RESUMO
Cancer involves a series of diseases where cellular growth is not controlled. Cancer is a leading cause of death worldwide, and the burden of cancer incidence and mortality is rapidly growing, mainly in developing countries. Many drugs are currently used, from chemotherapeutic agents to immunotherapy, among others, along with organ transplantation. Treatments can cause severe side effects, including remission and progression of the disease with serious consequences. Increased glycolytic activity is characteristic of cancer cells. Triosephosphate isomerase is essential for net ATP production in the glycolytic pathway. Notably, some post-translational events have been described that occur in human triosephosphate isomerase in which functional and structural alterations are provoked. This is considered a window of opportunity, given the differences that may exist between cancer cells and their counterpart in normal cells concerning the glycolytic enzymes. Here, we provide elements that bring out the potential of triosephosphate isomerase, under post-translational modifications, to be considered an efficacious target for treating cancer.
Assuntos
Neoplasias , Triose-Fosfato Isomerase , Humanos , Triose-Fosfato Isomerase/genética , Neoplasias/tratamento farmacológico , Processamento de Proteína Pós-Traducional , Ciclo Celular , Proliferação de CélulasRESUMO
Beyond the problem in public health that protist-generated diseases represent, understanding the variety of mechanisms used by these parasites to interact with the human immune system is of biological and medical relevance. Giardia lamblia is an early divergent eukaryotic microorganism showing remarkable pathogenic strategies for evading the immune system of vertebrates. Among various multifunctional proteins in Giardia, arginine deiminase is considered an enzyme that plays multiple regulatory roles during the life cycle of this parasite. One of its most important roles is the crosstalk between the parasite and host. Such a molecular "chat" is mediated in human cells by membrane receptors called Toll-like receptors (TLRs). Here, we studied the importance of the 3D structure of giardial arginine deiminase (GlADI) to immunomodulate the human immune response through TLRs. We demonstrated the direct effect of GlADI on human TLR signaling. We predicted its mode of interaction with TLRs two and four by using the AlphaFold-predicted structure of GlADI and molecular docking. Furthermore, we showed that the immunomodulatory capacity of this virulent factor of Giardia depends on the maintenance of its 3D structure. Finally, we also showed the influence of this enzyme to exert specific responses on infant-like dendritic cells.
Assuntos
Giardia , Giardíase , Animais , Humanos , Hidrolases , Imunidade , Imunomodulação , Simulação de Acoplamento Molecular , Receptores Toll-LikeRESUMO
Human triosephosphate isomerase (HsTIM) is a central glycolytic enzyme and is overexpressed in cancer cells with accelerated glycolysis. Triple-negative breast cancer is highly dependent on glycolysis and is typically treated with a combination of surgery, radiation therapy, and chemotherapy. Deamidated HsTIM was recently proposed as a druggable target. Although thiol-reactive drugs affect cell growth in deamidated HsTIM-complemented cells, the role of this protein as a selective target has not been demonstrated. To delve into the usefulness of deamidated HsTIM as a selective target, we assessed its natural accumulation in breast cancer cells. We found that deamidated HsTIM accumulates in breast cancer cells but not in noncancerous cells. The cancer cells are selectively programmed to undergo cell death with thiol-reactive drugs that induced the production of methylglyoxal (MGO) and advanced glycation-end products (AGEs). In vivo, a thiol-reactive drug effectively inhibits the growth of xenograft tumors with an underlying mechanism involving deamidated HsTIM. Our findings demonstrate the usefulness of deamidated HsTIM as target to develop new therapeutic strategies for the treatment of cancers and other pathologies in which this post translationally modified protein accumulates.
Assuntos
Neoplasias da Mama , Triose-Fosfato Isomerase , Feminino , Glicólise , Humanos , Proteínas/metabolismo , Aldeído Pirúvico/metabolismo , Compostos de Sulfidrila , Triose-Fosfato Isomerase/metabolismoRESUMO
Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.
Assuntos
Giardia lamblia/efeitos dos fármacos , Giardíase/tratamento farmacológico , Hidrolases/metabolismo , Animais , Antiprotozoários/farmacologia , Simulação por Computador , Cisteína/química , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Giardia lamblia/patogenicidade , Giardíase/imunologia , Tiomalato Sódico de Ouro/farmacologia , Humanos , Hidrolases/efeitos dos fármacos , Hidrolases/ultraestrutura , Omeprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol , Tiamina/análogos & derivados , Tiamina/farmacologia , Trofozoítos/efeitos dos fármacosRESUMO
Delivery methods during childbirth and their related gut microbiota profiles have important impacts on health later in life, they can contribute to the development of diseases such as obesity, whose highest prevalence rate is found among the Mexican child population. Coincidentally, Mexico has one of the highest global average annual rate increase in cesarean births (C-section). Since Mexico leads the world in childhood obesity, studying the relationship between childbirth delivery methods and gut microbiota profiles in this vulnerable population may be used to identify early risk factors for obesity in other developed and developing countries. The objective of this study is to determine the association between child delivery method and gut microbiota profiles in healthy Mexican newborns.Fecal samples of 57 term infants who participated in a randomized clinical trial in 2013 to study the safety of Agave fructans in newborns, were used in this study. DNA samples were extracted and used to characterize the microbiota composition using high-throughput 16S rRNA gene sequencing. The samples were further divided based on childbirth delivery method, as well as early diet. Gut microbiota profiles were determined and analyzed using cluster analysis followed by multiple correspondence analysis.An unusual high abundance of Proteobacteria was found in the gut microbiota of all Mexican infants studied, regardless of delivery method. Feces from infants born by C-section had low levels of Bacteroidetes, high levels of Firmicutes, especially Clostridium and Enterococcus, and a strikingly high ratio of Firmicutes/Bacteroidetes (F:B). Profiles enriched in Bacteroidetes and low F:B ratios, were strongly associated with vaginal delivery.The profile of gut microbiota associated with feces from Mexican infants born by C-section, may be added to the list of boosting factors for the worrying obesity epidemic in Mexico.
Assuntos
Cesárea/estatística & dados numéricos , Microbioma Gastrointestinal , Obesidade/epidemiologia , Cesárea/efeitos adversos , Fezes/microbiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , México/epidemiologia , Fatores de RiscoRESUMO
Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.
Assuntos
Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Amidas/antagonistas & inibidores , Amidas/metabolismo , COVID-19 , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Mutação , Pandemias , Proteoma/antagonistas & inibidores , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismoRESUMO
The microsporidia are a large group of intracellular parasites with a broad range of hosts, including humans. Encephalitozoon intestinalis is the second microsporidia species most frequently associated with gastrointestinal disease in humans, especially immunocompromised or immunosuppressed individuals, including children and the elderly. The prevalence reported worldwide in these groups ranges from 0 to 60%. Currently, albendazole is most commonly used to treat microsporidiosis caused by Encephalitozoon species. However, the results of treatment are variable, and relapse can occur. Consequently, efforts are being directed toward identifying more effective drugs for treating microsporidiosis, and the study of new molecular targets appears promising. These parasites lack mitochondria, and oxidative phosphorylation therefore does not occur, which suggests the enzymes involved in glycolysis as potential drug targets. Here, we have for the first time characterized the glycolytic enzyme triosephosphate isomerase of E. intestinalis at the functional and structural levels. Our results demonstrate the mechanisms of inactivation of this enzyme by thiol-reactive compounds. The most striking result of this study is the demonstration that established safe drugs such as omeprazole, rabeprazole and sulbutiamine can effectively inactivate this microsporidial enzyme and might be considered as potential drugs for treating this important disease.
Assuntos
Albendazol/uso terapêutico , Proteínas Fúngicas/antagonistas & inibidores , Microsporídios/efeitos dos fármacos , Microsporidiose/tratamento farmacológico , Triose-Fosfato Isomerase/antagonistas & inibidores , Sequência de Aminoácidos , Encephalitozoon/efeitos dos fármacos , Encephalitozoon/enzimologia , Encephalitozoon/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/microbiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Microsporídios/enzimologia , Microsporídios/genética , Microsporidiose/microbiologia , Omeprazol/uso terapêutico , Rabeprazol/uso terapêutico , Homologia de Sequência de Aminoácidos , Tiamina/análogos & derivados , Tiamina/uso terapêutico , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/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.
Assuntos
Variação Genética , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Modelos Moleculares , Conformação Molecular , Mutação , Catálise , Ativação Enzimática , Expressão Gênica , Glucosefosfato Desidrogenase/metabolismo , Humanos , Cinética , Estabilidade Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , TermodinâmicaRESUMO
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.
Assuntos
Álcool Desidrogenase/metabolismo , Etanol/metabolismo , Gluconacetobacter/enzimologia , Acetatos/análise , Álcool Desidrogenase/química , Álcool Desidrogenase/isolamento & purificação , Aldeídos/análise , Sequência de Aminoácidos , Biocatálise , Radioisótopos de Carbono/química , Cromatografia Gasosa-Espectrometria de Massas , Marcação por Isótopo , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oxirredução , Desnaturação Proteica , TemperaturaRESUMO
La deficiencia de Glucosa-6-fosfato deshidrogenasa (G6PD) es la enzimopatíamás frecuente, con una prevalencia global del 4,9% y con alrededor de 330 a 400 millones de personas afectadas en el mundo. La G6PD desempeña un papel fundamental en el equilibrio redox intracelular, especialmente en los eritrocitos; en condiciones de estrés oxidativo inducido (por ejemplo,por exposición a agentes externos como fármacos, alimentos o infecciones),los hematíes portadores de la variante enzimática y con deficiencia de la actividad enzimática, sufren daños irreversibles que condicionan su destrucción acelerada. La hemólisis explica el espectro de manifestaciones clínicas de esta enfermedad, que incluyen ictericia neonatal, episodios de hemólisis aguda inducida por agentes externos o anemia hemolítica crónica. El presente trabajo hace una reseña de los aspectos epidemiológicos y clínicos de esta enfermedad y revisa los aspectos fisiopatológicos a nivel bioquímico-molecular, con particular énfasis en la caracterización genética,estructural y funcional de las variantes asociadas a la deficiencia de G6PD.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent enzymopathy in humans with a global prevalence of 4.9 % and around 330 to 400 million patients affected worldwide. G6PD plays a fundamental role in the intracellular redox equilibrium, especially in red blood cells (RBC). Under oxidative stress (induced by exposure to external agents like drugs, infections or diet) RBC carrying the deficient variant suffer irreversible damage resulting in their accelerated destruction. This hemolysis explains the clinical manifestations of the disease that include neonatal jaundice, inducedacute hemolysis or chronic hemolytic anemia. This work summarizes the epidemiologic and clinical features of G6PD deficiency, and reviews the molecular pathophysiology of this disease with special emphasis on the genetical, structural and functional characterization of variants causing this pathology.
A deficiência da Glicose-6-FosFato desidrogenase (G6PD) é a enzimopatia mais Frequente, com uma prevalência global do 4,9%, e com aproximadamente 330 a 400 milhões de pessoas afetadas no mundo. A G6PD tem um importante papel no equilíbrio celular redox intracelular, especialmente nos eritrócitos; em condições de estresse oxidativo induzido, (por exemplo, pela exposição a agentes externos como Fármacos, alimentos, ou infecções) as hemácias portadoras da variante enzimática e com defciência da atividade enzimática, sofrem danos irreversíveis que condicionam a sua destruição acelerada. A hemólise explica o espectro de manifestações clínicas desta doença, que incluem icterícia neonatal, episódios de hemólise aguda induzida por agentes externos ou anemia hemolítica crônica. Este trabalho faz uma resenha dos aspectos epidemiológicos e clínicos desta doença, e revisa os aspectos fsiopatológicos no nível bioquímico-molecular, com ênfase especial na caracterização genética, estrutural e funcional das variantes associadas à defciência de G6PD.
Assuntos
Humanos , Glucosefosfato Desidrogenase , Deficiência de Glucosefosfato Desidrogenase , Anemia Hemolítica Congênita , Erros Inatos do MetabolismoRESUMO
La deficiencia de Glucosa-6-fosfato deshidrogenasa (G6PD) es la enzimopatía más frecuente, con una prevalencia global del 4,9% y con alrededor de 330 a 400 millones de personas afectadas en el mundo. La G6PD desempeña un papel fundamental en el equilibrio redox intracelular, especialmente en los eritrocitos; en condiciones de estrés oxidativo inducido (por ejemplo, por exposición a agentes externos como fármacos, alimentos o infecciones), los hematíes portadores de la variante enzimática y con deficiencia de la actividad enzimática, sufren daños irreversibles que condicionan su destrucción acelerada. La hemólisis explica el espectro de manifestaciones clínicas de esta enfermedad, que incluyen ictericia neonatal, episodios de hemólisis aguda inducida por agentes externos o anemia hemolítica crónica. El presente trabajo hace una reseña de los aspectos epidemiológicos y clínicos de esta enfermedad y revisa los aspectos fisiopatológicos a nivel bioquímico-molecular, con particular énfasis en la caracterización genética, estructural y funcional de las variantes asociadas a la deficiencia de G6PD.(AU)
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent enzymopathy in humans with a global prevalence of 4.9 % and around 330 to 400 million patients affected worldwide. G6PD plays a fundamental role in the intracellular redox equilibrium, especially in red blood cells (RBC). Under oxidative stress (induced by exposure to external agents like drugs, infections or diet) RBC carrying the deficient variant suffer irreversible damage resulting in their accelerated destruction. This hemolysis explains the clinical manifestations of the disease that include neonatal jaundice, induced acute hemolysis or chronic hemolytic anemia. This work summarizes the epidemiologic and clinical features of G6PD deficiency, and reviews the molecular pathophysiology of this disease with special emphasis on the genetical, structural and functional characterization of variants causing this pathology.(AU)
A deficiÛncia da Glicose-6-fosfato desidrogenase (G6PD) é a enzimopatia mais frequente, com uma prevalÛncia global do 4,9%, e com aproximadamente 330 a 400 milh§es de pessoas afetadas no mundo. A G6PD tem um importante papel no equilíbrio celular redox intracelular, especialmente nos eritrócitos; em condiþ§es de estresse oxidativo induzido, (por exemplo, pela exposiþÒo a agentes externos como fármacos, alimentos, ou infecþ§es) as hemácias portadoras da variante enzimática e com deficiÛncia da atividade enzimática, sofrem danos irreversíveis que condicionam a sua destruiþÒo acelerada. A hemólise explica o espectro de manifestaþ§es clínicas desta doenþa, que incluem icterícia neonatal, episódios de hemólise aguda induzida por agentes externos ou anemia hemolítica cr¶nica. Este trabalho faz uma resenha dos aspectos epidemiológicos e clínicos desta doenþa, e revisa os aspectos fisiopatológicos no nível bioquímico-molecular, com Ûnfase especial na caracterizaþÒo genética, estrutural e funcional das variantes associadas O deficiÛncia de G6PD.(AU)
RESUMO
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide, causing a wide spectrum of conditions with severity classified from the mildest (Class IV) to the most severe (Class I). To correlate mutation sites in the G6PD with the resulting phenotypes, we studied four naturally occurring G6PD variants: Yucatan, Nashville, Valladolid and Mexico City. For this purpose, we developed a successful over-expression method that constitutes an easier and more precise method for obtaining and characterizing these enzymes. The k(cat) (catalytic constant) of all the studied variants was lower than in the wild-type. The structural rigidity might be the cause and the most evident consequence of the mutations is their impact on protein stability and folding, as can be observed from the protein yield, the T50 (temperature where 50% of its original activity is retained) values, and differences on hydrophobic regions. The mutations corresponding to more severe phenotypes are related to the structural NADP+ region. This was clearly observed for the Classes III and II variants, which became more thermostable with increasing NADP+, whereas the Class I variants remained thermolabile. The mutations produce repulsive electric charges that, in the case of the Yucatan variant, promote increased disorder of the C-terminus and consequently affect the binding of NADP+, leading to enzyme instability.
Assuntos
Deficiência de Glucosefosfato Desidrogenase/enzimologia , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/genética , Mutação , Estabilidade Enzimática , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Humanos , Modelos Moleculares , Fenótipo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Giardia lamblia/efeitos dos fármacos , Omeprazol/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Triose-Fosfato Isomerase/antagonistas & inibidores , Trofozoítos/efeitos dos fármacos , Albendazol/farmacologia , Cultura Axênica , Cisteína/química , Cisteína/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Giardia lamblia/enzimologia , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/isolamento & purificação , Humanos , Metronidazol/farmacologia , Mutação , Nitrocompostos , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Tiazóis/farmacologia , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismo , Trofozoítos/enzimologia , Trofozoítos/crescimento & desenvolvimentoRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway. In erythrocytes, the functionality of the pathway is crucial to protect these cells against oxidative damage. G6PD deficiency is the most frequent enzymopathy in humans with a global prevalence of 4.9 %. The clinical picture is characterized by chronic or acute hemolysis in response to oxidative stress, which is related to the low cellular activity of G6PD in red blood cells. The disease is heterogeneous at genetic level with around 160 mutations described, mostly point mutations causing single amino acid substitutions. The biochemical studies aimed to describe the detrimental effects of mutations on the functional and structural properties of human G6PD are indispensable to understand the molecular physiopathology of this disease. Therefore, reliable systems for efficient expression and purification of the protein are highly desirable. In this work, human G6PD was heterologously expressed in Escherichia coli and purified by immobilized metal affinity chromatography in a single chromatographic step. The structural and functional characterization indicates that His-tagged G6PD resembles previous preparations of recombinant G6PD. In contrast with previous protein yield systems, our method is based on commonly available resources and fully accessible laboratory equipment; therefore, it can be readily implemented.
Assuntos
Glucosefosfato Desidrogenase/isolamento & purificação , Glucosefosfato Desidrogenase/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Cromatografia de Afinidade , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/genética , Histidina , Humanos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND: We have previously proposed triosephosphate isomerase of Giardia lamblia (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation. RESULTS: In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design. CONCLUSIONS: The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.
Assuntos
Giardia lamblia/enzimologia , Mutagênese Sítio-Dirigida , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo , Biocatálise , Sequência Conservada , Cristalografia por Raios X , Estabilidade Enzimática , Glicolatos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espectral , Triose-Fosfato Isomerase/genéticaRESUMO
Giardia lamblia depends on glycolysis to obtain ATP, highlighting the suitability of glycolytic enzymes as targets for drug design. We studied triosephosphate isomerase from G. lamblia (GlTIM) as a potential species-specific drug target. Cysteine-reactive agents were used as probes, in order to test those regions near to cysteine residues as targets to perturb enzyme structure and activity. Methyl methanethiosulfonate (MMTS) derivatized three of the five Cys per subunit of dimeric GlTIM and induced 50% of inactivation. The 2-carboxyethyl methanethiosulfonate (MTSCE) modified four Cys and induced 97% of inactivation. Inactivation by MMTS or MTSCE did not affect secondary structure, nor induce dimer dissociation; however, Cys modification decreased thermal stability of enzyme. Inactivation and dissociation of the dimer to stable monomers were reached when four Cys were derivatized by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). The effects of DTNB were completely abolished when GlTIM was first treated with MMTS. The effect of thiol reagents on human TIM was also assayed; it is 180-fold less sensitive than GlTIM. Collectively, the data illustrate GlTIM as a good target for drug design.