Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 721
Filtrar
1.
Proc Natl Acad Sci U S A ; 117(25): 14243-14250, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32518113

RESUMO

Cells must couple cell-cycle progress to their growth rate to restrict the spread of cell sizes present throughout a population. Linear, rather than exponential, accumulation of Whi5, was proposed to provide this coordination by causing a higher Whi5 concentration in cells born at a smaller size. We tested this model using the inducible GAL1 promoter to make the Whi5 concentration independent of cell size. At an expression level that equalizes the mean cell size with that of wild-type cells, the size distributions of cells with galactose-induced Whi5 expression and wild-type cells are indistinguishable. Fluorescence microscopy confirms that the endogenous and GAL1 promoters produce different relationships between Whi5 concentration and cell volume without diminishing size control in the G1 phase. We also expressed Cln3 from the GAL1 promoter, finding that the spread in cell sizes for an asynchronous population is unaffected by this perturbation. Our findings indicate that size control in budding yeast does not fundamentally originate from the linear accumulation of Whi5, contradicting a previous claim and demonstrating the need for further models of cell-cycle regulation to explain how cell size controls passage through Start.


Assuntos
Tamanho Celular , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/fisiologia , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Fase G1 , Galactoquinase/genética , Galactoquinase/metabolismo , Galactose , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
2.
Carbohydr Res ; 486: 107839, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704571

RESUMO

Galactokinase catalyses the ATP-dependent phosphorylation of galactose and structurally related sugars. The enzyme has attracted interest as a potential biocatalyst for the production of sugar 1-phosphates and several attempts have been made to broaden its specificity. In general, bacterial galactokinases have wider substrate ranges than mammalian ones. The enzymes from Escherichia coli and Lactococcus lactis have received particular attention and a number of variants with increased promiscuity have been identified. Here, we present a molecular dynamics study designed to investigate the molecular causes of the wider substrate ranges of these enzymes and their variants with particular reference to protein mobility. Some regions close to the active site of the enzyme have different structures in the bacterial enzymes compared to the human one. Alterations known to increase the substrate range (e.g. Y371H in the E. coli enzyme), tend to alter the conformation of a key α-helical region (residues 216-232 in the E. coli enzyme). The equivalent helix in the human enzyme has previously been predicted to be altered in variants which affect catalytic activity or protein stability. This helix appears to be a key region in galactokinases from a range of species and may represent an interesting target for future attempts to broaden the specificity of galactokinases.


Assuntos
Escherichia coli/enzimologia , Galactoquinase/química , Galactoquinase/metabolismo , Biocatálise , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Especificidade por Substrato
3.
Nat Commun ; 10(1): 3657, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31413260

RESUMO

We lack an understanding of how the full range of genetic variants that occur in individuals can interact. To address this shortcoming, here we combine diverse mutations between genes in a model regulatory network, the galactose (GAL) switch of budding yeast. The effects of thousands of pairs of mutations fall into a limited number of phenotypic classes. While these effects are mostly predictable using simple rules that capture the 'stereotypical' genetic interactions of the network, some double mutants have unexpected outcomes including constituting alternative functional switches. Each of these 'harmonious' genetic combinations exhibits altered dependency on other regulatory genes. These cases illustrate how both pairwise and higher epistasis determines gene essentiality and how combinations of mutations rewire regulatory networks. Together, our results provide an overview of how broad spectra of mutations interact, how these interactions can be predicted, and how diverse genetic solutions can achieve 'wild-type' phenotypic behavior.


Assuntos
Proteínas de Ligação a DNA/genética , Galactose/metabolismo , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Epistasia Genética , Galactoquinase/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Saccharomyces cerevisiae/genética , Biologia de Sistemas , Transativadores/genética
4.
Int J Oral Sci ; 11(2): 19, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31148553

RESUMO

Streptococcus mutans is a well-known cause of dental caries, due to its acidogenicity, aciduricity, and ability to synthesize exopolysaccharides in dental plaques. Intriguingly, not all children who carry S. mutans manifest caries, even with similar characteristics in oral hygiene, diet, and other environmental factors. This phenomenon suggests that host susceptibility potentially plays a role in the development of dental caries; however, the association between host genetics, S. mutans, and dental caries remains unclear. Therefore, this study examined the influence of host gene-by-S. mutans interaction on dental caries. Genome-wide association analyses were conducted in 709 US children (<13 years old), using the dbGap database acquired from the center for oral health research in appalachia (COHRA) and the Iowa Head Start programmes (GEIRS). A generalized estimating equation was used to examine the gene-by-S. mutans interaction effects on the outcomes (decayed and missing/filled primary teeth due to caries). Sequentially, the COHRA and GEIRS data were used to identify potential interactions and replicate the findings. Three loci at the genes interleukin 32 (IL32), galactokinase 2 (GALK2), and CUGBP, Elav-like family member 4 (CELF4) were linked to S. mutans carriage, and there was a severity of caries at a suggestive significance level among COHRA children (P < 9 × 10-5), and at a nominal significance level among GEIRS children (P = 0.047-0.001). The genetic risk score that combined the three loci also significantly interacted with S. mutans (P < 0.000 1). Functional analyses indicated that the identified genes are involved in the host immune response, galactose carbohydrate metabolism, and food-rewarding system, which could potentially be used to identify children at high risk for caries and to develop personalized caries prevention strategies.


Assuntos
Suscetibilidade à Cárie Dentária/genética , Cárie Dentária/microbiologia , Streptococcus mutans/genética , Streptococcus mutans/isolamento & purificação , Adolescente , Criança , Índice CPO , Galactoquinase , Estudo de Associação Genômica Ampla , Humanos , Dente Decíduo
5.
EMBO J ; 38(12)2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101674

RESUMO

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform called orbital tracking, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation.


Assuntos
Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sítios de Ligação , Metabolismo dos Carboidratos/genética , Galactoquinase/genética , Galactoquinase/metabolismo , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Imagem Molecular/métodos , Organismos Geneticamente Modificados , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única/métodos , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Transcrição Genética , Ativação Transcricional/genética
6.
Biotechnol Lett ; 41(6-7): 779-788, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31065855

RESUMO

OBJECTIVE: Over-express galactokinase (Galk1) in tissue plasminogen activator (tPA) producing CHO cells as a potential strategy to improve cell growth and product synthesis. RESULTS: tPA producing CHO cells were transfected with the galactokinase (Galk1) gene. CHO-Galk1 cells showed a 39% increase of the specific growth rate in galactose. Moreover, clones were able to use this hexose as their main carbon source to sustain growth contrary to their parental cell line. Metabolic Flux Analysis revealed that the CHO-Galk1 selected clone shows an active metabolism towards biomass and product synthesis, characterized by higher fluxes in the TCA cycle, which is consistent with increased cellular densities and final product concentration. CONCLUSION: This cellular engineering strategy, where modifications of key points of alternative carbon sources metabolism lead to an improved metabolism of these sugars, is a starting point towards the generation of new cell lines with reduced lactate synthesis and increased cell growth and productivity.


Assuntos
Células CHO/metabolismo , Engenharia Celular/métodos , Galactose/metabolismo , Lactatos/metabolismo , Proteínas Recombinantes/biossíntese , Ativador de Plasminogênio Tecidual/biossíntese , Animais , Carbono/metabolismo , Cricetulus , Galactoquinase/genética , Galactoquinase/metabolismo , Expressão Gênica
7.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30837296

RESUMO

The 1:1 balance between the numbers of large and small ribosomal subunits can be disturbed by mutations that inhibit the assembly of only one of the subunits. Here, we have investigated if the cell can counteract an imbalance of the number of the two subunits. We show that abrogating 60S assembly blocks 40S subunit accumulation. In contrast, cessation of the 40S pathways does not prevent 60S accumulation, but does, however, lead to fragmentation of the 25S rRNA in 60S subunits and formation of a 55S ribosomal particle derived from the 60S. We also present evidence suggesting that these events occur post assembly and discuss the possibility that the turnover of subunits is due to vulnerability of free subunits not paired with the other subunit to form 80S ribosomes.


Assuntos
Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sobrevivência Celular/fisiologia , Galactoquinase/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Estabilidade Proteica , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética
8.
Enzyme Microb Technol ; 123: 8-14, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30686349

RESUMO

In order to display xylose reductase at the surface of S. cerevisiae cells two different gene constructs have been prepared. In the first, xylose reductase gene GRE3 was fused with two parts of the CCW12 gene, the N-terminal one coding for the secretion signal sequence, and the C-terminal coding for the glycosylphosphatidylinositol anchoring signal. Transformed cells synthesized xylose reductase and incorporated it in the cell wall through the remnant of the glycosylphosphatidylinositol anchor. The other construct was prepared by fusing the GRE3 with the PIR4 gene coding for one of the proteins of the Pir-family containing the characteristic N-terminal repetitive sequence that anchors Pir proteins to ß-1,3-glucan. In this way xylose reductase was covalently attached to glucan through its N-terminus. For the expression of the constructs either the GAL1, or the PHO5 promoters have been used. Both strains displayed active xylose reductases and their enzyme properties were compared with the control enzyme bearing the secretion signal sequence but no anchoring signals, thus secreted into the medium. The enzyme displayed through the N-terminal fusion with PIR4 had higher affinity for xylose than the other construct, but they both expressed somewhat lower affinity than the control enzyme. Similarly, the Km values for NADPH of both immobilized enzymes were somewhat higher than the Km of the control XR. Both displayed enzymes, especially the one fused with Pir4, had higher thermal and pH stability than the control, while other enzymatic properties were not significantly impaired by surface immobilization.


Assuntos
Parede Celular/enzimologia , D-Xilulose Redutase/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , D-Xilulose Redutase/química , D-Xilulose Redutase/genética , Galactoquinase/genética , Galactoquinase/metabolismo , Glucanos/metabolismo , Plasmídeos , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Xilose/metabolismo
9.
BMB Rep ; 52(2): 127-132, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30463643

RESUMO

Cells must fine-tune their gene expression programs for optimal cellular activities in their natural growth conditions. Transcriptional memory, a unique transcriptional response, plays a pivotal role in faster reactivation of genes upon environmental changes, and is facilitated if genes were previously in an active state. Hyper-activation of gene expression by transcriptional memory is critical for cellular differentiation, development, and adaptation. TREM (Transcriptional REpression Memory), a distinct type of transcriptional memory, promoting hyper-repression of unnecessary genes, upon environmental changes has been recently reported. These two transcriptional responses may optimize specific gene expression patterns, in rapidly changing environments. Emerging evidence suggests that they are also critical for immune responses. In addition to memory B and T cells, innate immune cells are transcriptionally hyperactivated by restimulation, with the same or different pathogens known as trained immunity. In this review, we briefly summarize recent progress in chromatin-based regulation of transcriptional memory, and its potential role in immune responses. [BMB Reports 2019; 52(2): 127-132].


Assuntos
Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Animais , Cromatina , Epigenômica/métodos , Galactoquinase/genética , Galactoquinase/fisiologia , Expressão Gênica/genética , Expressão Gênica/fisiologia , Humanos , Elementos Reguladores de Transcrição/genética , Elementos Reguladores de Transcrição/fisiologia
10.
Carbohydr Res ; 472: 132-137, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30593944

RESUMO

Promiscuous galactokinases (GalKs), which catalyse the ATP dependent phosphorylation of galactose in nature, have been widely exploited in biotechnology for the rapid synthesis of diverse sugar-1-phosphates. This work focuses on the characterisation of a bacterial GalK from Streptomyces coelicolor (ScGalK), which was overproduced in Escherichia coli and shown to phosphorylate galactose. ScGalK displayed a broad substrate tolerance, with activity towards Gal, GalN, Gal3D, GalNAc, Man and L-Ara. Most interestingly, ScGalK demonstrated a high activity over a broad pH and temperature range, suggesting that the enzyme could be highly amenable to multi-enzyme systems.


Assuntos
Galactoquinase/genética , Galactoquinase/metabolismo , Streptomyces coelicolor/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Galactoquinase/química , Concentração de Íons de Hidrogênio , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Especificidade por Substrato , Termodinâmica
12.
Plant Cell Environ ; 42(4): 1139-1157, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30156702

RESUMO

The purple acid phosphatase AtPAP26 plays a central role in Pi-scavenging by Pi-starved (-Pi) Arabidopsis. Mass spectrometry (MS) of AtPAP26-S1 and AtPAP26-S2 glycoforms secreted by -Pi suspension cells demonstrated that N-glycans at Asn365 and Asn422 were modified in AtPAP26-S2 to form high-mannose glycans. A 55-kDa protein that co-purified with AtPAP26-S2 was identified as a Galanthus nivalis agglutinin-related and apple domain lectin-1 (AtGAL1; At1g78850). MS revealed that AtGAL1 was bisphosphorylated at Tyr38 and Thr39 and glycosylated at four conserved Asn residues. When AtGAL was incubated in the presence of a thiol-reducing reagent prior to immunoblotting, its cross-reactivity with anti-AtGAL1-IgG was markedly attenuated (consistent with three predicted disulfide bonds in AtGAL1's apple domain). Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating posttranscriptional control of AtGAL1 expression. Growth of a -Pi atgal1 mutant was unaffected, possibly due to compensation by AtGAL1's closest paralog, AtGAL2 (At1g78860). Nevertheless, AtGAL1's induction by numerous stresses combined with the broad distribution of AtGAL1-like lectins in diverse species implies an important function for AtGAL1 orthologs within the plant kingdom. We hypothesize that binding of AtPAP26-S2's high-mannose glycans by AtGAL1 enhances AtPAP26 function to facilitate Pi-scavenging by -Pi Arabidopsis.


Assuntos
Fosfatase Ácida/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Galactoquinase/metabolismo , Fosfatos/deficiência , Fosfatase Ácida/isolamento & purificação , Proteínas de Arabidopsis/isolamento & purificação , Células Cultivadas , Cromatografia em Gel , Dissacarídeos , Galactoquinase/isolamento & purificação , Glucuronatos , Fosfatos/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Regulação para Cima
14.
Cell Rep ; 25(3): 737-748.e4, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30332652

RESUMO

Despite advances made in understanding the effects of promoter structure on transcriptional activity, limited knowledge exists regarding the role played by chromatin architecture in transcription. Previous work hypothesized that transcription from the bidirectional GAL1/GAL10 promoter is controlled through looping of its UAS region around a nonstandard nucleosome. Here, by editing the GAL1/GAL10 promoter at high resolution, we provide insights into bidirectional expression control. We demonstrate that the first and fourth Gal4 binding sites within the UAS do not functionally contribute to promoter activation. Instead, these sites, along with nearby regulatory regions, contribute to the directional regulation of gene expression. Furthermore, Gal4 binding to the third binding site is critical for gene expression, while binding to the other three sites is not sufficient for transcriptional activation. Because the GAL1/GAL10 UAS can activate gene expression in many eukaryotes, the regulatory mechanism presented is expected to operate broadly across the eukaryotic clade.


Assuntos
Galactoquinase/genética , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/genética , DNA Fúngico , Galactoquinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Transcrição Genética
15.
Bioorg Chem ; 81: 649-657, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30253338

RESUMO

Galactokinase catalyses the phosphorylation of α-d-galactose and some structurally related monosaccharides. The enzyme is of interest due to its potential as a biocatalyst for the production of sugar 1-phosphates and due to its involvement in the inherited metabolic disease type II galactosemia. It has been previously shown that a region (residues 231-245) in human galactokinase often has altered mobility when active site residues are varied. We hypothesised that the reverse may be true and that designing changes to this region might affect the functioning of the active site of the enzyme. Focussing on four residues (Leu-231, Gln-242, Glu-244 and Glu-245) we conducted molecular dynamics simulations to explore the effects of changing these residues to glycine or serine. In most cases the variations resulted in local changes to the 231-245 region and global changes to the root mean squared fluctuation (RMSF) of the protein. The four serine variants were expressed as recombinant proteins. All had altered steady state enzyme kinetic parameters with α-d-galactose as a substrate. However, these changes were generally less than ten-fold in magnitude. Changes were also observed with 2-deoxy-α-d-galactose, α-d-galactosamine and α-d-talose as substrates, including (in some cases) loss of detectable activity, suggesting that these variations can tune the specificity of the enzyme. This study demonstrates that activity and specificity of human galactokinase can be modulated by variations designed to affect active site flexibility. It is likely that this principle can be generalised to other enzymes.


Assuntos
Galactoquinase/genética , Galactoquinase/metabolismo , Substituição de Aminoácidos , Domínio Catalítico , Estabilidade Enzimática , Galactoquinase/química , Galactose/análogos & derivados , Galactose/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação Puntual , Conformação Proteica , Especificidade por Substrato
16.
Appl Microbiol Biotechnol ; 102(23): 10027-10041, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30215129

RESUMO

Degradation of mannans is a key process in the production of foods and prebiotics. ß-Mannanase is the key enzyme that hydrolyzes 1,4-ß-D-mannosidic linkages in mannans. Heterogeneous expression of ß-mannanase in Pichia pastoris systems is widely used; however, Saccharomyces cerevisiae expression systems are more reliable and safer. We optimized ß-mannanase gene from Aspergillus sulphureus and expressed it in five S. cerevisiae strains. Haploid and diploid strains, and strains with constitutive promoter TEF1 or inducible promoter GAL1, were tested for enzyme expression in synthetic auxotrophic or complex medium. Highest efficiency expression was observed for haploid strain BY4741 integrated with ß-mannanase gene under constitutive promoter TEF1, cultured in complex medium. In fed-batch culture in a fermentor, enzyme activity reached ~ 24 U/mL after 36 h, and production efficiency reached 16 U/mL/day. Optimal enzyme pH was 2.0-7.0, and optimal temperature was 60 °C. In studies of ß-mannanase kinetic parameters for two substrates, locust bean gum galactomannan (LBG) gave Km = 24.13 mg/mL and Vmax = 715 U/mg, while konjac glucomannan (KGM) gave Km = 33 mg/mL and Vmax = 625 U/mg. One-hour hydrolysis efficiency values were 57% for 1% LBG, 74% for 1% KGM, 39% for 10% LBG, and 53% for 10% KGM. HPLC analysis revealed that the major hydrolysis products were the oligosaccharides mannose, mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexaose. Our findings show that this ß-mannanase has high efficiency for hydrolysis of mannans to mannooligosaccharides, a type of prebiotic, suggesting strong potential application in food industries.


Assuntos
Aspergillus/enzimologia , Mananas/metabolismo , Saccharomyces cerevisiae/metabolismo , beta-Manosidase/metabolismo , Técnicas de Cultura Celular por Lotes , DNA Fúngico/genética , Galactanos/química , Galactoquinase/genética , Galactoquinase/metabolismo , Dosagem de Genes , Regulação Enzimológica da Expressão Gênica , Hidrólise , Microbiologia Industrial , Mananas/química , Manose/metabolismo , Oligossacarídeos/metabolismo , Pichia , Gomas Vegetais/química , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Trissacarídeos/metabolismo , beta-Manosidase/genética
17.
J Microbiol Biotechnol ; 28(9): 1473-1481, 2018 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-30111071

RESUMO

A cellulase hyperproducing mutant strain, JNDY-13, was obtained using the ARTP mutation system and with Trichoderma reesei RUT-C30 as the parent strain. Whole-genome sequencing of JNDY-13 confirmed that 105 of the 653 SNPs were point mutations, 336 mutations were deletions and 165 were insertions. Moreover, 99 mutations were insertions and duplications. Among all the mutations, the one that occurred in the galactokinase gene might be related to the production of cellulases in T. reesei JNDY-13. Moreover, the up-regulation of cellulase and hemicellulase genes in JNDY-13 might contribute to higher cellulases production. Under optimal conditions, the highest cellulase activity by batch fermentation reached 4.35 U/ml, and the highest activity of fed-batch fermentation achieved was 5.40 U/ml.


Assuntos
Celulases/metabolismo , Trichoderma , Celulases/genética , DNA Fúngico/genética , Fermentação , Galactoquinase/genética , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Mutação , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Trichoderma/enzimologia , Trichoderma/genética , Trichoderma/isolamento & purificação
18.
Sci Rep ; 8(1): 11154, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042439

RESUMO

Cargo is transported from the trans-Golgi Network to the plasma membrane by adaptor complexes, which are pan-eukaryotic components. However, in yeast, cargo can also be exported by the exomer complex, a heterotetrameric protein complex consisting of two copies of Chs5, and any two members of four paralogous proteins (ChAPs). To understand the larger relevance of exomer, its phylogenetic distribution and function outside of yeast need to be explored. We find that the four ChAP proteins are derived from gene duplications after the divergence of Yarrowia from the remaining Saccharomycotina, with BC8 paralogues (Bch2 and Chs6) being more diverged relative to the BB8 paralogues (Bch1 and Bud7), suggesting neofunctionalization. Outside Ascomycota, a single preduplicate ChAP is present in nearly all Fungi and in diverse eukaryotes, but has been repeatedly lost. Chs5, however, is a fungal specific feature, appearing coincidentally with the loss of AP-4. In contrast, the ChAP protein is a wide-spread, yet uncharacterized, membrane-trafficking component, adding one more piece to the increasingly complex machinery deduced as being present in our ancient eukaryotic ancestor.


Assuntos
Evolução Biológica , Biologia Celular , Células Eucarióticas/metabolismo , Complexo de Golgi/metabolismo , Filogenia , Saccharomyces cerevisiae/metabolismo , Rede trans-Golgi/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Galactoquinase/metabolismo , Duplicação Gênica , Microscopia de Fluorescência , Fenótipo , Ligação Proteica , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência
19.
Exp Parasitol ; 192: 65-72, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30040960

RESUMO

Galactokinase catalyses the ATP-dependent phosphorylation of galactose. A galactokinase-like sequence was identified in a Fasciola hepatica EST library. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein of approximately 50 kDa. The protein is monomeric, like galactokinases from higher animals, yeasts and some bacteria. The protein has no detectable enzymatic activity with galactose or N-acetylgalactosamine as a substrate. However, it does bind to ATP. Molecular modelling predicted that the protein adopts a similar fold to galactokinase and other GHMP kinases. However, a key loop in the active site was identified which may influence the lack of activity. Sequence analysis strongly suggested that this protein (and other proteins annotated as "galactokinase" in the trematodes Schistosoma mansoni and Clonorchis sinensis) are closer to N-acetylgalactosamine kinases. No other galactokinase-like sequences appear to be present in the genomes of these three species. This raises the intriguing possibility that these (and possibly other) trematodes are unable to catabolise galactose through the Leloir pathway due to the lack of a functional galactokinase.


Assuntos
Fasciola hepatica/enzimologia , Galactoquinase/metabolismo , Galactose/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Fluorometria , Galactoquinase/genética , Galactoquinase/isolamento & purificação , Galactose/química , Modelos Moleculares , Fosforilação , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
20.
J Cell Biochem ; 119(9): 7585-7598, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29893426

RESUMO

Galactosemia type 2 is an autosomal recessive disorder characterized by the deficiency of galactokinase (GALK) enzyme due to missense mutations in GALK1 gene, which is associated with various manifestations such as hyper galactosemia and formation of cataracts. GALK enzyme catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of α-d-galactose to galactose-1-phosphate. We searched 4 different literature databases (Google Scholar, PubMed, PubMed Central, and Science Direct) and 3 gene-variant databases (Online Mendelian Inheritance in Man, Human Gene Mutation Database, and UniProt) to collect all the reported missense mutations associated with GALK deficiency. Our search strategy yielded 32 missense mutations. We used several computational tools (pathogenicity and stability, biophysical characterization, and physiochemical analyses) to prioritize the most significant mutations for further analyses. On the basis of the pathogenicity and stability predictions, 3 mutations (P28T, A198V, and L139P) were chosen to be tested further for physicochemical characterization, molecular docking, and simulation analyses. Molecular docking analysis revealed a decrease in interaction between the protein and ATP in all the 3 mutations, and molecular dynamic simulations of 50 ns showed a loss of stability and compactness in the mutant proteins. As the next step, comparative physicochemical changes of the native and the mutant proteins were carried out using essential dynamics. Overall, P28T and A198V were predicted to alter the structure and function of GALK protein when compared to the mutant L139P. This study demonstrates the power of computational analysis in variant classification and interpretation and provides a platform for developing targeted therapeutics.


Assuntos
Galactoquinase/genética , Galactosemias/genética , Simulação de Acoplamento Molecular , Mutação de Sentido Incorreto , Trifosfato de Adenosina/metabolismo , Galactoquinase/metabolismo , Galactosemias/metabolismo , Humanos , Ligação Proteica , Conformação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA