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-XRESUMO
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 SubstratoRESUMO
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 5ARESUMO
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/genéticaRESUMO
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/genéticaRESUMO
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/químicaRESUMO
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 5ARESUMO
Mx proteins are evolutionarily conserved dynamin-like large GTPases involved in viral resistance triggered by types I and III interferons. The human MxA is a cytoplasmic protein that confers resistance to a large number of viruses. The MxA protein is also known to self-assembly into high molecular weight homo-oligomers. Using a yeast two-hybrid screen, we identified 27 MxA binding partners, some of which are related to the SUMOylation machinery. The interaction of MxA with Small-Ubiquitin MOdifier 1 (SUMO1) and Ubiquitin conjugating enzyme 9 (Ubc9) was confirmed by co-immunoprecipitation and co-localization by confocal microscopy. We identified one SUMO conjugation site at lysine 48 and two putative SUMO interacting motifs (SIMa and SIMb). We showed that MxA interacts with the EIL loop of SUMO1 in a SIM-independent manner via its CID-GED domain. The yeast two-hybrid mapping also revealed that Ubc9 binds to the MxA GTPase domain. Mutation in the putative SIMa and SIMb, which are located in the GTPase binding domain, reduced MxA antiviral activity. In addition, we showed that MxA can be conjugated to SUMO2 or SUMO3 at lysine 48 and that the SUMOylation-deficient mutant of MxA (MxAK48R) retained its capacity to oligomerize and to inhibit Vesicular Stomatitis Virus (VSV) and Influenza A Virus replication, suggesting that MxA SUMOylation is not essential for its antiviral activity.
Assuntos
Proteínas de Resistência a Myxovirus/metabolismo , Sumoilação , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células HeLa , Humanos , Camundongos , Proteínas de Resistência a Myxovirus/química , Células NIH 3T3 , Ligação Proteica , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
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/metabolismoRESUMO
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 5ARESUMO
OBJECTIVE: Forgerini et al. investigated the role of seven genetic variants in the risk of upper gastrointestinal bleeding as an adverse drug reaction. In 289 participants (50 cases and 189 controls), the presence of seven variants in the IL-1ß, IL-1RN, and TNF-α genes was not associated with susceptibility to acetylsalicylic acid-induced upper gastrointestinal bleeding. The use of acetylsalicylic acid, even in low doses, may be associated with the onset of upper gastrointestinal bleeding as an idiosyncratic response. Considering the role of the genetic background in inter-individual responses to pharmacotherapy, we aimed to investigate the role of seven variants in the TNF-α, IL-ß, and IL-1RN genes in association with the risk of upper gastrointestinal bleeding in users of low-dose acetylsalicylic acid for the prevention of cardiovascular events. METHODS: A case-control study was conducted in a Brazilian hospital complex. The Case Group comprised patients diagnosed with upper gastrointestinal bleeding who were administered a low dose of acetylsalicylic acid (n=50). Two Control Groups were recruited: 1) low-dose acetylsalicylic acid users without gastrointestinal complaints and under the supervision of a cardiologist (n=50) and 2) healthy controls (n=189). Sociodemographic, clinical, pharmacotherapeutic, and lifestyle data were recorded through face-to-face interviews. Genomic DNA from all participants was genotyped for rs16944 and rs1143634 (IL-ß gene), rs4251961 (IL-1RN gene), and rs1799964, rs1799724, rs361525, and rs1800629 (TNF-α gene). RESULTS: No significant difference was noted in the genotypic frequencies of TNF-α, IL-ß, and IL-1RN variants between the Case and Control Groups of low-dose acetylsalicylic acid users (p>0.05). The frequency of rs1800629 genotypes (TNF-α gene) differed significantly between the Case Group and healthy controls (p=0.003). None of the evaluated variants were associated with a risk of upper gastrointestinal bleeding. CONCLUSION: This study aimed to explore pharmacogenomics biomarkers in low-dose acetylsalicylic acid users. Our data suggest that the presence of IL-1ß, IL-1RN, and TNF-α variants was not associated with an increased risk of upper gastrointestinal bleeding.
Assuntos
Aspirina , Hemorragia Gastrointestinal , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1beta , Fator de Necrose Tumoral alfa , Humanos , Masculino , Estudos de Casos e Controles , Feminino , Aspirina/efeitos adversos , Fator de Necrose Tumoral alfa/genética , Interleucina-1beta/genética , Hemorragia Gastrointestinal/induzido quimicamente , Hemorragia Gastrointestinal/genética , Pessoa de Meia-Idade , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/efeitos adversos , Idoso , Fatores de Risco , Genótipo , Predisposição Genética para Doença/genética , Adulto , Polimorfismo de Nucleotídeo ÚnicoRESUMO
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-HíbridoRESUMO
The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNA(Phe) tertiary structure, analogously to the EF-P monomer.
Assuntos
Fatores de Alongamento de Peptídeos/química , Fatores de Iniciação de Peptídeos/química , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Dimerização , Humanos , Modelos Moleculares , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers - alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor) - and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)ß was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERß expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERß, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus.
Assuntos
Receptor beta de Estrogênio/metabolismo , Rana catesbeiana/fisiologia , Receptores Androgênicos/metabolismo , Globulina de Ligação a Hormônio Sexual/biossíntese , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Globulina de Ligação a Hormônio Sexual/metabolismo , Espermatogênese , Espermatogônias/citologia , Células-Tronco/citologiaRESUMO
Bullfrog stem spermatogonia, also named primordial germ cells (PGCs), show strong testosterone immunolabeling in winter, but no or weak testosterone immunoexpression in summer. Thus, the role of testosterone in these cells needs to be clarified. In this study, we proposed to evaluate whether PGCs express aromatase and estrogen receptors, and verify a possible role of estrogen in PGCs seasonal proliferation. Testes of male adult bullfrogs, collected in winter (WG) and summer (SG), were fixed and embedded in historesin, for quantitative analysis, or paraffin for immunohistochemistry (IHC). The number of haematoxylin/eosin stained PGCs/lobular area was obtained. Proliferating cell nuclear antigen (PCNA), aromatase, estrogen receptor ß (ERß) and PCNA/ERß double immunolabeling were detected by IHC. The number of PCNA-positive PGCs and the histological score (HSCORE) of aromatase and ERß immunolabeled PGCs were obtained. Although the number of PGCs increased significantly in WG, a high number of PCNA-positive PGCs was observed in summer. Moreover, aromatase and ERß HSCORE was higher in SG than WG. The results indicate that PGCs express a seasonal proliferative activity; the low mitotic activity in winter is related to the maximal limit of germ cells which can be supported in the large lobules. In SG, the increased ERß and aromatase HSCORE suggests that testosterone is converted into estrogen from winter to summer. Moreover, the parallelism between the high PGCs mitotic activity and ERß immunoexpression suggest a participation of estrogen in the control of the PGCs seasonal proliferative activity which guarantee the formation of new germ cysts from summer to next autumn.
Assuntos
Aromatase/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Rana catesbeiana/metabolismo , Espermatogônias/metabolismo , Animais , Imuno-Histoquímica , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Testosterona/metabolismoRESUMO
The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.
Assuntos
Núcleo Celular/metabolismo , Desenvolvimento Muscular , Proteína II de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , RNA Mensageiro/biossíntese , Animais , Diferenciação Celular , Proliferação de Células , Camundongos , Camundongos Endogâmicos BALB C , Mioblastos/citologia , Mioblastos/metabolismo , Poli A/metabolismo , Poliadenilação , Transporte de RNARESUMO
BACKGROUND: Recently, attenuation of anti-inflammatory and increase of pro-inflammatory mediators was demonstrated in individuals with Down syndrome (DS) in comparison with euploid patients during periodontal disease (PD), suggesting a shift to a more aggressive inflammation in DS. AIM: To determine the influence of DS in the modulation of interferons (IFNs) signaling pathway in PD. MATERIALS AND METHODS: Clinical periodontal assessment was performed and gingival tissue samples obtained from a total of 51 subjects, including 19 DS individuals with PD, 20 euploid individuals with PD and 12 euploid individuals without PD. Expression levels of interferon-gamma (IFNG) and interferon-alpha (IFNA), and their receptors IFNGR1, IFNGR2, IFNAR1 and IFNAR2, the signaling intermediates Janus kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1) were determined using real time quantitative polymerase chain reaction (qPCR). RESULTS: Clinical signs of periodontal disease were markedly more severe in DS and euploid patients with PD in comparison to euploid and periodontally healthy patients. There was no difference on mRNA levels of IFNA, IFNG, INFGR2, IFNAR1 and IFNAR2 between DS and euploid individuals, even though some of these genes are located on chromosome 21. STAT1 and IRF1 mRNA levels were significantly lower in DS patients in comparison with euploid individuals with PD. In euploid individuals, PD was associated with an increased expression of IFNGR1, IFNGR2, IFNAR1, STAT1 and IRF1. CONCLUSIONS: Reduced expression of STAT1 and IRF1 genes indicate an impaired activation of IFNs signaling in individuals with DS and PD. Expression of IFNA, IFNG and IFN receptors was not altered in DS patients, indicating that indirect mechanisms are involved in the reduced activation of IFN signaling.
Assuntos
Síndrome de Down/genética , Regulação da Expressão Gênica , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Periodontite/genética , Adulto , Síndrome de Down/complicações , Síndrome de Down/metabolismo , Feminino , Humanos , Fator Regulador 1 de Interferon/metabolismo , Janus Quinase 1/metabolismo , Masculino , Pessoa de Meia-Idade , Periodontite/complicações , Periodontite/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta/análise , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Adulto Jovem , Receptor de Interferon gamaRESUMO
eIF5A is highly conserved from archaea to mammals, essential for cell viability and the only protein known to contain the essential amino acid residue hypusine, generated by a unique posttranslational modification. eIF5A was originally identified as a translation initiation factor due to its ability to stimulate the formation of the first peptide bond. However, recent studies have shown that depletion of eIF5A causes a significant decrease in polysome run-off and an increase in the ribosome transit time, suggesting that eIF5A is actually involved in the elongation step of protein synthesis. We have previously shown that the depletion mutant tif51A-3 (eIF5A(C39Y/G118D)) shows a sicker phenotype when combined with the dominant negative mutant eft2 ( H699K ) of the elongation factor eEF2. In this study, we used the eIF5A(K56A) mutant to further investigate the relationship between eIF5A and eEF2. The eIF5A(K56A) mutant is temperature sensitive and has a defect in protein synthesis, but instead of causing depletion of the eIF5A protein, this mutant has a defect in hypusine modification. Like the mutant tif51A-3, the eIF5A(K56A) mutant is synthetic sick with the mutant eft2 ( H699K ) of eEF2. High-copy eEF2 not only improves cell growth of the eIF5A(K56A) mutant, but also corrects its increased cell size defect. Moreover, eEF2 suppression of the eIF5A(K56A) mutant is correlated with the improvement of total protein synthesis and with the increased resistance to the protein synthesis inhibitor hygromycin B. Finally, the polysome profile defect of the eIF5A(K56A) mutant is largely corrected by high-copy eEF2. Therefore, these results demonstrate that eIF5A is closely related to eEF2 function during translation elongation.
Assuntos
Fator 2 de Elongação de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Citometria de Fluxo , Ligação Proteica , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
BACKGROUND: Intracanal medication is important for endodontic treatment success as it eliminates microorganisms that persist after biomechanical preparation. Aim. To evaluate the effect of two intracanal medications against Porphyromonas gingivalis and Enterococcus faecalis in the root canals of human primary teeth with necrotic pulp with and without furcal/periapical lesion, using quantitative real-time polymerase chain reaction (qRT-PCR). DESIGN: Thirty-two teeth with necrotic pulp were used. Twelve teeth did not present lesion, and 20 teeth presented radiographically visible furca/periapical lesion. Microbiological samples were collected after coronal access and biomechanical preparation. The teeth were medicated with calcium hydroxide pastes prepared with either polyethylene glycol or chlorhexidine. After 30days, the medication was removed and a third collection was performed. Microbiological samples were processed using qRT-PCR. Data were analysed by Wilcoxon and Mann-Whitney tests (α=0.05). RESULTS: There was no significant difference in the microbiota present in the primary teeth with and without furcal/periapical lesion. Biomechanical preparation was effective in reducing the number of microorganisms (P<0.05). The intracanal medications had similar antibacterial activity. CONCLUSION: The association of chlorhexidine with calcium hydroxide did not increase the antibacterial activity of the intracanal medication in the treatment of primary teeth with necrotic pulp with and without furcal/periapical lesion.
Assuntos
Hidróxido de Cálcio/administração & dosagem , Clorexidina/administração & dosagem , Necrose da Polpa Dentária/terapia , Irrigantes do Canal Radicular/administração & dosagem , Dente Decíduo/patologia , Anti-Infecciosos/administração & dosagem , Criança , Pré-Escolar , Contagem de Colônia Microbiana , Assistência Odontológica para Crianças/métodos , Cavidade Pulpar/microbiologia , Necrose da Polpa Dentária/complicações , Necrose da Polpa Dentária/microbiologia , Método Duplo-Cego , Combinação de Medicamentos , Enterococcus faecalis/efeitos dos fármacos , Feminino , Defeitos da Furca/complicações , Defeitos da Furca/terapia , Humanos , Masculino , Pomadas , Doenças Periapicais/complicações , Doenças Periapicais/terapia , Polietilenoglicóis/administração & dosagem , Porphyromonas gingivalis/efeitos dos fármacos , Estatísticas não Paramétricas , Resultado do TratamentoRESUMO
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".