RESUMO

SGTs (small glutamine-rich TPR-containing proteins) are dimeric proteins that belong to the class of co-chaperones characterized by the presence of TPR domains (containing tetratricopeptide repeats). Human (SGTA) and yeast (Sgt2) SGTs are characterized by three distinct domains: an N-terminal dimerization domain, a central TPR-domain important for binding to other proteins (chaperones included) and a C-terminal domain involved in hydrophobic interactions. Both these SGTs are involved in the cellular PQC (protein quality control) system, as they interact with chaperones and have functions that aid stress recovery. However, there are differences between them, such as structural features and binding specificities, that could be better understood if other orthologous proteins were studied. Therefore, we produced and characterized a putative SGT protein, designated AaSGT, from the mosquito Aedes aegypti, which is a vector of several diseases, such as dengue and Zika. The protein was produced as a folded dimer which was stable up to 40 °C and was capable of binding to AaHsp90 and fully protecting a model protein, α-synuclein, from aggregation. The conformation of AaSGT was investigated by biophysical tools and small angle X-ray scattering, which showed that the protein had an elongated conformation and that its C-terminal domain was mainly disordered. The results with a C-terminal deletion mutant supported these observations. Altogether, these results are consistent with those from other functional SGT proteins and add to the understanding of the PQC system in Aedes aegypti, an important aim that may help to develop inhibitory strategies against this vector of neglected diseases.

Assuntos

RESUMO

Cellular proteostasis is maintained by a system consisting of molecular chaperones, heat shock proteins (Hsps) and proteins involved with degradation. Among the proteins that play important roles in the function of this system is Hsp90, which acts as a node of this network, interacting with at least 10% of the proteome. Hsp90 is ATP-dependent, participates in critical cell events and protein maturation and interacts with large numbers of co-chaperones. The study of Hsp90 orthologs is justified by their differences in ATPase activity levels and conformational changes caused by Hsp90 interaction with nucleotides. This study reports the characterization of Hsp90 from Aedes aegypti, a vector of several diseases in many regions of the planet. Aedes aegypti Hsp90, AaHsp90, was cloned, purified and characterized for its ATPase and chaperone activities and structural conformation. These parameters indicate that it has the characteristics of eukaryotic Hsp90s and resembles orthologs from yeast rather than from human. Finally, constitutive and increased stress expression in Aedes cells was confirmed. Taken together, the results presented here help to understand the relationship between structure and function in the Hsp90 family and have strong potential to form the basis for studies on the network of chaperone and Hsps in Aedes.

Assuntos

RESUMO

O gene floR descrito é descrito pela literatura como o responsável pela resistência ao florfenicol, que é um antimicrobiano amplamente utilizado na aquicultura. Esse gene já foi relatado em muitas espécies de bactérias, inclusive no gênero Aeromonas. Essas bactérias causam alta mortalidade na piscicultura trazendo prejuízos econômicos. É importante que haja estudos sobre esse gene e possíveis mutações que possam levar a alterações na estrutura e função da proteína. Os objetivos desse estudo foram caracterizar o gene floR em isolados de Aeromonas spp. obtidas do Vale do São Francisco e verificar se a presença desse gene está associada com a resistência ao florfenicol. Foram realizadas reações em cadeia da polimerase (PCR) para a presença do gene floR em 27 isolados de Aeromonas spp.. Amostras positivas para a presença do gene foram sequenciadas e analisadas quanto à presença de polimorfismos por meio de alinhamentos. Os diferentes haplótipos detectados foram utilizados para análises com os programas SIFT e PolyPhen para predição de alteração de função proteica. A modelagem estrutural da proteina codificada pelo gene floR foi realizada com o programa Modeller e, os modelos foram avaliados pelo Procheck, Verify3D e Whatif. A similaridade da estrutura tridimensional da proteína referência com as estruturas tridimensionais das proteínas codificadas pelos diferentes haplótipos foi comparada através do TM-align. A resistência das bactérias ao florfenicol foi avaliada através do teste de microdiluição em caldo, o qual também foi realizado na presença do carbonil cianeto m-clorofenil hidrazona para verificar o efeito da inibição da bomba de efluxo sobre tal resistência. Dos vinte e sete isolados avaliados quanto a presença do gene floR, 14 isolados foram positivos e 10 foram sequenciados, o que permitiu a identificação de três polimorfismos no gene floR, que levaram a construção de três haplótipos diferentes (TAA, TTA e CTG). As análises realizadas com os programas SIFT e PolyPhen apontaram que os haplótipos TTA e TAA provavelmente poderiam alterar a estrutura e função da proteína. As proteínas modeladas para os três haplótipos demonstraram possuir praticamente a mesma conformação estrutural entre si. Todos os isolados que apresentaram o gene foram resistentes ao florfenicol e aqueles que não apresentavam foram sensíveis. O teste na presença do Carbonil Cianeto m-Clorofenil Hidrazona foi realizado para três isolados, cada isolado representando um haplótipo, sendo possível observar a inibição do crescimento bacteriano em todas as concentrações independente do haplótipo. Os resultados obtidos nesse estudo mostram que a resistência ao flofenicol em Aeromonas spp. pode ser explicada pela presença do gene floR, e que esse gene está relacionado com uma bomba de efluxo. As mutações verificadas no gene floR, parecem não estar envolvidas com alteração de estrutura e função da proteína codificada por esse gene.(AU)

The floR gene is described in related literature as responsible for resistance to florfenicol, which is a widely used antimicrobial agent in aquaculture. This gene has been reported in many species of bacteria, including the genus Aeromonas. These bacteria cause high mortality in fish farming bringing economic losses. It is important that studies of this gene and possible mutations that can lead to changes in the structure and function of the protein. The aim of this study was to characterize the floR gene in isolates of Aeromonas spp. and check if the presence of this gene is associated with resistance to florfenicol in Aeromonas spp. obtained from the San Francisco Valley. PCR (Polymerase Chain Reaction) were also performed to verify the presence of the floR gene in 27 isolates of Aeromonas spp. Positive samples for the presence of the gene were sequenced and analyzed for the presence of polymorphisms using alignments. Different haplotypes detected were used for analysis with the SIFT and PolyPhen programs for prediction of changes in protein function. The structural modeling of protein encoded by the floR gene was performed using the Modeller software, and the models were evaluated by Procheck, Verify3D and Whatif. The similarity of the dimensional structure of reference protein with the dimensional structures of the proteins encoded by the different haplotypes was compared by TM-align. Bacterial resistance to florfenicol was assessed by the microdilution test, which was also performed in the presence of carbonyl cyanide m-chlorophenyl hydrazone to verify the effect of inhibiting the efflux pump. 14 isolates were positive for the presence of floR gene and 10 were sequenced and allowed the identification of three polymorphisms in the floR gene, which led to construction of three different haplotypes (TAA TTA and CTG). The analyzes carried out with the SIFT and PolyPhen programs showed that the TTA and TAA haplotypes could probably change the protein structure-function. Proteins modeled for the three haplotypes were found to have substantially the same structural conformation with each other. All isolates presenting the gene were resistant to florfenicol and those who did not have were sensitive. The test in the presence of carbonyl cyanide m-chlorophenylhydrazone was conducted for three isolates, representing each single haplotype and was observed inhibition of bacterial growth at all concentrations independent of the haplotype. The results of this study show that resistance to flofenicol in Aeromonas spp. may be explained by the presence of floR gene and that this gene is associated with an efflux pump. Mutations observed in floR gene do not appear to be involved with chenges in structure and function of the protein encoded by gene.(AU)

Assuntos

RESUMO

O gene floR descrito é descrito pela literatura como o responsável pela resistência ao florfenicol, que é um antimicrobiano amplamente utilizado na aquicultura. Esse gene já foi relatado em muitas espécies de bactérias, inclusive no gênero Aeromonas. Essas bactérias causam alta mortalidade na piscicultura trazendo prejuízos econômicos. É importante que haja estudos sobre esse gene e possíveis mutações que possam levar a alterações na estrutura e função da proteína. Os objetivos desse estudo foram caracterizar o gene floR em isolados de Aeromonas spp. obtidas do Vale do São Francisco e verificar se a presença desse gene está associada com a resistência ao florfenicol. Foram realizadas reações em cadeia da polimerase (PCR) para a presença do gene floR em 27 isolados de Aeromonas spp.. Amostras positivas para a presença do gene foram sequenciadas e analisadas quanto à presença de polimorfismos por meio de alinhamentos. Os diferentes haplótipos detectados foram utilizados para análises com os programas SIFT e PolyPhen para predição de alteração de função proteica. A modelagem estrutural da proteina codificada pelo gene floR foi realizada com o programa Modeller e, os modelos foram avaliados pelo Procheck, Verify3D e Whatif. A similaridade da estrutura tridimensional da proteína referência com as estruturas tridimensionais das proteínas codificadas pelos diferentes haplótipos foi comparada através do TM-align. A resistência das bactérias ao florfenicol foi avaliada através do teste de microdiluição em caldo, o qual também foi realizado na presença do carbonil cianeto m-clorofenil hidrazona para verificar o efeito da inibição da bomba de efluxo sobre tal resistência. Dos vinte e sete isolados avaliados quanto a presença do gene floR, 14 isolados foram positivos e 10 foram sequenciados, o que permitiu a identificação de três polimorfismos no gene floR, que levaram a construção de três haplótipos diferentes (TAA, TTA e CTG)...(AU)

The floR gene is described in related literature as responsible for resistance to florfenicol, which is a widely used antimicrobial agent in aquaculture. This gene has been reported in many species of bacteria, including the genus Aeromonas. These bacteria cause high mortality in fish farming bringing economic losses. It is important that studies of this gene and possible mutations that can lead to changes in the structure and function of the protein. The aim of this study was to characterize the floR gene in isolates of Aeromonas spp. and check if the presence of this gene is associated with resistance to florfenicol in Aeromonas spp. obtained from the San Francisco Valley. PCR (Polymerase Chain Reaction) were also performed to verify the presence of the floR gene in 27 isolates of Aeromonas spp. Positive samples for the presence of the gene were sequenced and analyzed for the presence of polymorphisms using alignments. Different haplotypes detected were used for analysis with the SIFT and PolyPhen programs for prediction of changes in protein function. The structural modeling of protein encoded by the floR gene was performed using the Modeller software, and the models were evaluated by Procheck, Verify3D and Whatif. The similarity of the dimensional structure of reference protein with the dimensional structures of the proteins encoded by the different haplotypes was compared by TM-align. Bacterial resistance to florfenicol was assessed by the microdilution test, which was also performed in the presence of carbonyl cyanide m-chlorophenyl hydrazone to verify the effect of inhibiting the efflux pump. 14 isolates were positive for the presence of floR gene and 10 were sequenced and allowed the identification of three polymorphisms in the floR gene, which led to construction of three different haplotypes (TAA TTA and CTG). The analyzes carried out with the SIFT and PolyPhen...(AU)

Assuntos

RESUMO

Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25°C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.