RESUMO
Amylomyces rouxii is a zygomycete that produces extracellular protease and tyrosinase. The tyrosinase activity is negatively regulated by the proteases and, which attempts to purify the tyrosinase (tyr) enzyme that has been hampered by the presence of a protease that co-purified with it. In this work we identified genes encoding aspartic protease II (aspII) and VI of A. rouxii. Using an RNAi strategy based on the generation of a siRNA by transcription from two opposite-orientated promoters, the expression of these two proteases was silenced, showing that this molecular tool is suitable for gene silencing in Amylomyces. The transformant strains showed a significant attenuation of the transcripts (determined by RT-qPCR), with respective inhibition of the protease activity. In the case of aspII, inhibition was in the range of 43-90 % in different transformants, which correlated well with up to a five-fold increase in tyr activity with respect to the wild type and control strains. In contrast, silencing of aspVI caused a 43-65 % decrease in protease activity but had no significant effect on the tyr activity. The results show that aspII has a negative effect on tyr activity, and that the silencing of this protease is important to obtain strains with high levels of tyr activity.
Assuntos
Ácido Aspártico Proteases , Mucorales , RNA Interferente Pequeno , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Mucorales/genéticaRESUMO
A wide variety of biological functions, including those involved in the morphogenesis process of basidiomycete fungi, have been attributed to laccase enzymes. In this work, RNA interference (RNAi) was used to evaluate the role of the laccase (lacc2) gene of Pleurotus ostreatus PoB. Previously, transformant strains of P. ostreatus were obtained and according to their level of silencing they were classified as light (T7), medium (T21) or severe (T26 and T27). The attenuation of the lacc2 gene in these transformants was determined by RT-PCR. Silencing of lacc2 resulted in a decrease in laccase activity between 30 and 55%, which depended on the level of laccase expression achieved. The silenced strains (T21, T26, and T27) displayed a delay in the development of mycelium on potato dextrose agar (PDA) medium, whereas in the cultures grown on wheat straw, we found that these strains were incapable of producing aerial mycelium, primordia, and fruiting bodies. Scanning electron microscopy (SEM) showed the presence of toxocyst-like structures. The highest abundance of these structures was observed in the wild-type (PoB) and T7 strains. However, the abundance of toxocysts decreased in the T21 and T26 strains, and in T27 they were not detected. These results suggest that the presence and abundance of toxocyst-like structures are directly related to the development of fruiting bodies. Furthermore, our data confirm that lacc2 is involved in the morphogenesis process of P. ostreatus.
Assuntos
Ascomicetos , Pleurotus , Lacase/genética , Lacase/metabolismo , Ascomicetos/metabolismoRESUMO
BACKGROUND: The pentose phosphate pathway (PPP) has received significant attention because of the role of NADPH and R-5-P in the maintenance of cancer cells, which are necessary for the synthesis of fatty acids and contribute to uncontrollable proliferation. The HsG6PD enzyme is the rate-limiting step in the oxidative branch of the PPP, leading to an increase in the expression levels in tumor cells; therefore, the protein has been proposed as a target for the development of new molecules for use in cancer. METHODS: Through in vitro studies, we assayed the effects of 55 chemical compounds against recombinant HsG6PD. Here, we present the kinetic characterization of four new HsG6PD inhibitors as well as their functional and structural effects on the protein. Furthermore, molecular docking was performed to determine the interaction of the best hits with HsG6PD. RESULTS: Four compounds, JMM-2, CCM-4, CNZ-3, and CNZ-7, were capable of reducing HsG6PD activity and showed noncompetitive and uncompetitive inhibition. Moreover, experiments using circular dichroism and fluorescence spectroscopy showed that the molecules affect the structure (secondary and tertiary) of the protein as well as its thermal stability. Computational docking analysis revealed that the interaction of the compounds with the protein does not occur at the active site. CONCLUSIONS: We identified two new compounds (CNZ-3 and JMM-2) capable of inhibiting HsG6PD that, compared to other previously known HsG6PD inhibitors, showed different mechanisms of inhibition. GENERAL SIGNIFICANCE: Screening of new inhibitors for HsG6PD with a future pharmacological approach for the study and treatment of cancer.
Assuntos
Inibidores Enzimáticos/química , Glucosefosfato Desidrogenase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Domínio Catalítico , Ensaios Enzimáticos , Expressão Gênica , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
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.
Assuntos
Hidrolases de Éster Carboxílico/genética , Estabilidade Enzimática/genética , Glucosefosfato Desidrogenase/genética , NADP/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Trichomonas vaginalis/genética , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Estabilidade Proteica , Alinhamento de Sequência , TemperaturaRESUMO
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.
Assuntos
Deficiência de Glucosefosfato Desidrogenase/enzimologia , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Naftalenossulfonato de Anilina/química , Catálise , Dicroísmo Circular , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/isolamento & purificação , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Guanidina , Humanos , Cinética , México , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Software , Temperatura , Tripsina/químicaRESUMO
Triosephosphate isomerase (TPI) is a glycolysis enzyme, which catalyzes the reversible isomerization between dihydroxyactetone-3-phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). In pathogenic organisms, TPI is essential to obtain the energy used to survive and infect. Fusarium oxisporum (Fox) is a fungus of biotechnological importance due to its pathogenicity in different organisms, that is why the relevance of also biochemically analyzing its TPI, being the first report of its kind in a Fusarium. Moreover, the kinetic characteristics or structural determinants related to its function remain unknown. Here, the Tpi gene from F. oxysporum was isolated, cloned, and overexpressed. The recombinant protein named FoxTPI was purified (97% purity) showing a molecular mass of 27 kDa, with optimal activity at pH 8.0 and and temperature of 37 °C. The values obtained for Km and Vmax using the substrate GAP were 0.47 ± 0.1 mM, and 5331 µmol min-1 mg-1, respectively. Furthemore, a protein structural modeling showed that FoxTPI has the classical topology of TPIs conserved in other organisms, including the catalytic residues conserved in the active site (Lys12, His94 and Glu164). Finally, when FoxTPI was analyzed with inhibitors, it was found that one of them inhibits its activity, which gives us the perspective of future studies and its potential use against this pathogen.
RESUMO
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.
Assuntos
Clonagem Molecular , Gluconacetobacter/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Escherichia coli/metabolismo , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Concentração de Íons de Hidrogênio , Cinética , NADP/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , TemperaturaRESUMO
Giardia lambia is a flagellated protozoan parasite that lives in the small intestine and is the causal agent of giardiasis. It has been reported that G. lamblia exhibits glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway (PPP). Our group work demonstrated that the g6pd and 6pgl genes are present in the open frame that gives rise to the fused G6PD::6PGL protein; where the G6PD region is similar to the 3D structure of G6PD in Homo sapiens. The objective of the present work was to show the presence of the structural NADP+ binding site on the fused G6PD::6PGL protein and evaluate the effect of the NADP+ molecule on protein stability using biochemical and computational analysis. A protective effect was observed on the thermal inactivation, thermal stability, and trypsin digestions assays when the protein was incubated with NADP+. By molecular docking, we determined the possible structural-NADP+ binding site, which is located between the Rossmann fold of G6PD and 6PGL. Finally, molecular dynamic (MD) simulation was used to test the stability of this complex; it was determined that the presence of both NADP+ structural and cofactor increased the stability of the enzyme, which is in agreement with our experimental results.
Assuntos
Giardia lamblia/enzimologia , Glucosefosfato Desidrogenase/química , NADP/química , NADP/metabolismo , Fosfogluconato Desidrogenase/química , Sítios de Ligação , Glucosefosfato Desidrogenase/metabolismo , Humanos , Modelos Moleculares , Fosfogluconato Desidrogenase/metabolismo , Conformação Proteica , Estabilidade Proteica , TemperaturaRESUMO
El síndrome renal del cascanueces es una enfermedad rara, más frecuente en mujeres entre la tercera y cuarta décadas de la vida. Consiste en la compresión de la vena renal izquierda, entre la arteria mesentérica superior y la aorta abdominal, que provoca un aumento en el gradiente presión de la vena renal izquierda y produce hematuria renal unilateral izquierda. Se presenta una paciente de 55 años de edad con antecedentes patológicos personales de hipertensión arterial hace 15 años, que comienza con cuadro de dolor lumbar izquierdo, asociado a hematuria microscópica y leucocituria. Se interpreta como infección del tracto urinario, recibió tratamiento antibiótico por 10 días, aunque con persistencia de la hematuria, sin otra manifestación clínica. Se le realizó angio-TAC (fase simple, arterial, venosa, excretora) que mostró arterias únicas y normales. Existe ligera compresión de la vena renal izquierda, por disminución del ángulo aorto mesentérico (ángulo 25°), compatible con síndrome renal del cascanueces. Se presenta este caso, dado lo poco frecuente del reporte de este síndrome en Cuba(AU)
Nutcracker syndrome is a rare disease, more frequent in in women between the third and fourth decades of life. It consists of the compression of the left renal vein between the superior mesenteric artery and the abdominal aorta, causing an increase in the pressure gradient of the left renal vein and producing left unilateral renal hematuria. We present a 55-year-old patient with a personal pathological history of arterial hypertension for 15 years, who began with left lumbar pain, associated with microscopic hematuria and leukocyturia. It was interpreted as a urinary tract infection, the patient received antibiotic treatment for 10 days , although hematuria continued, until now, with no other clinical manifestation. The patient underwent a angio-CT (simple, arterial, venous, excretory phase) that showed single and normal arteries. There is slight compression of the left renal vein, due to reduction of the mesenteric aorto angle (angle 25°), compatible with renal nutcracker syndrome. We present this case since this syndrome is rare in Cuba(AU)
Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Flebografia/métodos , Stents , Síndrome do Quebra-NozesRESUMO
El síndrome renal del cascanueces es una enfermedad rara, más frecuente en mujeres entre la tercera y cuarta décadas de la vida. Consiste en la compresión de la vena renal izquierda, entre la arteria mesentérica superior y la aorta abdominal, que provoca un aumento en el gradiente presión de la vena renal izquierda y produce hematuria renal unilateral izquierda. Se presenta una paciente de 55 años de edad con antecedentes patológicos personales de hipertensión arterial hace 15 años, que comienza con cuadro de dolor lumbar izquierdo, asociado a hematuria microscópica y leucocituria. Se interpreta como infección del tracto urinario, recibió tratamiento antibiótico por 10 días, aunque con persistencia de la hematuria, sin otra manifestación clínica. Se le realizó angio-TAC (fase simple, arterial, venosa, excretora) que mostró arterias únicas y normales. Existe ligera compresión de la vena renal izquierda, por disminución del ángulo aorto mesentérico (ángulo 25°), compatible con síndrome renal del cascanueces. Se presenta este caso, dado lo poco frecuente del reporte de este síndrome en Cuba(AU)
Nutcracker syndrome is a rare disease, more frequent in in women between the third and fourth decades of life. It consists of the compression of the left renal vein between the superior mesenteric artery and the abdominal aorta, causing an increase in the pressure gradient of the left renal vein and producing left unilateral renal hematuria. We present a 55-year-old patient with a personal pathological history of arterial hypertension for 15 years, who began with left lumbar pain, associated with microscopic hematuria and leukocyturia. It was interpreted as a urinary tract infection, the patient received antibiotic treatment for 10 days , although hematuria continued, until now, with no other clinical manifestation. The patient underwent a angio-CT (simple, arterial, venous, excretory phase) that showed single and normal arteries. There is slight compression of the left renal vein, due to reduction of the mesenteric aorto angle (angle 25°), compatible with renal nutcracker syndrome. We present this case since this syndrome is rare in Cuba(AU)
Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Flebografia/métodos , Stents , Síndrome do Quebra-Nozes/diagnóstico por imagem , CubaRESUMO
The deficiency of glucose6phosphate dehydrogenase (G6PD) is one of the most common inborn errors of metabolism worldwide. This congenital disorder generally results from mutations that are spread throughout the entire gene of G6PD. Three single-point mutations for G6PD have been reported in the Mexican population and named Veracruz (Arg365His), G6PD Seattle (Asp282His), and G6PD Mexico DF (Thr65Ala), whose biochemical characterization have not yet been studied. For this reason, in this work we analyzed the putative role of the three mutations to uncover the functional consequences on G6PD activity. To this end, was developed a method to clone, overexpress, and purify recombinant human G6PD. The results obtained from all variants showed a loss of catalysis by 80 to 97% and had a decrease in affinity for both physiological substrates with respect to the wild type (WT) G6PD. Our results also showed that the three mutations affected three-dimensional structure and protein stability, suggesting an unstable structure with low conformational stability that affected its G6PD functionality. Finally, based on the biochemical characterization of the unclassified G6PD Mexico DF, we suggest that this variant could be grouped as a Class I variant, because biochemical data are similar with other Class I G6PDs.
Assuntos
Clonagem Molecular , Genética Populacional , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Mutação , Dicroísmo Circular , Ativação Enzimática , Estabilidade Enzimática , Glucosefosfato Desidrogenase/isolamento & purificação , Humanos , Cinética , México , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardialamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene (6pgl). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardialamblia. First, the gene of g6pd fused with the 6pgl gene (6gpd::6pgl) was isolated from trophozoites of Giardialamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli. Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target.
Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Giardia lamblia/enzimologia , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Hidrolases de Éster Carboxílico/genética , DNA Complementar/química , DNA Complementar/genética , Ativação Enzimática , Estabilidade Enzimática , Expressão Gênica , Giardia lamblia/genética , Glucosefosfato Desidrogenase/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , TemperaturaRESUMO
The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100-200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways.
RESUMO
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.
Assuntos
Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/genética , Mutação , Alelos , Substituição de Aminoácidos , Ativação Enzimática/efeitos dos fármacos , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/isolamento & purificação , Humanos , Cinética , Modelos Moleculares , Mutagênese , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Análise Espectral , TermodinâmicaRESUMO
Purification of nucleic acids is an essential procedure for most experiments in molecular biology. In this paper, the freeze-squeeze method with some modifications is proposed as an alternative methodology for the purification, concentration and recovery of small DNA fragments from agarose gels. The advantage of this alternative methodology is that it enables the recovery of fragments that are less than 100 bp in length and enables suspension of products in smaller volumes compared to several commercially available kits. In addition, the purified fragments were re-amplified by PCR and used for cloning and sequencing. Moreover, this protocol was used to perform the isolation and identification of microRNAs from Giardia lamblia, as previously reported. This protocol has the advantage of being inexpensive and easy and can be employed for various molecular applications. The advantages of this protocol include â¢A modified classical method was used for purification of small DNA fragments from G. lamblia.â¢The modified freeze-squeeze method was more efficient in cleaning up small DNA fragments from agarose gels compared to commercial kits.â¢The modified method allows concentration and recovery of fragments up to 60 bp in length.â¢The modified freeze-squeeze method allows re-suspension of the products in volumes of up to 2.5 µL.
RESUMO
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.
Assuntos
Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Biologia Computacional , Glucosefosfato Desidrogenase/química , Humanos , Mutação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect-in a rapid, sensitive, specific, and reproducible way-the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia.
RESUMO
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.
Assuntos
Indígena Americano ou Nativo do Alasca/genética , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Mutação , Domínio Catalítico , Clonagem Molecular , Biologia Computacional/métodos , Cristalografia por Raios X , Glucosefosfato Desidrogenase/metabolismo , Humanos , Cinética , México , Modelos Moleculares , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
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.