowed us to propose a detailed enzymatic mechanism for 6PGL enzymes.
Asunto(s)
Hidrolasas de Éster Carboxílico/química , Simulación por Computador , Trypanosoma brucei brucei/enzimología , Animales , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Ácido Cítrico/química , Ácido Cítrico/metabolismo , Cristalografía por Rayos X , Gluconatos/química , Gluconatos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Tripanosomiasis Africana , Difracción de Rayos XRESUMEN
The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26. The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PPi)-dependent PFKs, despite being ATP-dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei, Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica. It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6-phosphate, showed slight positive cooperativity. PPi, even at high concentrations, did not have any effect. AMP acted as an activator of PFK, shifting its kinetics for fructose 6-phosphate from slightly sigmoid to hyperbolic, and increasing considerably the affinity for this substrate, whereas GDP did not have any effect. Modelling studies and site-directed mutagenesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PPi specificity among PFKs.
Asunto(s)
Leishmania donovani/enzimología , Fosfofructoquinasa-1/genética , Proteínas Protozoarias/genética , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Proteínas de Escherichia coli/química , Fructosadifosfatos/metabolismo , Fructosafosfatos/metabolismo , Genes Protozoarios , Glucólisis , Kinetoplastida/enzimología , Kinetoplastida/genética , Leishmania donovani/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfofructoquinasa-1/química , Fosfofructoquinasa-1/metabolismo , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Relación Estructura-Actividad , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.