RESUMO

BACKGROUND AND AIMS: Considering the lack of knowledge regarding the influence of the variable number of repeats of 27 pb in intron 4 (4b/4a VNTR - rs61722009) of the endothelial nitric oxide synthase (eNOS) on the drug response, we assessed the influence of this polymorphism for the risk of upper gastrointestinal bleeding (UGIB). METHODS: A case-control study, including 200 cases and 706 controls, was conducted in a Brazilian hospital complex. Cases were participants with UGIB diagnosis. Controls were participants admitted to surgical procedures not related to gastrointestinal problems. The 4b/4a VNTR was determined through polymerase chain reaction followed by fragment analysis. Conditional logistic regression models were designed. The additive interaction between the presence of the 4b/4a VNTR variant and the use of low-dose aspirin (LDA) and nonsteroidal anti-inflammatory drugs (NSAIDs) was calculated by fitting the Cox regression model through the parameters of Synergism index (S) and Relative Excess Risk Due To Interaction (RERI). RESULTS: The presence of the 4b/4a VNTR variant did not increase the risk of UGIB: carriers of the 4a/4a genotype (OR=0.37, 95%CI: 0.09-1.45) and of the variant allele "4a" (OR=0.91, 95%CI: 0.55-1.51). The risk of UGIB in LDA users carriers of the wild genotype (OR=4.96, 95%CI: 2.04- 2.06) and the variant allele "4a" (OR=3.49, 95%CI: 1.18-10.38) is similar, as well as for NSAID users carriers of the wild genotype (OR=5.73, 95%CI: 2.61-12.60) and variant allele "4a" (OR=5.51, 95%CI: 1.42-15.82). No additive interaction was identified between the presence of the genetic variant and the use of LDA [RERI: -1.44 (95%CI: -6.02-3.14; S: 0.63 (95%CI: -1.97-1.15)] and NSAIDs [RERI: -0.13 (95%CI: -6.79-6.53; S: 0.97 (95%CI: -0.23-4.19)] on the UGIB risk. CONCLUSION: Our data suggests that there is no increase in the magnitude of UGIB risk in LDA and NSAIDs users' carrying the variant allele "4a".

Assuntos

Hemorragia Gastrointestinal , Íntrons , Óxido Nítrico Sintase Tipo III , Proteínas de Transporte de Nucleotídeos , Anti-Inflamatórios não Esteroides/administração & dosagem , Aspirina/administração & dosagem , Estudos de Casos e Controles , Hemorragia Gastrointestinal/induzido quimicamente , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/genética , Predisposição Genética para Doença , Genótipo , Humanos , Repetições Minissatélites , Óxido Nítrico Sintase Tipo III/genética , Proteínas de Transporte de Nucleotídeos/genética , Polimorfismo Genético

RESUMO

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and ß-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

Assuntos

Alquil e Aril Transferases/metabolismo , Maytenus/enzimologia , Proteínas de Plantas/metabolismo , Triterpenos/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Substituição de Aminoácidos , Vias Biossintéticas , Ciclização , Genes de Plantas , Leucina/química , Maytenus/genética , Metionina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/biossíntese , Triterpenos Pentacíclicos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato

RESUMO

OBJECTIVES: The aim of this study was to evaluate the effect of specific inhibition of MMP-13 on inflammation and inflammatory bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontitis. MATERIALS AND METHODS: Periodontitis was induced in mice by micro-injections of LPS into the gingival tissues adjacent to the palatal surfaces of maxillary molars twice a week for 15 days. Matrix metalloproteinase-13 (Mmp-13) shRNA or a specific biochemical inhibitor were also injected into the same sites in alternating days with the LPS injections. Efficacy of shRNA-mediated silencing of Mmp-13 was verified by quantitative real-time polymerase chain reaction (qPCR) and immunoblot. Bone resorption was assessed by microcomputed tomography (uCT). Histological sections stained with hematoxylin/eosin (H/E) were used in the stereometric analysis of the inflammatory infiltrate. Gingival tissues were used to evaluate expression of Mmp-13, Il-6, Tnf-α, Ptgs2, and Rankl (qPCR). Protein levels of TGF-ß and IL-10 in the tissues were determined by enzyme-linked immunosorbent assays (ELISA) or by MMP-13 and p38 immunoblot. RESULTS: Silencing Mmp-13 expression reduced bone resorption significantly. Expression of Mmp-13, Il-6, and Tnf-α, as well as the protein levels of IL-6 and TNF-α, was reduced in the animals treated with adenovirus-delivered shRNA; however, these effects were not associated with modulation of p38 MAPK signaling. Interestingly, inhibition Mmp-13 did not affect the severity of inflammatory infiltrate. CONCLUSIONS: Site-specific inhibition of MMP-13 reduced bone resorption and production of inflammatory mediators associated with periodontal disease. CLINICAL RELEVANCE: The results suggest that site-specific inhibition of MMP-13 may be an interesting strategy to modulate inflammation and reduce bone resorption in osteolytic inflammatory diseases.

Assuntos

Reabsorção Óssea , Doenças Periodontais , Animais , Lipopolissacarídeos , Metaloproteinase 13 da Matriz/genética , Camundongos , Microtomografia por Raio-X

RESUMO

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

Assuntos

Anti-Helmínticos/farmacologia , Antiprotozoários/farmacologia , Brugia Malayi/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Helminto/antagonistas & inibidores , Leishmania major/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anti-Helmínticos/química , Antiprotozoários/química , Brugia Malayi/enzimologia , Brugia Malayi/genética , Brugia Malayi/crescimento & desenvolvimento , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Ensaios de Triagem em Larga Escala , Leishmania major/enzimologia , Leishmania major/genética , Leishmania major/crescimento & desenvolvimento , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência

RESUMO

Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.

Assuntos

Liases/metabolismo , Fosfotransferases/metabolismo , Selenocisteína/biossíntese , Selenoproteínas/metabolismo , Trypanosoma brucei brucei/enzimologia , Conformação Proteica , Proteínas de Protozoários/metabolismo , Selênio/metabolismo

RESUMO

Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite’s ER stress response.

RESUMO

OBJECTIVE: The aim of this study was to investigate the functionality of ATC/TTC (Hap-1) and ATT/TTC (Hap-2) Interleukin (IL) 8 gene haplotypes in the response of neutrophils to Gram-negative bacteria associated with periodontitis. DESIGN: Neutrophils were isolated by gradient centrifugation from whole peripheral blood of systemically healthy individuals presenting the two IL8 gene haplotypes. Neutrophils were stimulated with P. gingivalis, A. actinomycetemcomitans and PMA/ionomycin. Cytokine gene expression (RT-qPCR) and migration/chemotaxis (boyden chamber assay) were compared according to the presence of Hap-1 or Hap-2 haplotypes. Protein production was also evaluted in the multiplex assay using the mixed population of leukocytes present in the whole blood from the same individuals. The influence of these two haplotypes on the IL8 promoter activity was assessed in gene-reporter experiments. RESULTS: Hap-1 haplotype in neutrophils and leukocytes exacerbated the response to stimulation with Gram-negative bacteria, with higher levels of TNF-α (mRNA and protein), IL-1ß, IL-2R and IFN-γ (protein) and with increased chemotaxis. Presence of the T allele at the rs4071 polymorphism (alias -251) was associated with increased activity of IL8 proximal promoter. CONCLUSIONS: Neutrophils and leukocytes carrying the Hap-1 haplotype (ATC/TTC) in the IL8 gene present an enhanced response to stimulation with Gram-negative bacteria associated with periodontitis. Presence of the T allele (rs4073) in the IL8 proximal promoter increases transcription activity.

Assuntos

Bactérias Gram-Negativas , Interleucina-8/genética , Neutrófilos/imunologia , Periodontite/genética , Citocinas/imunologia , Predisposição Genética para Doença , Haplótipos , Humanos , Periodontite/microbiologia , Projetos Piloto , Regiões Promotoras Genéticas

RESUMO

Green fluorescent protein (GFP) is a globular protein used as biosensor and biomarker in medical and industrial fields. However, due to the expensive production costs of expressing proteins using high-cost inducers like isopropyl-ß-d-1-thiogalactopyranoside (IPTG), the number of GFP applications are still scarce. This work studied the production of enhanced GFP (EGFP) using Escherichia coli BL21 (DE3) [pLysS; pET28(a)], aiming to increase its yield and reduce costs. First, the influence of agitation rate, induction time, and concentration of IPTG in the production of EGFP was evaluated, but only the first two parameters were significant. Subsequently, aiming to reduce costs related to the use of inducer, the IPTG concentration (0.005, 0.010, and 0.025 mM) was decreased and, interestingly, the production levels were maintained or increased. These results show that a proper choice of production conditions, particularly through the decrease of inducer concentration, is effective to reduce the upstream production costs and guarantee high EGFP expression.

Assuntos

Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/economia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/economia , Proteínas Recombinantes/genética

RESUMO

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

Assuntos

Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Peptídeos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Inativação Gênica , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Fatores de Iniciação de Peptídeos/genética , Fosforilação , Reação em Cadeia da Polimerase , Ligação Proteica , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA/genética , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A

RESUMO

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2?Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

RESUMO

A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.

Assuntos

Expressão Gênica , Engenharia Metabólica , Peptídeos/genética , Saccharomyces cerevisiae/genética , Vírus/genética , Vetores Genéticos , Maytenus/enzimologia , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Triterpenos/metabolismo , Proteínas Virais/genética

RESUMO

Triterpenes are interesting compounds because they play an important role in cell homeostasis and a wide variety exhibiting defense functions is produced by plant secondary metabolism. Those same plant secondary metabolites also exhibit biological properties with promising therapeutic potential as anti-inflammatory and antitumor agents. Friedelin is a triterpene ketone with anti-inflammatory and gastroprotective activities and it is a precursor of relevant antitumor quinonemethides. Although many triterpene synthases have been described, only two friedelin synthases were characterized and there is no information about their genomic features and alleles. In the present work, we aimed to identify the gene and new isoforms of friedelin synthase in Maytenus ilicifolia leaves to be functionally characterized in Saccharomyces cerevisiae. The gene sequence analysis elucidated the exon/intron structure and confirmed the presence of single nucleotide polymorphisms with four non-synonymous mutations outside the active site of the enzyme. Therefore, two new isoforms were observed and the heterologous production of the enzymes in yeast showed similar production of friedelin. This first description of different alleles of the gene of friedelin synthase in M. ilicifolia can guide their validation as markers for friedelin-producer specimens.

Assuntos

Maytenus/enzimologia , Oxirredutases/metabolismo , Triterpenos/metabolismo , Sequência de Aminoácidos , Éxons/genética , Genes de Plantas , Íntrons/genética , Isoenzimas/metabolismo , Maytenus/genética , Fases de Leitura Aberta/genética , Oxirredutases/química , Oxirredutases/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Triterpenos/química

RESUMO

BACKGROUND: Some probiotic strains have the potential to assist in relieving the symptoms of inflammatory bowel disease. The impact of daily ingestion of a soy-based product fermented by Enterococcus faecium CRL 183 and Lactobacillus helveticus 416 with the addition of Bifidobacterium longum ATCC 15707 on chemically induced colitis has been investigated thereof within a period of 30 days. METHODS: Colitis was induced by dextran sulfate sodium. The animals were randomly assigned into five groups: Group C: negative control; Group CL: positive control; Group CLF: DSS with the fermented product; Group CLP: DSS with the non-fermented product (placebo); Group CLS: DSS with sulfasalazine. The following parameters were monitored: disease activity index, fecal microbial analyses, gastrointestinal survival of probiotic microorganisms and short-chain fatty acids concentration in the feces. At the end of the protocol the animals' colons were removed so as to conduct a macroscopical and histopathological analysis, cytokines and nitrite quantification. RESULTS: Animals belonging to the CLF group showed fewer symptoms of colitis during the induction period and a lower degree of inflammation and ulceration in their colon compared to the CL, CLS and CLP groups (p<0.05). The colon of the animals in groups CL and CLS presented severe crypt damage, which was absent in CLF and CLP groups. A significant increase in the population of Lactobacillus spp. and Bifidobacterium spp. at the end of the protocol was verified only in the CLF animals (p<0.05). This group also showed an increase in short-chain fatty acids (propionate and acetate). Furthermore, the intestinal survival of E. faecium CRL 183 and B. longum ATCC 15707 in the CLF group has been confirmed by biochemical and molecular analyzes. CONCLUSIONS: The obtained results suggest that a regular intake of the probiotic product, and placebo to a lesser extent, can reduce the severity of DSS-induced colitis on rats.

Assuntos

Bifidobacterium longum , Colite/tratamento farmacológico , Enterococcus faecium , Fezes/microbiologia , Intestinos/microbiologia , Probióticos/uso terapêutico , Animais , Bebidas , Colite/induzido quimicamente , Colite/microbiologia , Sulfato de Dextrana , Modelos Animais de Doenças , Ácidos Graxos Voláteis/análise , Fezes/química , Ratos , Resultado do Tratamento

RESUMO

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to ß-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

Assuntos

Transferases Intramoleculares/metabolismo , Maytenus/enzimologia , Proteínas de Plantas/metabolismo , Triterpenos/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Transferases Intramoleculares/química , Transferases Intramoleculares/classificação , Transferases Intramoleculares/genética , Leucina/química , Leucina/metabolismo , Maytenus/genética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Alinhamento de Sequência , Triterpenos/análise , Triterpenos/química

RESUMO

The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A-4, tif51A-6, tif51A-7 and tif51A-11, and complementation analysis revealed that tif51A-4 and tif51A-7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A-4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A-7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A-4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A-7. Taking into account the modeling eIF5A + 80S + P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A-4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype.

Assuntos

Mutagênese , Fatores de Iniciação de Peptídeos , Proteínas de Ligação a RNA , Proteínas Ribossômicas , Ribossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A

RESUMO

BACKGROUND: Cardiovascular disease is the leading cause of worldwide morbidity and mortality. Several studies have demonstrated that specific probiotics affect the host's metabolism and may influence the cardiovascular disease risk. OBJECTIVES: The aim of this study was to investigate the influence of an isoflavone-supplemented soy product fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416 on cardiovascular risk markers in moderately hypercholesterolemic subjects. DESIGN: Randomized placebo-controlled double-blind trial Setting: São Paulo State University in Araraquara, SP, Brazil. PARTICIPANTS: 49 male healthy men with total cholesterol (TC) >5.17 mmol/L and <6.21 mmol/L Intervention: The volunteers have consumed 200 mL of the probiotic soy product (group SP-10(10) CFU/day), isoflavone-supplemented probiotic soy product (group ISP-probiotic plus 50 mg of total isoflavones/100 g) or unfermented soy product (group USP-placebo) for 42 days in a randomized, double-blind study. MAIN OUTCOME MEASURES: Lipid profile and additional cardiovascular biomarkers were analyzed on days 0, 30 and 42. Urine samples (24 h) were collected at baseline and at the end of the experiment so as to determine the isoflavones profile. RESULTS: After 42 days, the ISP consumption led to improved total cholesterol, non-HDL-C (LDL + IDL + VLDL cholesterol fractions) and electronegative LDL concentrations (reduction of 13.8%, 14.7% and 24.2%, respectively, p < 0.05). The ISP and SP have prevented the reduction of HDL-C level after 42 days. The C-reactive protein and fibrinogen levels were not improved. The equol production by the ISP group subjects was inversely correlated with electronegative LDL concentration. CONCLUSIONS: The results suggest that a regular consumption of this probiotic soy product, supplemented with isoflavones, could contribute to reducing the risk of cardiovascular diseases in moderately hypercholesterolemic men, through the an improvement in lipid profile and antioxidant properties.

Assuntos

Suplementos Nutricionais , Hipercolesterolemia/dietoterapia , Isoflavonas/administração & dosagem , Lipídeos/sangue , Probióticos/administração & dosagem , Alimentos de Soja/microbiologia , Adulto , Antioxidantes/administração & dosagem , Biomarcadores/sangue , Biomarcadores/urina , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/prevenção & controle , Colesterol/sangue , HDL-Colesterol/efeitos dos fármacos , LDL-Colesterol/efeitos dos fármacos , Método Duplo-Cego , Voluntários Saudáveis , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/complicações , Isoflavonas/urina , Masculino , Pessoa de Meia-Idade , Fatores de Risco

RESUMO

BACKGROUND: 14-3-3 proteins comprise a family of eukaryotic multifunctional proteins involved in several cellular processes. The Pb14-3-3 of Paracoccidioides brasiliensis seems to play an important role in the Paracoccidioides-host interaction. Paracoccidioides brasiliensis is an etiological agent of paracoccidioidomycosis, which is a systemic mycosis that is endemic in Latin America. In the initial steps of the infection, Paracoccidioides spp. synthetizes adhesins that allow it to adhere and invade host cells. Therefore, the aim of this work was to perform a functional analysis of Pb14-3-3 using Saccharomyces cerevisiae as a model. RESULTS: The functional analysis of Pb14-3-3 was performed in S. cerevisiae, and it was found that Pb14-3-3 partially complemented S. cerevisiae proteins Bmh1p and Bmh2p, which are recognized as two yeast 14-3-3 homologues. When we evaluated the adhesion profile of S. cerevisiae transformants, Pb14-3-3 acted as an adhesin in S. cerevisiae; however, Bmh1p did not show this function. The influence of Pb14-3-3 in S. cerevisiae ergosterol pathway was also evaluated and our results showed that Pb14-3-3 up-regulates genes involved in ergosterol biosynthesis. CONCLUSIONS: Our data showed that Pb14-3-3 was able to partially complement Bmh1p and Bmh2p proteins in S. cerevisiae; however, we suggest that Pb14-3-3 has a differential role as an adhesin. In addition, Pb-14-3-3 may be involved in Paracoccidioides spp. ergosterol biosynthesis which makes it an interest as a therapeutic target.

Assuntos

Proteínas 14-3-3/metabolismo , Proteínas Fúngicas/metabolismo , Paracoccidioides/genética , Saccharomyces cerevisiae/genética , Proteínas 14-3-3/genética , Clonagem Molecular , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Paracoccidioides/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMO

Translational control is extremely important in all organisms, and some of its aspects are highly conserved among all primary kingdoms, such as those related to the translation elongation step. The previously classified translation initiation factor 5A (eIF5A) and its bacterial homologue elongation factor P (EF-P) were discovered in the late 70's and have recently been the object of many studies. eIF5A and EF-P are the only cellular proteins that undergo hypusination and lysinylation, respectively, both of which are unique posttranslational modifications. Herein, we review all the important discoveries related to the biochemical and functional characterization of these factors, highlighting the implication of eIF5A in translation elongation instead of initiation. The findings that eIF5A and EF-P are important for specific cellular processes and play a role in the relief of ribosome stalling caused by specific amino acid sequences, such as those containing prolines reinforce the hypothesis that these factors are involved in specialized translation. Although there are some divergences between these unique factors, recent studies have clarified that they act similarly during protein synthesis. Further studies may reveal their precise mechanism of ribosome activity modulation as well as the mRNA targets that require eIF5A and EF-P for their proper translation.

Assuntos

Elongação Traducional da Cadeia Peptídica/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Modificação Traducional de Proteínas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Animais , Humanos , Fatores de Alongamento de Peptídeos/genética , Fatores de Iniciação de Peptídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Fator de Iniciação de Tradução Eucariótico 5A

RESUMO

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved and essential protein present in all organisms except bacteria. To be activated, eIF5A requires the conversion of a specific residue of lysine into hypusine. This hypusine modification occurs posttranslationally in two enzymatic steps, and the polyamine spermidine is the substrate. Despite having an essential function in translation elongation, the critical role played by eIF5A remains unclear. In addition to demonstrating genetic interactions with translation factors, eIF5A mutants genetically interact with mutations in YPT1, which encodes an essential protein involved in endoplasmic reticulum (ER)-to-Golgi vesicle transport. In this study, we investigated the correlation between the function of eIF5A in translation and secretion in yeast. The results of in vivo translocation assays and genetic interaction analyses suggest a specific role for eIF5A in the cotranslational translocation of proteins into the ER, but not in the posttranslational pathway. Additionally, we observed that a block in eIF5A activation up-regulates stress-induced chaperones, which also occurs when SRP function is lost. Finally, loss of eIF5A function affects binding of the ribosome-nascent chain complex to SRP. These results link eIF5A function in translation with a role of SRP in the cell and may help explain the dual effects of eIF5A in differential and general translation.

Assuntos

Retículo Endoplasmático/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Fator de Iniciação de Tradução Eucariótico 5A

RESUMO

BACKGROUND: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. METHODS: To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. RESULTS: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. CONCLUSIONS: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication.

Assuntos

Fator de Iniciação 3 em Eucariotos/metabolismo , Interações Hospedeiro-Patógeno , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Vírus da Febre Amarela/fisiologia , Análise Mutacional de DNA , Humanos , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido