RESUMEN
Metronidazole (MTZ) is the most common drug used against Trichomonas vaginalis (T. vaginalis) infections; however, treatment failures and high rates of recurrence of trichomoniasis have been reported, suggesting the presence of resistance in T. vaginalis to MTZ. Therefore, research into new therapeutic options against T. vaginalis infections has become increasingly urgent. This study investigated the trichomonacidal activity of a series of five imidazole carbamate compounds (AGR-1, AGR-2, AGR-3, AGR-4, and AGR-5) through in vitro susceptibility assays to determine the IC50 value of each compound. All five compounds demonstrated potent trichomonacidal activity, with IC50 values in the nanomolar range and AGR-2 being the most potent (IC50 400 nM). To gain insight into molecular events related to AGR-induced cell death in T. vaginalis, we analyzed the expression profiles of some metabolic genes in the trophozoites exposed to AGR compounds and MTZ. It was found that both AGR and MTZ compounds reduced the expression of the glycolytic genes (CK, PFK, TPI, and ENOL) and genes involved in metabolism (G6PD, TKT, TALDO, NADHOX, ACT, and TUB), suggesting that disturbing these key metabolic genes alters the survival of the T. vaginalis parasite and that they probably share a similar mechanism of action. Additionally, the compounds showed low cytotoxicity in the Caco-2 and HT29 cell lines, and the results of the ADMET analysis indicated that these compounds have pharmacokinetic properties similar to those of MTZ. The findings offer significant insights that can serve as a basis for future in vivo studies of the compounds as a potential new treatment against T. vaginalis.
Asunto(s)
Carbamatos , Imidazoles , Trichomonas vaginalis , Trichomonas vaginalis/efectos de los fármacos , Trichomonas vaginalis/genética , Trichomonas vaginalis/crecimiento & desarrollo , Imidazoles/farmacología , Imidazoles/química , Humanos , Carbamatos/farmacología , Carbamatos/química , Metronidazol/farmacología , Metronidazol/química , Regulación de la Expresión Génica/efectos de los fármacos , Trofozoítos/efectos de los fármacosRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting an estimated 500 million people worldwide, is a genetic disorder that causes human enzymopathies. Biochemical and genetic studies have identified several variants that produce different ranges of phenotypes; thus, depending on its severity, this enzymopathy is classified from the mildest (Class IV) to the most severe (Class I). Therefore, understanding the correlation between the mutation sites of G6PD and the resulting phenotype greatly enhances the current knowledge of enzymopathies' phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments for patients with G6PD deficiency. In this review, we analyzed and compared the structural and functional data from 21 characterized G6PD variants found in the Mexican population that we previously characterized. In order to contribute to the knowledge regarding the function and structure of the variants associated with G6PD deficiency, this review aimed to determine the molecular basis of G6PD and identify how these mutations could impact the structure, stability, and function of the enzyme and its relation with the clinical manifestations of this disease.
Asunto(s)
Deficiencia de Glucosafosfato Deshidrogenasa , Glucosafosfato Deshidrogenasa , Humanos , Glucosafosfato Deshidrogenasa/genética , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Genotipo , Mutación , FenotipoRESUMEN
Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP+ binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.
Asunto(s)
Giardia lamblia , Giardiasis , Humanos , Giardiasis/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Tiazoles/farmacología , Tiazoles/uso terapéuticoRESUMEN
Treatments to combat giardiasis have been reported to have several drawbacks, partly due to the drug resistance and toxicity of current antiparasitic agents. These constraints have prompted many researchers to investigate new drugs that act against protozoan parasites. Enzyme inhibition is an important means of regulating pathogen metabolism and has recently been identified as a significant alternative target in the search for new treatments. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase (G6PD::6PGL) is a bifunctional enzyme involved in the pentose phosphate pathway (PPP) in Giardia lamblia (G. lamblia). The G. lamblia enzyme is unusual since, unlike the human enzyme, it is a fused enzyme. Here, we show, through inhibition assays, that an in-house chemical library of 120 compounds and four target compounds, named CNZ-7, CNZ-8, CMC-1, and FLP-2, are potent inhibitors of the G. lamblia G6PD::6PGL fused enzyme. With a constant (k2) of 2.3, 3.2, and 2.8 M−1 s−1, respectively, they provoke alterations in the secondary and tertiary protein structure and global stability. As a novel approach, target compounds show antigiardial activity, with IC50 values of 8.7, 15.2, 15.3, and 24.1 µM in trophozoites from G. lamblia. Moreover, these compounds show selectivity against G. lamblia, since, through counter-screening in Caco-2 and HT29 human cells, they were found to have low toxicity. This finding positions these compounds as a potential and attractive starting point for new antigiardial drugs.
Asunto(s)
Giardia lamblia , Giardiasis , Animales , Humanos , Giardiasis/tratamiento farmacológico , Giardiasis/parasitología , Trofozoítos/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Células CACO-2RESUMEN
Protozoan parasites, such as Giardia lamblia and Trichomonas vaginalis, cause the most prevalent infections in humans in developing countries and provoke significant morbidity and mortality in endemic countries. Despite its side-effects, metronidazole is still the drug of choice as a giardiacidal and trichomonacidal tissue-active agent. However, the emergence of metronidazole resistance and its evolved strategies of parasites to evade innate host defenses have hindered the identification and development of new therapeutic strategies against these parasites. Here, we tested five synthesized benzimidazole derivatives as possible drugs for treating giardiasis and trichomoniasis, probing the bifunctional enzyme glucose 6-phosphate dehydrogenase::6-phosphogluconolactone from G. lamblia (GlG6PD::6PGL) and T. vaginalis (TvG6PD::6PGL) as a drug target. The investigated benzimidazole derivatives were H-B2M1, H-B2M2, H2N-BZM6, O2N-BZM7, and O2N-BZM9. The recombinant enzymes were used in inhibition assays, and in silico computational predictions and spectroscopic studies were applied to follow the structural alteration of the enzymes and identify the possible mechanism of inhibition. We identified two potent benzimidazole compounds (O2N-BZM7 and O2N-BZM9), which are capable of inhibiting both protozoan G6PD::6PGL enzymes and in vitro assays with these parasites, showing that these compounds also affect their viability. These results demonstrate that other therapeutic targets of the compounds are the enzymes GlG6PD::6PGL and TvG6PD::6PGL, which contribute to their antiparasitic effect and their possible use in antigiardial and trichomonacidal therapies.
Asunto(s)
Antiprotozoarios , Giardia lamblia , Parásitos , Trichomonas vaginalis , Animales , Humanos , Metronidazol/farmacología , Antiparasitarios/farmacología , Bencimidazoles/farmacología , Antiprotozoarios/farmacologíaRESUMEN
Trichomoniasis is a sexually transmitted disease with a high incidence worldwide, affecting 270 million people. Despite the existence of a catalog of available drugs to combat this infection, their extensive use promotes the appearance of resistant Trichomonas vaginalis (T. vaginalis), and some side effects in treated people, which are reasons why it is necessary to find new alternatives to combat this infection. In this study, we investigated the impact of an in-house library comprising 55 compounds on the activity of the fused T. vaginalis G6PD::6PGL (TvG6PD::6PGL) protein, a protein mediating the first reaction step of the pentose phosphate pathway (PPP), a crucial pathway involved in the parasite's energy production. We found four compounds: JMM-3, CNZ-3, CNZ-17, and MCC-7, which inhibited the TvG6PD::6PGL protein by more than 50%. Furthermore, we determined the IC50, the inactivation constants, and the type of inhibition. Our results showed that these inhibitors induced catalytic function loss of the TvG6PD::6PGL enzyme by altering its secondary and tertiary structures. Finally, molecular docking was performed for the best inhibitors, JMM-3 and MCC-7. All our findings demonstrate the potential role of these selected hit compounds as TvG6PD::6PGL enzyme selective inhibitors.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas , Inhibidores Enzimáticos/química , Glucosafosfato Deshidrogenasa , Simulación del Acoplamiento Molecular , Trichomonas vaginalis/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/química , CinéticaRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency.
Asunto(s)
Deficiencia de Glucosafosfato Deshidrogenasa/enzimología , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/metabolismo , Naftalenosulfonatos de Anilina/química , Catálisis , Dicroismo Circular , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Deficiencia de Glucosafosfato Deshidrogenasa/metabolismo , Guanidina , Humanos , Cinética , México , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Programas Informáticos , Temperatura , Tripsina/químicaRESUMEN
This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.
Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Estabilidad de Enzimas/genética , Glucosafosfato Deshidrogenasa/genética , NADP/genética , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Trichomonas vaginalis/genética , Secuencia de Aminoácidos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Estabilidad Proteica , Alineación de Secuencia , TemperaturaRESUMEN
Giardiasis is a diarrheal disease that is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; however, failures in drug therapy are common, and have adverse effects and increased resistance of the parasite to the drug, generating the need to find new alternative treatments. In this study, we synthesized a series of 2-mercaptobenzimidazoles that are derivatives of omeprazole, and the chemical structures were confirmed through mass, 1H NMR, and 13C NMR techniques. The in vitro efficacy compounds against Giardia, as well as its effect on the inhibition of triosephosphate isomerase (TPI) recombinant, were investigated, the inactivation assays were performed with 0.2 mg/mL of the enzyme incubating for 2 h at 37 °C in TE buffer, pH 7.4 with increasing concentrations of the compounds. Among the target compounds, H-BZM2, O2N-BZM7, and O2N-BZM9 had greater antigiardial activity (IC50: 36, 14, and 17 µM on trophozoites), and inhibited the TPI enzyme (K2: 2.3, 3.2, and 2.8 M-1 s-1) respectively, loading alterations on the secondary structure, global stability, and tertiary structure of the TPI protein. Finally, we demonstrated that it had low toxicity on Caco-2 and HT29 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.
Asunto(s)
Antiprotozoarios/farmacología , Bencimidazoles/farmacología , Giardia lamblia/efectos de los fármacos , Omeprazol/farmacología , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Células CACO-2 , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Giardia lamblia/enzimología , Células HT29 , Humanos , Cinética , Lansoprazol/farmacología , Simulación del Acoplamiento Molecular , Omeprazol/síntesis química , Omeprazol/química , Espectrometría de Fluorescencia , Triosa-Fosfato Isomerasa/antagonistas & inhibidores , Triosa-Fosfato Isomerasa/química , Trofozoítos/efectos de los fármacosRESUMEN
BACKGROUND: Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein. RESULTS: The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb. CONCLUSIONS: rPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Pichia/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Aldehído Oxidasa/genética , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Reactores Biológicos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Glicosilación , Humanos , Pichia/crecimiento & desarrollo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Gluconacetobacter diazotrophicus PAL5 (GDI) is an endophytic bacterium with potential biotechnological applications in industry and agronomy. The recent description of its complete genome and its principal metabolic enzymes suggests that glucose metabolism is accomplished through the pentose phosphate pathway (PPP); however, the enzymes participating in this pathway have not yet been characterized in detail. The objective of the present work was to clone, purify, and biochemically and physicochemically characterize glucose-6-phosphate dehydrogenase (G6PD) from GDI. The gene was cloned and expressed as a tagged protein in E. coli to be purified by affinity chromatography. The native state of the G6PD protein in the solution was found to be a tetramer with optimal activity at pH 8.8 and a temperature between 37 and 50 °C. The apparent Km values for G6P and nicotinamide adenine dinucleotide phosphate (NADP+) were 63 and 7.2 µM, respectively. Finally, from the amino acid sequence a three-dimensional (3D) model was obtained, which allowed the arrangement of the amino acids involved in the catalytic activity, which are conserved (RIDHYLGKE, GxGGDLT, and EKPxG) with those of other species, to be identified. This characterization of the enzyme could help to identify new environmental conditions for the knowledge of the plant-microorganism interactions and a better use of GDI in new technological applications.
Asunto(s)
Clonación Molecular , Gluconacetobacter/enzimología , Glucosafosfato Deshidrogenasa/metabolismo , Escherichia coli/metabolismo , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/genética , Concentración de Iones de Hidrógeno , Cinética , NADP/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , TemperaturaRESUMEN
We report a 23 year old woman presenting with a nephrotic syndrome due to minimal change disease, central diabetes insipidus, primary hypothyroidism, vitiligo and universal alopecia. Eleven years later, she presented secondary amenorrhea due to hypogonadotropic hypogonadism, with mild hyperprolactinemia and central adrenal insufficiency. A magnetic resonance imaging of the sella turcica showed a pituitary mass with suprasellar extension that was resected using a transsphenoidal approach. Pathology confirmed the presence of a lymphoplasmacytic hypophysitis. She needed a second surgical resection due to mass growth and neuro-ophthalmologic impairment. One year later, systemic lupus erythematosus, arterial hypertension and type 2 diabetes mellitus were diagnosed. Two years later, due to back pain, constipation and renal failure, retroperitoneal fibrosis was found, satisfactorily treated with glucocorticoids and colchicine. Hence, this clinical vignette shows the coexistence of autoimmune polyglandular syndrome with retroperitoneal fibrosis and lymphoplasmacytic hypophysitis. Tissue analysis showed the presence of IgG4 producing plasma cells in the pituitary and retroperitoneum, which constitute a basis for the diagnosis of IgG4 related disease.
Asunto(s)
Hipofisitis/complicaciones , Enfermedad Relacionada con Inmunoglobulina G4/complicaciones , Poliendocrinopatías Autoinmunes/complicaciones , Fibrosis Retroperitoneal/complicaciones , Femenino , Humanos , Hipofisitis/diagnóstico por imagen , Hipofisitis/patología , Enfermedad Relacionada con Inmunoglobulina G4/diagnóstico por imagen , Enfermedad Relacionada con Inmunoglobulina G4/patología , Imagen por Resonancia Magnética , Poliendocrinopatías Autoinmunes/diagnóstico por imagen , Poliendocrinopatías Autoinmunes/patología , Fibrosis Retroperitoneal/diagnóstico por imagen , Fibrosis Retroperitoneal/patología , Adulto JovenRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD) and G6PD Nefza (Leu323Pro), and the double mutant G6PD A- (Asn126Asp + Leu323Pro). The mutants showed lower residual activity (≤50% of WT G6PD) and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A-. Moreover, our study suggests that the G6PD Nefza and G6PD A- mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.
Asunto(s)
Deficiencia de Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/genética , Mutación , Alelos , Sustitución de Aminoácidos , Activación Enzimática/efectos de los fármacos , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Humanos , Cinética , Modelos Moleculares , Mutagénesis , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis Espectral , TermodinámicaRESUMEN
Congenital diarrheas correspond to a severe and low frequency digestive disease, with a high mortality. They start a few days or months after birth, leading to intestinal insufficiency and dependence on parenteral nutrition. It must be highly suspected in newborns or infants with diarrhea and severe electrolyte disorders. The diagnosis is based on clinical, endoscopic, histologic and eventually genetic findings. Treatment is supportive with intensive correction of electrolyte imbalances as well as parenteral nutrition. OBJECTIVE: To present a case report of congenital diarrhea identified as microvillous inclusion disease presenting in the neonatal period. CASE REPORT: Male patient currently 3 years of age, son of consanguineous parents. At 10 days of age presents a severe secretory diarrhea, requiring treatment in a critical care unit and parenteral nutrition. Initially he also presented with Fanconi syndrome, which improved afterwards. The suspicion of congenital microvillous inclusion was confirmed later by optic and electronic microscopy, and inmunohistochemistry. A succesful evolution was later achieved maintaining home parenteral nutrition after discharge. CONCLUSION: We present the first known case in Chile of congenital diarrhea due to microvillous inclusión disease and his evolution.
Asunto(s)
Diarrea/congénito , Síndromes de Malabsorción/diagnóstico , Microvellosidades/patología , Mucolipidosis/diagnóstico , Preescolar , Chile , Diarrea/etiología , Progresión de la Enfermedad , Humanos , Recién Nacido , Síndromes de Malabsorción/complicaciones , Masculino , Mucolipidosis/complicaciones , Índice de Severidad de la EnfermedadRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme in the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain an adequate reducing environment in the cells and is especially important in red blood cells (RBC). Given its central role in the regulation of redox state, it is understandable that mutations in the gene encoding G6PD can cause deficiency of the protein activity leading to clinical manifestations such as neonatal jaundice and acute hemolytic anemia. Recently, an extensive review has been published about variants in the g6pd gene; recognizing 186 mutations. In this work, we review the state of the art in G6PD deficiency, describing 217 mutations in the g6pd gene; we also compile information about 31 new mutations, 16 that were not recognized and 15 more that have recently been reported. In order to get a better picture of the effects of new described mutations in g6pd gene, we locate the point mutations in the solved three-dimensional structure of the human G6PD protein. We found that class I mutations have the most deleterious effects on the structure and stability of the protein.
Asunto(s)
Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Biología Computacional , Glucosafosfato Deshidrogenasa/química , Humanos , Mutación , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.
Asunto(s)
Indio Americano o Nativo de Alaska/genética , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/genética , Mutación , Dominio Catalítico , Clonación Molecular , Biología Computacional/métodos , Cristalografía por Rayos X , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Cinética , México , Modelos Moleculares , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the world. More than 160 mutations causing the disease have been identified, but only 10% of these variants have been studied at biochemical and biophysical levels. In this study we report on the functional and structural characterization of three naturally occurring variants corresponding to different classes of disease severity: Class I G6PD Durham, Class II G6PD Santa Maria, and Class III G6PD A+. The results showed that the G6PD Durham (severe deficiency), and the G6PD Santa Maria and A+ (less severe deficiency) (Class I, II and III, respectively) affect the catalytic efficiency of these enzymes, are more sensitive to temperature denaturing, and affect the stability of the overall protein when compared to the wild type WT-G6PD. In the variants, the exposure of more and buried hydrophobic pockets was induced and monitored with 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence, directly affecting the compaction of structure at different levels and probably reducing the stability of the protein. The degree of functional and structural perturbation by each variant correlates with the clinical severity reported in different patients.
Asunto(s)
Variación Genética , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/genética , Modelos Moleculares , Conformación Molecular , Mutación , Catálisis , Activación Enzimática , Expresión Génica , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Cinética , Estabilidad Proteica , Proteínas Recombinantes , Relación Estructura-Actividad , TermodinámicaRESUMEN
Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Etanol/metabolismo , Gluconacetobacter/enzimología , Acetatos/análisis , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/aislamiento & purificación , Aldehídos/análisis , Secuencia de Aminoácidos , Biocatálisis , Radioisótopos de Carbono/química , Cromatografía de Gases y Espectrometría de Masas , Marcaje Isotópico , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oxidación-Reducción , Desnaturalización Proteica , TemperaturaRESUMEN
In the pursuit of identifying the novel resin glycoside modulators glucose-6-phosphatase and α-glucosidase enzymes, associated with blood sugar regulation, methanol-soluble extracts from the flowers of Ipomoea murucoides (cazahuate, Nahuatl), renowned for its abundance of glycolipids, were employed. The methanol-soluble extracts were fractionated by applying the affinity-directed method with glucose-6-phosphatase enzymes from a rat's liver and α-glucosidase enzymes from its intestines. Mass spectrometry and nuclear magnetic resonance were employed to identify the high-affinity compound as a free ligand following the release from the enzymatic complex. Gel permeation through a spin size-exclusion column allowed the separated high-affinity molecules to bind to glucose-6-phosphatase and α-glucosidase enzymes in solution, which led to the identification of some previously reported resin glycosides in the flowers of cazahuate, where a glycolipid mainly structurally related to murucoidin XIV was observed. In vitro studies demonstrated the modulating properties of resin glycosides on the glucose-6-phosphatase enzyme. Dynamic light scattering revealed conformational variations induced by resin glycosides on α-glucosidase enzyme, causing them to become more compact, akin to observations with the positive control, acarbose. These findings suggest that resin glycosides may serve as a potential source for phytotherapeutic agents with antihyperglycemic properties.
RESUMEN
Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.