RESUMO
Rhipicephalus microplus, the "common cattle tick", is the most important ectoparasite in livestock worldwide due to the economic and health losses it produces. This tick is a vector for pathogens of several tick-borne diseases. In Latin American countries, damages reach approximately USD 500 million annually due to tick infections, as well as tick-borne diseases. Currently, resistant populations for every chemical group of acaricides have been reported, posing a serious problem for tick control. This study aims to find new alternatives for controlling resistant ticks with compounds derived from small synthetic organic molecules and natural origins. Using BME26 embryonic cells, we performed phenotypic screening of 44 natural extracts from 10 Mexican plants used in traditional medicine, and 33 compounds selected from our chemical collection. We found 10 extracts and 13 compounds that inhibited cell growth by 50% at 50 µg/mL and 100 µM, respectively; the dose-response profile of two of them was characterized, and these compounds were assayed in vitro against different life stages of Rhipicephalus microplus. We also performed a target-directed screening of the activity of triosephosphate isomerase, using 86 compounds selected from our chemical collection. In this collection, we found the most potent and selective inhibitor of tick triosephosphate isomerase reported until now. Two other compounds had a potent acaricidal effect in vitro using adults and larvae when compared with other acaricides such as ivermectin and Amitraz. Those compounds were also selective to the ticks compared with the cytotoxicity in mammalian cells like macrophages or bovine spermatozoids. They also had a good toxicological profile, resulting in promising acaricidal compounds for tick control in cattle raising.
Assuntos
Acaricidas , Doenças dos Bovinos , Rhipicephalus , Doenças Transmitidas por Carrapatos , Animais , Bovinos , Acaricidas/farmacologia , Triose-Fosfato Isomerase , Extratos Vegetais/química , Doenças dos Bovinos/parasitologia , Larva , MamíferosRESUMO
Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in Leishmania, a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in Leishmania mexicana (LmxPP2C). Recombinant LmxPP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn+2). LmxPP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti-LmxPP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized LmxPP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, LmxPP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms.
Assuntos
Leishmania mexicana , Leishmania , Leishmaniose , Humanos , Leishmania/metabolismo , Leishmania mexicana/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , SerinaRESUMO
Triosephosphate isomerase (TIM) is an enzyme of the glycolysis pathway which exists in almost all types of cells. Its structure is the prototype of a motif called TIM-barrel or (α/ß)8 barrel, which is the most common fold of all known enzyme structures. The simplest form in which TIM is catalytically active is a homodimer, in many species of bacteria and eukaryotes, or a homotetramer in some archaea. Here we show that the purified homodimeric TIMs from nine different species of eukaryotes and one of an extremophile bacterium spontaneously form higher order aggregates that can range from 3 to 21 dimers per macromolecular complex. We analysed these aggregates with clear native electrophoresis with normal and inverse polarity, blue native polyacrylamide gel electrophoresis, liquid chromatography, dynamic light scattering, thermal shift assay and transmission electron and fluorescence microscopies, we also performed bioinformatic analysis of the sequences of all enzymes to identify and predict regions that are prone to aggregation. Additionally, the capacity of TIM from Trypanosoma brucei to form fibrillar aggregates was characterized. Our results indicate that all the TIMs we studied are capable of forming oligomers of different sizes. This is significant because aggregation of TIM may be important in some of its non-catalytic moonlighting functions, like being a potent food allergen, or in its role associated with Alzheimer's disease.
Assuntos
Agregados Proteicos , Triose-Fosfato Isomerase/metabolismo , Cromatografia Líquida , Biologia Computacional/métodos , Difusão Dinâmica da Luz , Ativação Enzimática , Expressão Gênica , Cinética , Ligação Proteica , Multimerização Proteica , Sensibilidade e Especificidade , Especificidade da Espécie , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/isolamento & purificaçãoRESUMO
Experimentally relocating mitochondrial genes to the nucleus for functional expression (allotopic expression) is a challenging process. The high hydrophobicity of mitochondria-encoded proteins seems to be one of the main factors preventing this allotopic expression. We focused on subunit II of cytochrome c oxidase (Cox2) to study which modifications may enable or improve its allotopic expression in yeast. Cox2 can be imported from the cytosol into mitochondria in the presence of the W56R substitution, which decreases the protein hydrophobicity and allows partial respiratory rescue of a cox2-null strain. We show that the inclusion of a positive charge is more favorable than substitutions that only decrease the hydrophobicity. We also searched for other determinants enabling allotopic expression in yeast by examining the COX2 gene in organisms where it was transferred to the nucleus during evolution. We found that naturally occurring variations at within-membrane residues in the legume Glycine max Cox2 could enable yeast COX2 allotopic expression. We also evidence that directing high doses of allotopically synthesized Cox2 to mitochondria seems to be counterproductive because the subunit aggregates at the mitochondrial surface. Our findings are relevant to the design of allotopic expression strategies and contribute to the understanding of gene retention in organellar genomes.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genes Mitocondriais , Proteínas de Membrana/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Triosephosphate isomerase is the fifth enzyme in glycolysis and its canonical function is the reversible isomerization of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Within the last decade multiple other functions, that may not necessarily always involve catalysis, have been described. These include variations in the degree of its expression in many types of cancer and participation in the regulation of the cell cycle. Triosephosphate isomerase may function as an auto-antigen and in the evasion of the immune response, as a factor of virulence of some organisms, and also as an important allergen, mainly in a variety of seafoods. It is an important factor to consider in the cryopreservation of semen and seems to play a major role in some aspects of the development of Alzheimer's disease. It also seems to be responsible for neurodegenerative alterations in a few cases of human triosephosphate isomerase deficiency. Thus, triosephosphate isomerase is an excellent example of a moonlighting protein.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/veterinária , Doenças dos Animais/enzimologia , Erros Inatos do Metabolismo dos Carboidratos/veterinária , Triose-Fosfato Isomerase/deficiência , Triose-Fosfato Isomerase/metabolismo , Anemia Hemolítica Congênita não Esferocítica/tratamento farmacológico , Anemia Hemolítica Congênita não Esferocítica/metabolismo , Doenças dos Animais/tratamento farmacológico , Animais , Erros Inatos do Metabolismo dos Carboidratos/tratamento farmacológico , Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Glicólise , HumanosRESUMO
The cattle tick Rhipicephalus microplus is one of the most important ectoparasites causing significant economic losses for the cattle industry. The major tool of control is reducing the number of ticks, applying acaricides in cattle. However, overuse has led to selection of resistant populations of R. microplus to most of these products, some even to more than one active principle. Thus, exploration for new molecules with acaricidal activity in R. microplus has become necessary. Triosephosphate isomerase (TIM) is an essential enzyme in R. microplus metabolism and could be an interesting target for the development of new methods for tick control. In this work, we screened 227 compounds, from our in-house chemo-library, against TIM from R. microplus. Four compounds (50, 98, 14, and 161) selectively inhibited this enzyme with IC50 values between 25 and 50 µM. They were also able to diminish cellular viability of BME26 embryonic cells by more than 50% at 50 µM. A molecular docking study showed that the compounds bind in different regions of the protein; compound 14 interacts with the dimer interface. Furthermore, compound 14 affected the survival of partially engorged females, fed artificially, using the capillary technique. This molecule is simple, easy to produce, and important biological data-including toxicological information-are available for it. Our results imply a promising role for compound 14 as a prototype for development of a new acaricidal involving selective TIM inhibition.
RESUMO
Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four "unknown severity mutants" with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/enzimologia , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Mutação , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/deficiência , Triose-Fosfato Isomerase/metabolismo , Anemia Hemolítica Congênita não Esferocítica/genética , Erros Inatos do Metabolismo dos Carboidratos/genética , Estabilidade Enzimática , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Triose-Fosfato Isomerase/genéticaRESUMO
A series of fifty arylideneketones and thiazolidenehydrazines was evaluated against Leishmania infantum and Leishmania braziliensis. Furthermore, new simplified thiazolidenehydrazine derivatives were evaluated against Trypanosoma cruzi. The cytotoxicity of the active compounds on non-infected fibroblasts or macrophages was established in vitro to evaluate the selectivity of their anti-parasitic effects. Seven thiazolidenehydrazine derivatives and ten arylideneketones had good activity against the three parasites. The IC50 values for T. cruzi and Leishmania spp. ranged from 90 nM-25 µM. Eight compounds had multi-trypanocidal activity against T. cruzi and Leishmania spp. (the etiological agents of cutaneous and visceral forms). The selectivity of these active compounds was better than the three reference drugs: benznidazole, glucantime and miltefosine. They also had low toxicity when tested in vivo on zebrafish. Trying to understand the mechanism of action of these compounds, two possible molecular targets were investigated: triosephosphate isomerase and cruzipain. We also used a molecular stripping approach to elucidate the minimal structural requirements for their anti-T. cruzi activity.
Assuntos
Doença de Chagas/tratamento farmacológico , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Visceral/dietoterapia , Tripanossomicidas , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Linhagem Celular , Doença de Chagas/metabolismo , Doença de Chagas/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrazinas , Cetonas , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/patologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/patologia , Camundongos , Tiazolidinas , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Peixe-ZebraRESUMO
Rare arginine codons AGA and AGG affect the heterologous expression of proteins in Eschericha coli. The tRNAs necessary for protein synthesis are scarce in E. coli strain BL21(DE3) pLysS and plentiful in strain BL21(DE3) CodonPlus -RIL. We evaluated in both bacterial strains the effect of these rare codons on the expression of triosephosphate isomerases from 7 different species, whose sequences had different dispositions of rare arginine codons. The ratio of expressed protein (CP/Bl21) correlated with the number of rare codons. Our study shows that the number, position and particularities of the combination of rare Arg codons in the natural non-optimized sequences of the triosephosphate isomerases influence the synthesis of heterologous proteins in E. coli and could have implications in the selection of better sequences for engineering enzymes for novel or manipulated metabolic pathways or for the expression levels of non enzymatic proteins..
RESUMO
The receptor for advanced glycation end products (RAGE) is commonly involved in different neurodegenerative and inflammatory disorders. The cellular signaling associated to RAGE activation may occur upon binding to different ligands. In this study we investigated whether the toxic model produced by 6-hydroxydopamine (6-OHDA) in rats comprises early noxious responses related to RAGE-mediated signaling cascades. In order to explore a possible interaction between 6-OHDA and RAGE, affinity parameters of RAGE with 6-OHDA were estimated by different means. The possible binding sites of 6-OHDA with the VC1 homodimer for both rat and human RAGE were also modeled. Our results show that the striatal infusion of 6-OHDA recruits RAGE upregulation, as evidenced by an early expression of the receptor. 6-OHDA was also found to bind the VC1 homodimer, although its affinity was moderate when compared to other ligands. This work contributes to the understanding of the role of RAGE activation for 6-OHDA-induced neurotoxicity.
Assuntos
Corpo Estriado/metabolismo , Oxidopamina/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Sítios de Ligação/fisiologia , Ligantes , Masculino , Ligação Proteica/fisiologia , Ratos , Ratos WistarRESUMO
Triosephosphate isomerase (TIM) is an essential Trypanosoma cruzi enzyme and one of the few validated drug targets for Chagas disease. The known inhibitors of this enzyme behave poorly or have low activity in the parasite. In this work, we used symmetrical diarylideneketones derived from structures with trypanosomicidal activity. We obtained an enzymatic inhibitor with an IC50 value of 86â nm without inhibition effects on the mammalian enzyme. These molecules also affected cruzipain, another essential proteolytic enzyme of the parasite. This dual activity is important to avoid resistance problems. The compounds were studied in vitro against the epimastigote form of the parasite, and nonspecific toxicity to mammalian cells was also evaluated. As a proof of concept, three of the best derivatives were also assayed in vivo. Some of these derivatives showed higher in vitro trypanosomicidal activity than the reference drugs and were effective in protecting infected mice. In addition, these molecules could be obtained by a simple and economic green synthetic route, which is an important feature in the research and development of future drugs for neglected diseases.
Assuntos
Antiprotozoários/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Triose-Fosfato Isomerase/antagonistas & inibidores , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiprotozoários/química , Antiprotozoários/uso terapêutico , Sítios de Ligação , Doença de Chagas/tratamento farmacológico , Cisteína Endopeptidases/química , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Cetonas/química , Cetonas/farmacologia , Cetonas/uso terapêutico , Camundongos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Triose-Fosfato Isomerase/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM). Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.
Assuntos
Tolueno/análogos & derivados , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Macrófagos/efeitos dos fármacos , Camundongos , Esteróis/antagonistas & inibidores , Esteróis/biossíntese , Tolueno/síntese química , Tolueno/química , Tolueno/farmacologia , Tripanossomicidas/síntese química , Trypanosoma cruzi/metabolismoRESUMO
The neglected disease American trypanosomiasis is one of the major health problems in Latin America. Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), the etiologic agent of this disease, has been proposed as a druggable target. Some bis-benzothiazoles have been described as irreversible inhibitors of this enzyme. On the other hand, new bioactive furane-containing thiazoles have been described as excellent in vivo anti-T. cruzi agents. This encouraged us to design and develop new bis-thiazoles with potential use as drugs for American trypanosomiasis. The bis-thiazol 5, 3,3'-allyl-2,2'-bis[3-(2-furyl)-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, showed the best in vitro anti-T. cruzi profile with a higher selectivity index than the reference drugs Nifurtimox and Benznidazole against amastigote form of the parasite. This derivative displayed marginal activity against TcTIM however the bis-thiazol 14, 3-allyl-2-[3-(2-furyl)-2-propenylidenehydrazono]-3'-phenyl-2'-(3-phenyl-2-propenylidenehydrazono]-2,2',3,3'-tetrahydro-4,4'-bisthiazole, was an excellent inhibitor of the enzyme of the parasite. The absence of both in vitro mutagenic and in vivo toxicity effects, together with the activity of bis-thiazol 5in vivo, suggests that this compound is a promising anti-T. cruzi agent surpassing the "hit-to-lead" stage in the drug development process.
Assuntos
Inibidores Enzimáticos/farmacologia , Tiazóis/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Interações Hidrofóbicas e Hidrofílicas , Macrófagos , Camundongos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Triose-Fosfato Isomerase/metabolismo , Tripanossomicidas/síntese química , Tripanossomicidas/químicaRESUMO
CONTEXT: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of new small molecular weight compounds used against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified phenazine and 1,2,6-thiadiazine chemotypes as novel inhibitors of TIM from T. cruzi (TcTIM). OBJECTIVE: Study the mechanism of TcTIM inhibition by a phenazine derivative and by a 1,2,6-thiadiazine derivative. METHODS: We performed biochemical and theoretical molecular docking studies to characterize the interaction of the derivatives with wild-type and mutant TcTIM. RESULTS AND CONCLUSION: At low micromolar concentrations, the compounds induce highly selective irreversible inactivation of parasitic TIM. The molecular docking simulations indicate that the phenazine derivative likely interferes with the association of the two monomers of the dimeric enzyme by locating at the dimer interface, while 1,2,6-thiadiazine could act as an inhibitor binding to a region surrounding Cys-118.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Fenazinas/farmacologia , Tiadiazinas/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Trypanosoma cruzi/efeitos dos fármacos , Antiprotozoários/química , Ligação Competitiva , Doença de Chagas/tratamento farmacológico , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/química , Escherichia coli/genética , Modelos Biológicos , Simulação de Acoplamento Molecular , Estrutura Molecular , Testes de Sensibilidade Parasitária , Fenazinas/química , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Tiadiazinas/química , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética , Trypanosoma cruzi/enzimologiaRESUMO
CONTEXT: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of small molecular weight compounds with potential use against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified a new selective inhibitor chemotype of TIM from T. cruzi (TcTIM), 1,2,4-thiadiazol-5(4H)-one. OBJECTIVE: Study the mechanism of TcTIM inhibition by a 1,2,4-thiadiazol derivative. METHODS: We performed the biochemical characterization of the interaction of the 1,2,4-thiadiazol derivative with the wild-type and mutant TcTIMs, using DOSY-NMR and MS experiments. Studies of T. cruzi growth inhibition were additionally carried out. RESULTS AND CONCLUSION: At low micromolar concentrations, the compound induces highly selective irreversible inactivation of TcTIM through non-covalent binding. Our studies indicate that it interferes with the association of the two monomers of the dimeric enzyme. We also show that it inhibits T. cruzi growth in culture.
Assuntos
Inibidores Enzimáticos/farmacologia , Tiadiazóis/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Trypanosoma cruzi/enzimologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tiadiazóis/síntese química , Tiadiazóis/química , Triose-Fosfato Isomerase/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.
Assuntos
Arginina/genética , Escherichia coli/metabolismo , Lisina/genética , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo , Arginina/química , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Cromatografia Líquida , Biologia Computacional , Escherichia coli/genética , Humanos , Lisina/química , Polimorfismo Genético/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Triose-Fosfato Isomerase/genéticaRESUMO
For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.
Assuntos
Aminoácidos/química , Triose-Fosfato Isomerase/metabolismo , Trypanosoma/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Biocatálise , Primers do DNA , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genéticaRESUMO
Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 µmol min⻹ mg protein⻹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.
Assuntos
Embrião não Mamífero/enzimologia , Proteínas Recombinantes/metabolismo , Rhipicephalus/enzimologia , Triose-Fosfato Isomerase/metabolismo , Zigoto/enzimologia , Sequência de Aminoácidos , Animais , Catálise , Clonagem Molecular , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Dimerização , Escherichia coli , Gliceraldeído 3-Fosfato/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/genética , Rhipicephalus/embriologia , Alinhamento de Sequência , Reagentes de Sulfidrila/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/isolamento & purificaçãoRESUMO
Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), an enzyme in the glycolytic pathway that exhibits high catalytic rates of glyceraldehyde-3-phosphate- and dihydroxyacetone-phosphate-isomerization only in its dimeric form, was screened against an in-house chemical library containing nearly 230 compounds belonging to different chemotypes. After secondary screening, twenty-six compounds from eight different chemotypes were identified as screening positives. Four compounds displayed selectivity for TcTIM over TIM from Homo sapiens and, concomitantly, in vitro activity against T. cruzi.
Assuntos
Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Dimerização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Estereoisomerismo , Relação Estrutura-Atividade , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma cruzi/enzimologiaRESUMO
The PyrR transcriptional regulator is widely distributed in bacteria. This RNA-binding protein is involved in the control of genes involved in pyrimidine biosynthesis, in which uridyl and guanyl nucleotides function as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bacillus halodurans PyrR are reported. One of the forms belongs to the monoclinic space group P2(1) with unit-cell parameters a = 59.7, b = 87.4, c = 72.1 A, beta = 104.4 degrees , while the other form belongs to the orthorhombic space group P22(1)2(1) with unit-cell parameters a = 72.7, b = 95.9, c = 177.1 A. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of four and six monomers per asymmetric unit; a crystallographic tetramer is formed in both forms.