RESUMO
Selenium (Se) is an essential trace element primarily found in selenoproteins as the 21st amino acid (selenocysteine, Sec, or U). Selenoproteins play an important role in growth and proliferation and are typically involved in cellular redox balance. Selenocysteine is encoded by an in-frame UGA codon specified by a stem-loop structure, the Sec insertion sequence element (SECIS), which, in eukaryotes, is located in the 3'-untranslated region (UTR). The availability of the Naegleria gruberi (ATCC 30224) genome sequence and the use of this organism as a model system for the pathogenic amoeba N. fowleri allowed us to investigate the Sec incorporation pathway in this primitive eukaryote. Using bioinformatics tools, we identified gene sequences encoding PSTK (O-phosphoseryl-tRNA(Sec) kinase), SepSecS (O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase), SelD/SPS2 (selenophosphate synthetase), EFSec (selenocysteine-specific elongation factor) and SBP (SECIS binding protein). These findings were confirmed by RT-PCR and by sequencing. A potential tRNA(Ser)Sec (SelC) gene and a putative selenoprotein with sequence similarity to a mitochondrial thioredoxin reductase (TR3) were also identified. Our results show that the selenocysteine incorporation machinery is indeed present in N. gruberi. Interestingly, the SelD/SPS2 gene is 2214 bp in length and contains two distinct domains. The N-terminal region shows sequence similarity to predicted methyltransferase proteins, and the C-terminal region is homologous to prokaryotic SelD/SPS2. Our results suggest the possibility of novel selenoproteins.
Assuntos
Vias Biossintéticas/genética , Naegleria/genética , Naegleria/metabolismo , Selenocisteína/biossíntese , Selenoproteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biologia Computacional , Perfilação da Expressão Gênica , Genes de Protozoários , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Selenoproteins are characterized by the incorporation of at least one amino acid selenocysteine (Sec-U) encoded by in-frame UGA stop codons. These proteins, as well as the components of the Sec synthesis pathway, are present in members of the bacteria, archaea and eukaryote domains. Although not a ubiquitous pathway in all organisms, it was also identified in several protozoa, including the Kinetoplastida. Genetic evidence has indicated that the pathway is non-essential to the survival of Trypanosoma growing in non-stressed conditions. By analyzing the effects of RNA interference of the Trypanosoma brucei selenophosphate synthetase SPS2, we found a requirement under sub-optimal growth conditions. The present work shows that SPS2 is involved in oxidative stress protection of the parasite and its absence severely hampers the parasite survival in the presence of an oxidizing environment that results in an apoptotic-like phenotype and cell death.
Assuntos
Estresse Oxidativo , Fosfotransferases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Oxirredução , Fosfotransferases/genética , Proteínas de Protozoários/genética , Selênio/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
Selenophosphate synthetase (EC 2.7.9.3), the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide, for the synthesis of selenocysteine. The kinetoplastid Leishmania major and Trypanosoma brucei selD genes were cloned and the SELD protein overexpressed and purified to apparent homogeneity. The selD gene in L. major and T. brucei are respectively 1197 and 1179 bp long encoding proteins of 399 and 393 amino acids with molecular masses of 42.7 and 43 kDa. The molecular mass of 100 kDa for both (L. major and T. brucei) SELDs is consistent with dimeric proteins. The kinetoplastid selD complement Escherichia coli (WL400) selD deletion confirming it is a functional enzyme and the specific activity of these enzymes was determined. A conserved Cys residue was identified both by multiple sequence alignment as well as by functional complementation and activity assay of the mutant (Cys to Ala) forms of the SELD identifying this residue as essential for the catalytic function.
Assuntos
Leishmania major/enzimologia , Fosfotransferases/química , Proteínas de Protozoários/química , Selenocisteína/metabolismo , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cisteína/metabolismo , Teste de Complementação Genética , Leishmania major/metabolismo , Dados de Sequência Molecular , Fosfotransferases/isolamento & purificação , Fosfotransferases/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Trypanosoma brucei brucei/metabolismoRESUMO
Selenoproteins result from the incorporation of selenocysteine (Sec-U) at an UGA-stop codon positioned within a gene's open reading frame and directed by selenocysteine insertion sequence (SECIS) elements. Although the selenocysteine incorporation pathway has been identified in a wide range of organisms it has not yet been reported in the Kinetoplastida Leishmania and Trypanosoma. Here we present evidence consistent with the presence of a selenocysteine biosynthetic pathway in Kinetoplastida. These include the existence of SECIS-containing coding sequences in Leishmania major and Leishmania infantum, the incorporation of (75)Se into Leishmania proteins, the occurrence of selenocysteine-tRNA (tRNA (sec) (uca)) in both Leishmania and Trypanosoma and in addition the finding of all genes necessary for selenocysteine synthesis such as SELB, SELD, PSTK and SECp43. As in other eukaryotes, the Kinetoplastids have no identifiable SELA homologue. To our knowledge this is the first report on the identification of selenocysteine insertion machinery in Kinetoplastida, more specifically in Leishmania, at the sequence level.
Assuntos
Leishmania/metabolismo , Proteínas de Protozoários/metabolismo , Selenoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/genética , Leishmania/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA de Protozoário/química , RNA de Protozoário/genética , RNA de Transferência Aminoácido-Específico/química , RNA de Transferência Aminoácido-Específico/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Adenine phosphoribosyltransferase (APRT) enzyme from Leishmania tarentolae has been proposed as a target for the rational search of new leishmanicidal drugs. In this paper, we describe the evaluation of the inhibitory activity on L. tarentolae APRT enzyme of 46 crude extracts of Meliaceae and Rutaceae plants, besides three furoquinolone alkaloids. The results showed that 21 extracts were able to decrease the APRT enzymatic activity (IA% > or = 50). The methanolic extracts from roots and leaves of Cedrela fissilis and from fruits, branches and leaves of Cipadessa fruticosa have showed strong activities. Therefore, these species could be a promising source of lead compounds for the rational design of new leishmanicidal drugs. The phytochemical investigation of an active fraction from Almeidea rubra afforded the alkaloids isodutaduprine, isoskimmianine and isokokusagine, which showed low to moderate activity on APRT.
Assuntos
Adenina Fosforribosiltransferase/antagonistas & inibidores , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania/enzimologia , Meliaceae/química , Rutaceae/química , Alcaloides/química , Alcaloides/farmacologia , Animais , Extratos Vegetais/farmacologia , Folhas de Planta/química , Raízes de Plantas/química , Relação Quantitativa Estrutura-AtividadeRESUMO
Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P2(1)2(1)2 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 A. The crystals diffract to a maximum resolution of 3.3 A and a complete data set to 3.5 A resolution was collected and analysed.
Assuntos
Ribose-Fosfato Pirofosfoquinase/química , Ribose-Fosfato Pirofosfoquinase/isolamento & purificação , Saccharum/enzimologia , Escherichia coli/enzimologia , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ribose-Fosfato Pirofosfoquinase/genética , Transfecção , Difração de Raios XRESUMO
Several natural compounds have been identified for the treatment of leishmaniasis. Among them are some alkaloids, chalcones, lactones, tetralones, and saponins. The new compound reported here, 7-geranyloxycoumarin, called aurapten, belongs to the chemical class of the coumarins and has a molecular weight of 298.37. The compund was extracted from the Rutaceae species Esenbeckia febrifuga and was purified from a hexane extract starting from 407.7 g of dried leaves and followed by four silica gel chromatographic fractionation steps using different solvents as the mobile phase. The resulting compound (47 mg) of shows significant growth inhibition with an LD50 of 30 æM against the tropical parasite Leishmania major, which causes severe clinical manifestations in humans and is endemic in the tropical and subtropical regions. In the present study, we investigated the atomic structure of aurapten in order to determine the existence of common structural motifs that might be related to other coumarins and potentially to other identified inhibitors of Leishmania growth and viability. This compound has a comparable inhibitory activity of other isolated molecules. The aurapten is a planar molecule constituted of an aromatic system with electron delocalization. A hydrophobic side chain consisting of ten carbon atoms with two double bonds and negative density has been identified and may be relevant for further compound synthesis.
Assuntos
Animais , Antiprotozoários/farmacologia , Cumarínicos/farmacologia , Leishmaniose/tratamento farmacológico , Rutaceae , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Cumarínicos/química , Cumarínicos/isolamento & purificação , Testes de Sensibilidade Parasitária , Extratos Vegetais/farmacologiaRESUMO
Several natural compounds have been identified for the treatment of leishmaniasis. Among them are some alkaloids, chalcones, lactones, tetralones, and saponins. The new compound reported here, 7-geranyloxycoumarin, called aurapten, belongs to the chemical class of the coumarins and has a molecular weight of 298.37. The compound was extracted from the Rutaceae species Esenbeckia febrifuga and was purified from a hexane extract starting from 407.7 g of dried leaves and followed by four silica gel chromatographic fractionation steps using different solvents as the mobile phase. The resulting compound (47 mg) of shows significant growth inhibition with an LD50 of 30 microM against the tropical parasite Leishmania major, which causes severe clinical manifestations in humans and is endemic in the tropical and subtropical regions. In the present study, we investigated the atomic structure of aurapten in order to determine the existence of common structural motifs that might be related to other coumarins and potentially to other identified inhibitors of Leishmania growth and viability. This compound has a comparable inhibitory activity of other isolated molecules. The aurapten is a planar molecule constituted of an aromatic system with electron delocalization. A hydrophobic side chain consisting of ten carbon atoms with two double bonds and negative density has been identified and may be relevant for further compound synthesis.
Assuntos
Antiprotozoários/farmacologia , Cumarínicos/farmacologia , Leishmaniose/tratamento farmacológico , Rutaceae , Animais , Antiprotozoários/química , Antiprotozoários/isolamento & purificação , Cumarínicos/química , Cumarínicos/isolamento & purificação , Testes de Sensibilidade Parasitária , Extratos Vegetais/farmacologiaRESUMO
The three-dimensional structure of Leishmania tarentolae adenine phosphoribosyltransferase (APRT) in complex with adenosine-5-monophosphate (AMP) and a phosphate ion has been solved. Refinement against X-ray diffraction data extending to 2.2-A resolution led to a final crystallographic R factor of 18.3%. Structural comparisons amongst this APRT enzyme and other 'type I' PRTases whose structures have been determined reveal several important features of the PRTases catalytic mechanism. Based on structural superpositions and molecular interaction potential calculations, it was possible to suggest that the PRPP is the first substrate to bind, while the AMP is the last product to leave the active site, in accordance to recent kinetic studies performed with the Leishmania donovani APRT.
Assuntos
Adenina Fosforribosiltransferase/química , Leishmania/enzimologia , Adenina Fosforribosiltransferase/biossíntese , Adenina Fosforribosiltransferase/isolamento & purificação , Monofosfato de Adenosina/química , Animais , Sítios de Ligação , Cátions Bivalentes , Magnésio/química , Modelos Moleculares , Fosforribosil Pirofosfato/química , Difração de Raios XRESUMO
The second enzyme in the glycolytic pathway, phosphoglucose isomerase (PGI), catalyses an intracellular aldose-ketose isomerization. Here we describe the human recombinant PGI structure (hPGI) solved in the absence of active site ligands. Crystals isomorphous to those previously reported were used to collect a 94% complete data set to a limiting resolution of 2.1 A. From the comparison between the free active site hPGI structure and the available human and rabbit PGI (rPGI) structures, a mechanism for protein initial catalytic steps is proposed. Binding of the phosphate moiety of the substrate to two distinct elements of the active site is responsible for driving a series of structural changes resulting in the polarisation of the active site histidine, priming it for the initial ring-opening step of catalysis.
Assuntos
Glucose-6-Fosfato Isomerase/química , Sítios de Ligação , Catálise , Cristalização , Frutosefosfatos/metabolismo , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Difração de Raios XRESUMO
Plants possess several defense mechanisms against pathogenic attack. One of these defenses is the use of protease inhibitor proteins, which interfere in the development and growth of pathogens. Sugarcane productivity can be impacted by the plant's susceptibility to fungal diseases that result in production losses. A relevant line of investigation, therefore, is into the plant's natural defense mechanisms for the control of phytopathogens using cystatins-proteins that specifically inhibit cysteine proteases. In this paper, we discuss the expression, in Escherichia coli, of a sugarcane cystatin, its purification, antifungal activity, and circular dichroism to monitor correct folding. These studies revealed a secondary structure similar to that of the oryzacystatin I of rice. Moreover, the purified protein proved capable of inhibiting the growth of the filamentous fungus Trichoderma reesei, suggesting that it can also be employed to inhibit the growth of pathogenic sugarcane fungi.
Assuntos
Antifúngicos , Cistatinas , Inibidores de Cisteína Proteinase , Proteínas de Plantas , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Dicroísmo Circular , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , Cistatinas/farmacologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Trichoderma/citologia , Trichoderma/efeitos dos fármacos , Trichoderma/crescimento & desenvolvimentoRESUMO
Phosphoglucose isomerase (PGI) is the second enzyme in the glycolytic pathway and catalyzes an aldose-ketose isomerization. Outside the cell, PGI has been found to function as both a cytokine and as a growth factor. The human pgi gene was cloned and the expressed enzyme was purified to homogeneity. Isomorphous crystals were obtained under two conditions and belong to the P2(1)2(1)2(1) space group, with unit-cell parameters a = 80.37, b = 107.54, c = 270.33 A. A 94.7% complete data set was obtained and processed to a limiting resolution of 2.6 A. The asymmetric unit contains two hPGI dimers according to density calculations, a self-rotation function map and molecular-replacement solution.