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1.
Microb Cell Fact ; 18(1): 172, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601209

RESUMO

BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with ß-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.


Assuntos
Rafinose/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/enzimologia , alfa-Galactosidase/biossíntese , Cinética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Especificidade por Substrato , alfa-Galactosidase/química
2.
Elife ; 82019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31552827

RESUMO

Hsf1 is an ancient transcription factor that responds to protein folding stress by inducing the heat-shock response (HSR) that restore perturbed proteostasis. Hsp70 chaperones negatively regulate the activity of Hsf1 via stress-responsive mechanisms that are poorly understood. Here, we have reconstituted budding yeast Hsf1-Hsp70 activation complexes and find that surplus Hsp70 inhibits Hsf1 DNA-binding activity. Hsp70 binds Hsf1 via its canonical substrate binding domain and Hsp70 regulates Hsf1 DNA-binding activity. During heat shock, Hsp70 is out-titrated by misfolded proteins derived from ongoing translation in the cytosol. Pushing the boundaries of the regulatory system unveils a genetic hyperstress program that is triggered by proteostasis collapse and involves an enlarged Hsf1 regulon. The findings demonstrate how an apparently simple chaperone-titration mechanism produces diversified transcriptional output in response to distinct stress loads.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/biossíntese , DNA Fúngico/metabolismo , Temperatura Alta , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/efeitos da radiação
3.
Elife ; 82019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31124783

RESUMO

Ribosome biogenesis is a complex and energy-demanding process requiring tight coordination of ribosomal RNA (rRNA) and ribosomal protein (RP) production. Given the extremely high level of RP synthesis in rapidly growing cells, alteration of any step in the ribosome assembly process may impact growth by leading to proteotoxic stress. Although the transcription factor Hsf1 has emerged as a central regulator of proteostasis, how its activity is coordinated with ribosome biogenesis is unknown. Here, we show that arrest of ribosome biogenesis in the budding yeast Saccharomyces cerevisiae triggers rapid activation of a highly specific stress pathway that coordinately upregulates Hsf1 target genes and downregulates RP genes. Activation of Hsf1 target genes requires neo-synthesis of RPs, which accumulate in an insoluble fraction and presumably titrate a negative regulator of Hsf1, the Hsp70 chaperone. RP aggregation is also coincident with that of the RP gene activator Ifh1, a transcription factor that is rapidly released from RP gene promoters. Our data support a model in which the levels of newly synthetized RPs, imported into the nucleus but not yet assembled into ribosomes, work to continuously balance Hsf1 and Ifh1 activity, thus guarding against proteotoxic stress during ribosome assembly.


Assuntos
Biogênese de Organelas , Proteostase , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico , Transcrição Genética , Regulação Fúngica da Expressão Gênica
4.
Bioprocess Biosyst Eng ; 42(9): 1421-1433, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31055665

RESUMO

A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations.


Assuntos
Biocombustíveis , Etanol/metabolismo , Galactose/metabolismo , Gracilaria/química , Microrganismos Geneticamente Modificados , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sistemas CRISPR-Cas , Galactose/química , Expressão Gênica , Hidrólise , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
5.
Nucleic Acids Res ; 47(13): 7018-7034, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31114879

RESUMO

The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.


Assuntos
Códon/genética , Chaperonas Moleculares/fisiologia , Complexos Multiproteicos/fisiologia , Terminação Traducional da Cadeia Peptídica/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Códon de Terminação/genética , Conformação de Ácido Nucleico , Biogênese de Organelas , Paromomicina/metabolismo , Polilisina/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
6.
PLoS One ; 14(4): e0215009, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958856

RESUMO

Interorganelle phospholipid transfer is critical for eukaryotic membrane biogenesis. In the yeast Saccharomyces cerevisiae, phosphatidylserine (PS) synthesized by PS synthase, Pss1, in the endoplasmic reticulum (ER) is decarboxylated to phosphatidylethanolamine (PE) by PS decarboxylase, Psd1, in the ER and mitochondria or by Psd2 in the endosome, Golgi, and/or vacuole, but the mechanism of interorganelle PS transport remains to be elucidated. Here we report that Sfh1, a member of Sec14 family proteins of S. cerevisiae, possesses the ability to enhance PE production by Psd2. Overexpression of SFH1 in the strain defective in Psd1 restored its growth on non-fermentable carbon sources and increased the intracellular and mitochondrial PE levels. Sfh1 was found to bind various phospholipids, including PS, in vivo. Bacterially expressed and purified Sfh1 was suggested to have the ability to transport fluorescently labeled PS between liposomes by fluorescence dequenching assay in vitro. Biochemical subcellular fractionation suggested that a fraction of Sfh1 localizes to the endosome, Golgi, and/or vacuole. We propose a model that Sfh1 promotes PE production by Psd2 by transferring phospholipids between the ER and endosome.


Assuntos
Carboxiliases/deficiência , Proteínas de Ciclo Celular/biossíntese , Proteínas Cromossômicas não Histona/biossíntese , Mitocôndrias/metabolismo , Modelos Biológicos , Consumo de Oxigênio , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endossomos/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Mitocôndrias/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo
7.
Nat Cell Biol ; 21(4): 442-451, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30886345

RESUMO

The cytosolic accumulation of mitochondrial precursors is hazardous to cellular fitness and is associated with a number of diseases. However, it is not observed under physiological conditions. Individual mechanisms that allow cells to avoid cytosolic accumulation of mitochondrial precursors have recently been discovered, but their interplay and regulation remain elusive. Here, we show that cells rapidly launch a global transcriptional programme to restore cellular proteostasis after induction of a 'clogger' protein that reduces the number of available mitochondrial import sites. Cells upregulate the protein folding and proteolytic systems in the cytosol and downregulate both the cytosolic translation machinery and many mitochondrial metabolic enzymes, presumably to relieve the workload of the overstrained mitochondrial import system. We show that this transcriptional remodelling is a combination of a 'wideband' core response regulated by the transcription factors Hsf1 and Rpn4 and a unique mitoprotein-induced downregulation of the oxidative phosphorylation components, mediated by an inactivation of the HAP complex.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico/genética , Transcrição Genética , Citosol/enzimologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Fosforilação Oxidativa , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo
8.
Biochemistry ; 58(11): 1492-1500, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30817136

RESUMO

The field of synthetic biology is already beginning to realize its potential, with a wealth of examples showcasing the successful genetic engineering of microorganisms for the production of valuable compounds. The chassis Saccharomyces cerevisiae has been engineered to function as a microfactory for producing many of these economically and medically relevant compounds. However, strain construction and optimization to produce industrially relevant titers necessitate a wealth of underpinning biological knowledge alongside large investments of capital and time. Over the past decade, advances in DNA synthesis and editing tools have enabled multiplex genome engineering of yeast, permitting access to more complex modifications that could not have been easily generated in the past. These genome engineering efforts often result in large populations of strains with genetic diversity that can pose a significant challenge to screen individually via traditional methods such as mass spectrometry. The large number of samples generated would necessitate screening methods capable of analyzing all of the strains generated to maximize the explored genetic space. In this Perspective, we focus on recent innovations in multiplex genome engineering of S. cerevisiae, together with biosensors and high-throughput screening tools, such as droplet microfluidics, and their applications in accelerating chassis optimization.


Assuntos
Engenharia de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/biossíntese , Biologia Sintética/métodos , Sistemas CRISPR-Cas , Engenharia Genética/métodos , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Biochim Biophys Acta Mol Cell Res ; 1866(5): 806-818, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30759361

RESUMO

Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2. The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Fúngico/biossíntese , RNA Mitocondrial/biossíntese , RNA de Transferência/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , RNA Fúngico/genética , RNA Mitocondrial/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
10.
Appl Microbiol Biotechnol ; 103(5): 2277-2293, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30706115

RESUMO

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera ß-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.


Assuntos
Proteínas do Capsídeo/metabolismo , Quitina Sintase/biossíntese , Regulação Fúngica da Expressão Gênica/genética , Manosiltransferases/biossíntese , Glicoproteínas de Membrana/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas do Capsídeo/genética , Parede Celular/metabolismo , Quitina/biossíntese , Quitina Sintase/genética , Expressão Gênica/genética , Glutationa/biossíntese , Manosiltransferases/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Metionina/farmacologia , Nodaviridae/genética , Proteínas de Saccharomyces cerevisiae/genética , beta-Glucosidase/metabolismo
11.
FEMS Yeast Res ; 19(2)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629175

RESUMO

The 26S proteasome participates in cell stress responses via its ability to degrade regulatory and damaged proteins. In yeast, mutations in the subunits of the 19S proteasome regulatory subcomplex cause hyper-resistance to 4-nitroquinoline-1-oxide (4-NQO), a chemical mutagen and carcinogen. These data suggest a negative role for the 19S proteasome complex in the cellular response to 4-NQO, although the underlying mechanism is not clear. We proposed that decreased 19S subcomplex activity leads to the stabilisation of Rpn4p, a transcription factor and proteasome substrate. In turn, stabilised Rpn4p may upregulate stress-responsive genes that participate in the response to 4-NQO-induced stress. To test our hypothesis, we impaired the expression of the RPT5 gene, which encodes the ATPase subunit of the 19S subcomplex, by mutating the Rpn4p binding site in its promoter. The mutant strain accumulates polyubiquitinated proteins-a hallmark of compromised proteasome function-and shows hyper-resistance to 4-NQO. We found several groups of genes that conferred resistance to 4-NQO-induced stress and were overexpressed due to the Rpn4p stabilisation and impaired 19S subcomplex function. The upregulated genes are involved in the oxidative and proteotoxic stress response pathways, multidrug resistance and biosynthesis of cysteine and methionine. Consistently, the mutant strain was hyper-resistant to oxidative stress. Our data imply that the ubiquitin-proteasome system may regulate the cellular response to 4-NQO at the transcriptional level.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Quinolonas/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/biossíntese , Regulação para Cima , 4-Nitroquinolina-1-Óxido/metabolismo , Oxidantes/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Estresse Fisiológico
12.
Biochemistry ; 58(11): 1511-1520, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30618248

RESUMO

With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.


Assuntos
Proteínas de Saccharomyces cerevisiae/biossíntese , Biologia Sintética/métodos , Biologia Sintética/tendências , Sistemas CRISPR-Cas , Redes Reguladoras de Genes/genética , Engenharia Genética/métodos , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/genética , Engenharia de Proteínas/métodos , Engenharia de Proteínas/tendências , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Protein Expr Purif ; 154: 112-117, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30240633

RESUMO

Carboxyl-terminal repeat domain (CTD) of the largest subunit Rpb1 of RNA polymerace II is essential for transcription regulation. Heptapeptide repeat of CTD of Rpb1 is phosphorylated by carboxyl-terminal repeat domain kinase (CTDK-I), composed of CTK1, CTK2 and CTK3, in order to regulate transcription and transcription associated processes. The yeast specific protein CTK3 binds to cyclin CTK2 to form a heterodimer serving as a regulational factor to control CTK1 activity by binding to CTK1. Structural information of CTK2-CTK3 complex is yet to be elucidated. Here, we report the co-expression of CTK2-CTK3 complex from Saccharomyces cerevisiae with N-terminal His6-tag in CTK3 in Escherichia coli (E. coli), purification of the complex by four chromatographic steps and crystallization of the complex as well as the diffraction data collection and processing. This study provides some essential information and a guide for structural and functional study of CTK2-CTK3 complex and CTDK-I in the future.


Assuntos
Proteínas Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cristalografia por Raios X , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
14.
Yeast ; 36(2): 99-105, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30346046

RESUMO

The correct separation of chromosomes during mitosis is necessary to prevent genetic instability and aneuploidy, which are responsible for cancer and other diseases, and it depends on proper centrosome duplication. In a recent study, we found that Smy2 can suppress the essential role of Mps2 in the insertion of yeast centrosome into the nuclear membrane by interacting with Eap1, Scp160, and Asc1 and designated this network as SESA (Smy2, Eap1, Scp160, Asc1). Detailed analysis showed that the SESA network is part of a mechanism which regulates translation of POM34 mRNA. Thus, SESA is a system that suppresses spindle pole body duplication defects by repressing the translation of POM34 mRNA. In this study, we performed a genome-wide screening in order to identify new members of the SESA network and confirmed Dhh1 as a putative member. Dhh1 is a cytoplasmic DEAD-box helicase known to regulate translation. Therefore, we hypothesized that Dhh1 is responsible for the highly selective inhibition of POM34 mRNA by SESA.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/biossíntese , Mapas de Interação de Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Testes Genéticos
15.
Lett Appl Microbiol ; 68(1): 17-23, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30276838

RESUMO

Drug resistance commonly occurs when treating immunocompromised patients who have fungal infections. Curcumin, is a compound isolated from Curcuma longa, has been reported to inhibit drug efflux in several human cell lines and nonpathogenic budding yeast Saccharomyces cerevisiae cells that overexpresses the ATP-binding cassette (ABC) transporters S. cerevisiae Pdr5p and pathogenic Candida albicans Cdr1p and Cdr2p. The aim of this study was to examine the effects of curcumin on multidrug resistance in a wild-type strain of the budding yeast with an intrinsic expression system of multidrug efflux-related genes. The antifungal activity of dodecanol alone was temporary against S. cerevisiae; however, restoration of cell viability was completely inhibited when the cells were co-treated with dodecanol and curcumin. Furthermore, restriction of rhodamine 6G (R6G) efflux from the cells and intracellular accumulation of R6G were observed with curcumin treatment. Reverse transcription-polymerase chain reaction analysis revealed that curcumin reduced the dodecanol-induced overexpression of the ABC transporter-related genes PDR1, PDR3 and PDR5 to their control levels in untreated cells. Curcumin can directly restrict the glucose-induced drug efflux and inhibits the expression of the ABC transporter gene PDR5, and can thereby inhibit the efflux of dodecanol from S. cerevisiae cells. Curcumin is effective in potentiating the efficacy of antifungal drugs via its effects on ABC transporters. SIGNIFICANCE AND IMPACT OF THE STUDY: Drug resistance is common in immunocompromised patients with fungal infections. Curcumin, isolated from Curcuma longa, inhibits drug efflux in nonpathogenic budding yeast Saccharomyces cerevisiae cells overexpressing ABC transporters S. cerevisiae Pdr5p and pathogenic Candida albicans Cdr1p and Cdr2p. We examined the effects of curcumin on multidrug resistance in a wild-type strain of the budding yeast with an intrinsic expression system of multidrug efflux-related genes. Curcumin directly inhibited drug efflux and also suppressed the PDR5 expression, thereby enhancing the antifungal effects. Thus, curcumin potentially promotes the efficacy of antifungals via its effects on ABC transporters in wild-type fungal strains.


Assuntos
Antifúngicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Curcumina/farmacologia , Dodecanol/farmacologia , Farmacorresistência Fúngica Múltipla/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/biossíntese , Candida albicans/efeitos dos fármacos , Proteínas de Ligação a DNA/biossíntese , Sinergismo Farmacológico , Quimioterapia Combinada , Proteínas Fúngicas/biossíntese , Humanos , Proteínas de Membrana Transportadoras/biossíntese , Rodaminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomycetales/metabolismo , Fatores de Transcrição/biossíntese
16.
Sci Rep ; 8(1): 17809, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30546021

RESUMO

An imaging-integrated microfluidic cell volume sensor was used to evaluate the volumetric growth rate of single cells from a Saccharomyces cerevisiae population exhibiting two phenotypic expression states of the PDR5 gene. This gene grants multidrug resistance by transcribing a membrane transporter capable of pumping out cytotoxic compounds from the cell. Utilizing fluorescent markers, single cells were isolated and trapped, then their growth rates were measured in two on-chip environments: rich media and media dosed with the antibiotic cycloheximide. Approximating growth rates to first-order, we assessed the fitness of individual cells and found that those with low PDR5 expression had higher fitness in rich media whereas cells with high PDR5 expression had higher fitness in the presence of the drug. Moreover, the drug dramatically reduced the fitness of cells with low PDR5 expression but had comparatively minimal impact on the fitness of cells with high PDR5 expression. Our experiments show the utility of this imaging-integrated microfluidic cell volume sensor for high-resolution, single-cell analysis, as well as its potential application for studies that characterize and compare the fitness and morphology of individual cells from heterogeneous populations under different growth conditions.


Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Regulação Fúngica da Expressão Gênica , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
17.
Sci Rep ; 8(1): 13672, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30209405

RESUMO

Doxorubicin is one of the most effective chemotherapy drugs used against solid tumors in the treatment of several cancer types. Two different mechanisms, (i) intercalation of doxorubicin into DNA and inhibition of topoisomerase II leading to changes in chromatin structure, (ii) generation of free radicals and oxidative damage to biomolecules, have been proposed to explain the mode of action of this drug in cancer cells. A genome-wide integrative systems biology approach used in the present study to investigate the long-term effect of doxorubicin in Saccharomyces cerevisiae cells indicated the up-regulation of genes involved in response to oxidative stress as well as in Rad53 checkpoint sensing and signaling pathway. Modular analysis of the active sub-network has also revealed the induction of the genes significantly associated with nucleosome assembly/disassembly and DNA repair in response to doxorubicin. Furthermore, an extensive re-wiring of the metabolism was observed. In addition to glycolysis, and sulfate assimilation, several pathways related to ribosome biogenesis/translation, amino acid biosynthesis, nucleotide biosynthesis, de novo IMP biosynthesis and one-carbon metabolism were significantly repressed. Pentose phosphate pathway, MAPK signaling pathway biological processes associated with meiosis and sporulation were found to be induced in response to long-term exposure to doxorubicin in yeast cells.


Assuntos
DNA Fúngico/efeitos dos fármacos , Doxorrubicina/farmacologia , Saccharomyces cerevisiae/metabolismo , Inibidores da Topoisomerase II/farmacologia , Transcrição Genética/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/biossíntese , Quinase do Ponto de Checagem 2/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Reparo do DNA/genética , Fermentação/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Nucleossomos/metabolismo , Estresse Oxidativo/genética , Via de Pentose Fosfato/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
18.
ACS Nano ; 12(9): 9363-9371, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30207696

RESUMO

Amyloid nanofibrils are excellent scaffolds for designable materials that can be endowed with biotechnologically relevant functions. However, most of all excellent ideas and concepts that have been reported in the literature might never see real-world implementation in biotechnological applications. One bottleneck is the large-scale production of these materials. In this paper, we present an attempt to create a generic and scalable platform for producing ready-to-use functionalized nanofibrils directly from a eukaryotic organism. As a model material, we assembled Sup35(1-61) amyloid nanofibrils from Saccharomyces cerevisiae decorated with the Z-domain dimer, which has a high affinity toward antibody molecules. To this end, Komagataella pastoris was engineered by inserting gene copies of Sup35(1-61) and the protein chimera Sup35(1-61)-ZZ into the genome. This strain has the capability to constantly secrete amyloidogenic proteins into the extracellular medium, where the mature functionalized fibrils form, with a production yield of 35 mg/L culture. Another striking feature of this strategy is that the separation of the fibril material from the cells requires only centrifugation and resuspension in saline water. The fast production rates, minimal hands-on time, and high stability of the assembled material are some highlights that make the direct assembly of functionalized fibrils in the extracellular medium an alternative to production methods that are not suitable for large-scale production of designed amyloids.


Assuntos
Nanofibras/química , Fatores de Terminação de Peptídeos/biossíntese , Pichia/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Modelos Moleculares , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Pichia/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Lett Appl Microbiol ; 67(5): 484-490, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30098030

RESUMO

Ergosterol biosynthesis in Saccharomyces cerevisiae is complex and the underlying mechanism of regulation remains unclear. To clarify the influence of transcriptional regulation on the ergosterol content, transcription factor Ecm22 was overexpressed in S. cerevisiae. Results showed that the overexpression of ECM22 led to an increased invasive growth. Fluconazole susceptibility testing indicated that strains overexpressing ECM22 could grow at 20 µg(fluconazole)  ml-1 . By contrast, the control failed to grow at 16 µg(fluconazole)  ml-1 . Among truncated ECM22 fragments, only the 1440-bp DNA fragment exerted almost the same impact on ergosterol content as that of the full-length gene. In a 5-l bioreactor, the highest ergosterol yield of the recombinant reached 32∙7 mg g(dry cell weight) -1 , which was increased by about 20% compared with that of the control. In this work, a novel approach for enhancing the ergosterol production by overexpressing a transcription factor in S. cerevisiae was developed.


Assuntos
Antifúngicos/farmacologia , Ergosterol/biossíntese , Fluconazol/farmacologia , Regulação Fúngica da Expressão Gênica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Reatores Biológicos/microbiologia , DNA Fúngico/genética , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
20.
Protein Expr Purif ; 152: 56-63, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30030046

RESUMO

Chaperone proteins are required to maintain the overall fold and function of proteins in the cell. As part of the Hsp70 family, Ssa1 acts to maintain cellular proteostasis through a variety of diverse pathways aimed to preserve the native conformation of target proteins, thereby preventing aggregation and future states of cellular toxicity. Studying the structural dynamics of Ssa1 in vitro is essential to determining their precise mechanisms and requires the development of purification methods that result in highly pure chaperones. Current methods of expressing and purifying Ssa1 utilize affinity tagged constructs expressed in Escherichia coli, however, expression in an exogenous source produces proteins that lack post-translational modifications leading to undesired structural and functional effects. Current protocols to purify Ssa1 from Saccharomyces cerevisiae require large amounts of starting material, multiple steps of chromatography, and result in low yield. Our objective was to establish a small-scale purification of Ssa1 expressed from its endogenous source, Saccharomyces cerevisiae, with significant yield and purity. We utilized a protein A affinity tag that was previously used to purify large protein complexes from yeast, combined with magnetic Dynabeads that are conjugated with rabbit immunoglobulin G (IgG). Our results show that we can produce native, highly pure, active Ssa1 via this one-step purification with minimal amounts of starting material, and this Ssa1-protein A fusion does not alter cellular phenotypes. This methodology is a significant improvement in Ssa1 purification and will facilitate future experiments that will elucidate the biochemical and biophysical properties of Hsp70 chaperones.


Assuntos
Adenosina Trifosfatases/isolamento & purificação , Biotecnologia/métodos , Proteínas de Choque Térmico HSP70/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/genética , Proteína Estafilocócica A/isolamento & purificação , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/genética , Animais , Cromatografia de Afinidade/métodos , Clonagem Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Imunoglobulina G/química , Separação Imunomagnética/métodos , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo
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