RESUMO
Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at -70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts.
Assuntos
Cisticercose/genética , Interações Hospedeiro-Parasita , Taenia solium/genética , Tiorredoxinas/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cisticercose/patologia , Cisticercose/veterinária , Humanos , Estrutura Secundária de Proteína , Suínos , Taenia solium/patogenicidade , Tiorredoxinas/biossíntese , Tiorredoxinas/químicaRESUMO
Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide ((â¢)NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO(-) + ONOOH) formation, a strong oxidant arising from the reaction of (â¢)NO with superoxide radical (O(2)(-)). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O(2)(-) during a 60-90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained (â¢)NO production (â¼24 h). The major mechanism for infection control via reactive species formation occurred when (â¢)NO and O(2)() were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.
Assuntos
Citotoxinas/metabolismo , Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Peroxinitroso/metabolismo , Proteínas de Protozoários/biossíntese , Tiorredoxinas/biossíntese , Trypanosoma cruzi/enzimologia , Animais , Linhagem Celular , Doença de Chagas/enzimologia , Macrófagos/parasitologia , Macrófagos/ultraestrutura , Camundongos , Óxido Nítrico/metabolismo , Oxirredução , Superóxidos/metabolismo , Trypanosoma cruzi/ultraestruturaRESUMO
Insulin-like growth factor-I (IGF-I) and thioredoxin are regulated by gonadal steroids in the female reproductive tract of many species. Oestradiol regulates IGF-I and thioredoxin mRNA levels in the reproductive tract of prepubertal lambs. The physiological status (different endocrine environment) may affect the sensitivity of the reproductive tract to oestradiol and progesterone. We studied the effects of different endocrine milieus (late-follicular and luteal phases of the oestrous cycle, and ovariectomy before or after puberty) on the expression of IGF-I, thioredoxin, oestrogen receptor alpha (ER alpha) and progesterone receptor (PR) in sheep. The mRNA levels were determined by a solution hybridisation technique. In the uterus the levels of ER alpha, PR and thioredoxin mRNA were higher in the late-follicular phase group than in the other three groups, and IGF-I mRNA was high during both the late-follicular and the luteal phases. In the cervix only PR mRNA was significantly higher in the ewes in the late-follicular phase than in the other groups. In the oviducts the levels of thioredoxin and ER alpha mRNA were highest in the ovariectomised adult ewes, and thioredoxin mRNA was higher than the levels found in the ewes in the late-follicular phase. The IGF-I mRNA levels in the oviduct did not differ between any of the groups. The transcripts of IGF-I, thioredoxin, ER alpha and PR, varied according to the physiological status and also along the female reproductive tract, suggesting that the regulation of the mRNA levels of these factors by the steroid environment is tissue specific.
Assuntos
Ciclo Estral/fisiologia , Expressão Gênica/fisiologia , Fator de Crescimento Insulin-Like I/biossíntese , Ovinos/fisiologia , Tiorredoxinas/biossíntese , Animais , Peso Corporal , Estradiol/sangue , Feminino , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/genética , Tamanho do Órgão/fisiologia , Ovariectomia/veterinária , Progesterona/sangue , RNA Mensageiro/análise , Receptores de Estrogênio/análise , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Progesterona/análise , Receptores de Progesterona/biossíntese , Receptores de Progesterona/genética , Tiorredoxinas/análise , Tiorredoxinas/genética , Útero/fisiologiaRESUMO
The participation of thiol-oxidoreductases such as thioredoxin during implantation, embryogenesis and fetal development has been extensively studied. Here, we analyzed the expression of the thioredoxin superfamily enzyme quiescin Q6/sulfhydryl oxidase (QSOX) during development. Results show that QSOX is present in fetal bovine serum (4 months' gestation), but its levels decrease with time after birth (from P1 to P60). We also demonstrate that a sulfhydryl oxidase activity correlates with QSOX expression in such sera, suggesting a putative role in the redox modulation of developmental programs.
Assuntos
Sangue Fetal/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Tiorredoxinas/biossíntese , Animais , Bovinos , Oxirredução , Oxirredutases/biossíntese , Fatores de TempoRESUMO
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
Assuntos
Proteínas de Ligação a DNA/química , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas de Plantas/química , Tiorredoxinas/química , Fatores de Transcrição/química , Proteínas de Ligação a DNA/biossíntese , Proteínas Fúngicas/biossíntese , Zíper de Leucina , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/biossíntese , Rifampina/química , Rifampina/farmacologia , Tiorredoxinas/biossíntese , Fatores de Transcrição/biossínteseRESUMO
An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein ( approximately 62 kDa) consisting of 112 amino acids of thioredoxin and approximately 450 amino acids of fragment C. This fusion protein was recognized by anti-tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment-C-thioredoxin protein was purified significantly in one step by Ni(2+)-chelate Sepharose, the final yield being approximately 35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene-fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host.