RESUMO
Cold environments are more frequent than people think. They include deep oceans, cold lakes, snow, permafrost, sea ice, glaciers, cold soils, cold deserts, caves, areas at elevations greater than 3000 m, and also artificial refrigeration systems. These environments are inhabited by a diversity of eukaryotic and prokaryotic organisms that must adapt to the hard conditions imposed by cold. This adaptation is multifactorial and includes (i) sensing the cold, mainly through the modification of the liquid-crystalline membrane state, leading to the activation of a two-component system that transduce the signal; (ii) adapting the composition of membranes for proper functions mainly due to the production of double bonds in lipids, changes in hopanoid composition, and the inclusion of pigments; (iii) producing cold-adapted proteins, some of which show modifications in the composition of amino acids involved in stabilizing interactions and structural adaptations, e.g., enzymes with high catalytic efficiency; and (iv) producing ice-binding proteins and anti-freeze proteins, extracellular polysaccharides and compatible solutes that protect cells from intracellular and extracellular ice. However, organisms also respond by reprogramming their metabolism and specifically inducing cold-shock and cold-adaptation genes through strategies such as DNA supercoiling, distinctive signatures in promoter regions and/or the action of CSPs on mRNAs, among others. In this review, we describe the main findings about how organisms adapt to cold, with a focus in prokaryotes and linking the information with findings in eukaryotes.
Assuntos
Adaptação Fisiológica , Proteínas , Humanos , Adaptação Fisiológica/fisiologia , Proteínas/metabolismo , Aminoácidos , Oceanos e Mares , Solo , Temperatura BaixaRESUMO
Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.
Assuntos
Inteligência Artificial , Furilfuramida , Simulação de Acoplamento Molecular , Ureia/metabolismo , Tioureia , Acetamidas , Transportadores de UreiaRESUMO
In both prokaryotic and eukaryotic genomes, synonymous codons are unevenly used. Such differential usage of optimal or non-optimal codons has been suggested to play a role in the control of translation initiation and elongation, as well as at the level of transcription and mRNA stability. In the case of membrane proteins, codon usage has been proposed to assist in the establishment of a pause necessary for the correct targeting of the nascent chains to the translocon. By using as a model UreA, the Aspergillus nidulans urea transporter, we revealed that a pair of non-optimal codons encoding amino acids situated at the boundary between the N-terminus and the first transmembrane segment are necessary for proper biogenesis of the protein at 37°C. These codons presumably regulate the translation rate in a previously undescribed fashion, possibly contributing to the correct interaction of ureA-translating ribosome-nascent chain complexes with the signal recognition particle and/or other factors, while the polypeptide has not yet emerged from the ribosomal tunnel. Our results suggest that the presence of the pair of non-optimal codons would not be functionally important in all cellular conditions. Whether this mechanism would affect other proteins remains to be determined.
RESUMO
Psychrophilic and psychrotolerant bacteria from permanently cold environments may be the most abundant extremophiles on Earth and yet little is known on how they cope with temperature stress. Real-time reverse transcription PCR (RT-qPCR) is a powerful technique that could shed light on this matter but it requires pre-validated reference genes for normalization of data to get accurate results. In this study, we assessed the expression stability of eight candidate genes for the psychrotolerant Antarctic isolate Pseudomonas sp. AU10 during exponential growth under 4 °C and 30 °C, and after a cold-shock. Using the software programs BestKeeper and geNorm we validated recA, ftsZ, 16S rRNA, and rpoD as reference genes and we suggested the combination of recA and ftsZ for qPCR data normalization. Our results provide a starting point for gene expression studies in Antarctic Pseudomonas concerning temperature-related physiology and also for the validation of reference genes in other cold-adapted bacterial species.
Assuntos
Temperatura Baixa , Perfilação da Expressão Gênica/normas , Pseudomonas/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Estresse Fisiológico , Pseudomonas/metabolismo , Padrões de ReferênciaRESUMO
We present the first account of the structure-function relationships of a protein of the subfamily of urea/H(+) membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.
Assuntos
Aspergillus nidulans/genética , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Mutação/genética , Plantas/metabolismo , Ureia/metabolismo , Sequência de Aminoácidos , Aspergillus nidulans/metabolismo , Biologia Computacional , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Alinhamento de Sequência , Transportadores de UreiaRESUMO
The formation of inclusion bodies (IBs) constitute a frequent event during the production of heterologous proteins in bacterial hosts. Although the mechanisms leading to their formation are not completely understood, empirical data have been exploited trying to predict the aggregation propensity of specific proteins while a great number of strategies have been developed to avoid the generation of IBs. However, in many cases, the formation of such aggregates can be considered an advantage for basic research as for protein production. In this review, we focus on this positive side of IBs formation in bacteria. We present a compilation on recent advances on the understanding of IBs formation and their utilization as a model to understand protein aggregation and to explore strategies to control this process. We include recent information about their composition and structure, their use as an attractive approach to produce low cost proteins and other promising applications in Biomedicine.
RESUMO
Aspergillus is a genus of mold fungi that includes more than 200 described species. Many members of the group are relevant pathogens and other species are economically important. Only one species has been analyzed for codon usage, and this was performed with a small number of genes. In this paper, we report the codon usage patterns of eight completely sequenced genomes which belong to this genus. The results suggest that selection for translational efficiency and accuracy are the major factors shaping codon usage in all of the species studied so far, and therefore they were active in the last common ancestor of the group. Composition and molecular distances analyses show that highly expressed genes evolve slower at synonymous sites. We identified a conserved core of translationally optimal codons and study the tRNA gene pool in each genome. We found that the great majority of preferred triplets match the respective cognate tRNA with more copies in the respective genome. We discuss the possible scenarios that can explain the observed differences among the species analyzed. Finally we highlight the biotechnological application of this research regarding heterologous protein expression.
Assuntos
Aspergillus/genética , Códon/genética , RNA Fúngico/genética , Seleção Genética , Aspergillus/classificação , Sequência Conservada , Evolução Molecular , Genoma Fúngico , Modelos Genéticos , Mutação , Filogenia , Biossíntese de Proteínas , RNA de Transferência/genética , Especificidade da EspécieRESUMO
Membrane proteins play key roles in diverse cellular functions and have become the target for a large number of pharmacological drugs. Despite representing about 20-30% of cellular proteins, their characterization is long overdue since they are difficult to handle, to purify from their natural source or to obtain as recombinant proteins. Pichia pastoris is a methylotrophic yeast species increasingly used as a host for heterologous protein expression for both research and industrial purposes. Over the past few years many efforts have allowed important advances in the development of this expression system for the expression and production of membrane proteins. The most recent achievements in improving yield and proper folding of integral membrane proteins are summarized in this review.
Assuntos
Proteínas de Membrana , Pichia , Engenharia de Proteínas , Técnicas de Cultura de Células/métodos , Códon , Expressão Gênica , Vetores Genéticos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pichia/genética , Pichia/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
We report here the characterization of UreA, a high-affinity urea/H+ symporter of Aspergillus nidulans. The deletion of the encoding gene abolishes urea transport at low substrate concentrations, suggesting that in these conditions UreA is the sole transport system specific for urea in A. nidulans. The ureA gene is not inducible by urea or its precursors, but responds to nitrogen metabolite repression, necessitating for its expression the AreA GATA factor. In contrast to what was observed for other transporters in A. nidulans, repression by ammonium is also operative during the isotropic growth phase. The activity of UreA is down-regulated post-translationally by ammonium-promoted endocytosis. A number of homologues of UreA have been identified in A. nidulans and other Aspergilli, which cluster in four groups, two of which contain the urea transporters characterized so far in fungi and plants. This phylogeny may have arisen by gene duplication events, giving place to putative transport proteins that could have acquired novel, still unidentified functions.
Assuntos
Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Aspergillus nidulans/classificação , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Filogenia , Compostos de Amônio Quaternário/metabolismo , Ureia/metabolismo , Transportadores de UreiaRESUMO
We present the case of a patient referred to the gastroenterology service for investigation of abnormal liver function tests. She had been taking nitrofurantoin for 16 months as prophylaxis against urinary tract infections. CT scan showed evidence of lung pneumonitis and low attenuation in the liver parenchyma. Nitrofurantoin-induced pneumonitis and hepatotoxicity was diagnosed. The patient responded both clinically and biochemically to withdrawal of nitrofurantoin. This combination of adverse reaction to nitrofurantoin is rare.
Assuntos
Anti-Infecciosos Urinários/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico por imagem , Nitrofurantoína/efeitos adversos , Pneumonia/diagnóstico por imagem , Anti-Infecciosos Urinários/administração & dosagem , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Feminino , Humanos , Fígado/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Pessoa de Meia-Idade , Nitrofurantoína/administração & dosagem , Pneumonia/induzido quimicamente , Radiografia Torácica , Tomografia Computadorizada por Raios X , Infecções Urinárias/prevenção & controleRESUMO
We show that Neurospora crassa has a single histone H1 gene, hH1, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of hH1 by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless, hH1 mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene, cfp, was abnormally derepressed in hH1 mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the cfp gene promoter to the reporter gene hph was analyzed. Thus, Neurospora histone H1 is required for the proper regulation of cfp, a gene with a key role in the respiratory-fermentative pathway.
Assuntos
Respiração Celular/genética , Regulação Fúngica da Expressão Gênica/genética , Histonas/genética , Neurospora crassa/enzimologia , Neurospora crassa/genética , Piruvato Descarboxilase/biossíntese , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Respiração Celular/efeitos dos fármacos , Tamanho Celular/genética , Segregação de Cromossomos/genética , Metilação de DNA , Reparo do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Etanol/farmacologia , Dados de Sequência Molecular , Mutagênicos/farmacologia , Neurospora crassa/crescimento & desenvolvimento , Mutação Puntual/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/genética , Piruvato Descarboxilase/genéticaRESUMO
La información estadística que se presenta en ese documento está estructurada por Macro Regiones, Gerencias Departamentales y Centros Asistenciales y comprende básicamente, los servicios de Consulta Hospitalización, Emergencia, Centro Quirúrgico, Centro Obstétrico, Farmacia, Laboratorio y Rayos X(AU)
Assuntos
Acessibilidade aos Serviços de Saúde , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Cobertura de Serviços de Saúde , Interpretação Estatística de Dados , PeruRESUMO
La información estadística que se presenta en ese documento está estructurada por Macro Regiones, Gerencias Departamentales y Centros Asistenciales y comprende básicamente, los servicios de Consulta Hospitalización, Emergencia, Centro Quirúrgico, Centro Obstétrico, Farmacia, Laboratorio y Rayos X
Assuntos
Acessibilidade aos Serviços de Saúde , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Cobertura de Serviços de Saúde , Interpretação Estatística de Dados , PeruRESUMO
El objetivo de este estudio fue determinar la prevalencia de silicosis en una industria con riesgos reconocidos, como es la fabricación del vidrio, relacionandola con el tiempo de exposición al polvo e intensidad de dicha exposición y en forma retrospectiva a través de las cifras halladas evaluar las medidas de protección ambiental aplicadas a la industria. Se estudiaron 112 trabajadores entre los que se encontraron diez con silicosis. Fueron todas formas simples: cuatro con presentación típica y seis con localización no habitual, sólo en campos medios e inferiores del pulmón. Todos tuvieron opacidades pequeñas, redondeadas y la mayoría asoció opacidades lineales e irregulares que vinculamos a formas precoces de silicosis y al hábito de fumar. Se estableció una asociación significativa con los años y la intensidad de exposición al polvo. Se completó la evaluación con estudio funcional respiratorio que mostró obstrucción leve, restricción o patrón normal. La anatomía patológica mostró obstrucción leve, restricción o patrón normal. La anatomía patológica mostró desde nódulos silicóticos a fibrosis focales discretas. Los autores señalan la importancia de hacer mediciones correctas de la fracción inhalable y porcentaje de sílice libre para establecer una correlación con la prevalencia encontrada, a fin de ayudar a determinar los niveles de seguridad efectivos en las industrias que emplean sílice
Assuntos
Humanos , Masculino , Feminino , Silicose , Silicose/diagnóstico , Silicose/etiologiaRESUMO
El objetivo de este estudio fue determinar la prevalencia de silicosis en una industria con riesgos reconocidos, como es la fabricación del vidrio, relacionandola con el tiempo de exposición al polvo e intensidad de dicha exposición y en forma retrospectiva a través de las cifras halladas evaluar las medidas de protección ambiental aplicadas a la industria. Se estudiaron 112 trabajadores entre los que se encontraron diez con silicosis. Fueron todas formas simples: cuatro con presentación típica y seis con localización no habitual, sólo en campos medios e inferiores del pulmón. Todos tuvieron opacidades pequeñas, redondeadas y la mayoría asoció opacidades lineales e irregulares que vinculamos a formas precoces de silicosis y al hábito de fumar. Se estableció una asociación significativa con los años y la intensidad de exposición al polvo. Se completó la evaluación con estudio funcional respiratorio que mostró obstrucción leve, restricción o patrón normal. La anatomía patológica mostró obstrucción leve, restricción o patrón normal. La anatomía patológica mostró desde nódulos silicóticos a fibrosis focales discretas. Los autores señalan la importancia de hacer mediciones correctas de la fracción inhalable y porcentaje de sílice libre para establecer una correlación con la prevalencia encontrada, a fin de ayudar a determinar los niveles de seguridad efectivos en las industrias que emplean sílice (AU)