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1.
J Agric Food Chem ; 67(39): 10913-10920, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31532663

RESUMO

Copper in grape musts can influence the fermentation efficiency of Saccharomyces cerevisiae during winemaking. The present study revealed the impact of glutathione addition on yeast strains with variable copper sensitivity. The antioxidant glutathione increased yeast vitality and fastened sugar metabolism at copper concentrations up to 0.39 mM. A significant accumulation of acetaldehyde at high copper concentrations was mitigated by the addition of 20 mg L-1 glutathione. Low recovery of glutathione added implicated a complexation of both compounds. Specific alcohol dehydrogenase (ADH) activity was inhibited or reduced in the enzyme extracts of the copper-stressed yeast cells. The activity was restored in fermentations with glutathione at a copper concentration of 0.16 mM. At low copper concentrations, glutathione decreased ADH activity presumably due to complexation of essential copper amounts. Results provide important information on the use of glutathione as an antioxidant in winemaking to counteract negative effects of copper-rich musts on copper-sensitive yeast strains.


Assuntos
Cobre/metabolismo , Glutationa/metabolismo , Saccharomyces cerevisiae/metabolismo , Álcool Desidrogenase/metabolismo , Cobre/análise , Fermentação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Vitis/química , Vitis/metabolismo , Vitis/microbiologia , Vinho/análise , Vinho/microbiologia
2.
J Agric Food Chem ; 67(37): 10521-10533, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31461284

RESUMO

This work was designed to comparatively investigate 27 dietary flavonoids that act as α-glucosidase inhibitors and insulin sensitizers. On the basis of the results of an in vitro experiment of α-glucosidase inhibition, myricetin (IC50 = 11.63 ± 0.36 µM) possessed the strongest inhibitory effect, followed by apigenin-7-O-glucoside (IC50 = 22.80 ± 0.24 µM) and fisetin (IC50 = 46.39 ± 0.34 µM). A three-dimensional quantitative structure-activity relationship model of α-glucosidase inhibitors with good predictive capability [comparative molecular field analysis, q2 = 0.529, optimum number of components (ONC) = 10, R2 = 0.996, F = 250.843, standard error of estimation (SEE) = 0.064, and two descriptors; comparative similarity index analysis, q2 = 0.515, ONC = 10, R2 = 0.997, F = 348.301, SEE = 0.054, and four descriptors] was established and indicated that meta positions of ring B favored bulky and minor, electron-withdrawing, and hydrogen bond donor groups. The presence of electron-donating and hydrogen bond acceptor groups at position 4' of ring B could improve α-glucosidase activity. Position 3 of ring C favored minor, electron-donating, and hydrogen bond donor groups, whereas position 7 of ring A favored bulky and hydrogen bond acceptor groups. Molecular docking screened five flavonoids (baicalein, isorhamnetin-3-O-rutinoside, apigenin-7-O-glucoside, kaempferol-7-O-ß-glucoside, and cyanidin-3-O-glucoside) that can act as insulin sensitizers and form strong combinations with four key protein targets involved in the insulin signaling pathway. Apigenin-7-O-glucoside (60 µM) can effectively improve insulin resistance, and glucose uptake increased by approximately 73.06% relative to the model group of insulin-resistant HepG2 cells. Therefore, apigenin-7-O-glucoside might serve as the most effective α-glucosidase inhibitor and insulin sensitizer. This work may guide diabetes patients to improve their condition through dietary therapy.


Assuntos
Flavonoides/química , Inibidores de Glicosídeo Hidrolases/química , Insulina/metabolismo , Flavonoides/metabolismo , Flavonoides/farmacologia , Inibidores de Glicosídeo Hidrolases/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo
4.
J Agric Food Chem ; 67(31): 8590-8598, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287301

RESUMO

Patchoulol, a natural sesquiterpene compound, is widely used in perfumes and cosmetics. Several strategies were adopted to enhance patchoulol production in Saccharomyces cerevisiae: (i) farnesyl pyrophosphate (FPP) synthase and patchoulol synthase were fused to increase the utilization of FPP precursor; (ii) expression of the limiting genes of the mevalonate pathway was enhanced; (iii) squalene synthase was weakened by a glucose-inducible promoter of HXT1 (promoter for hexose transporter) to reduce metabolic flux from FPP to ergosterol; and (iv) farnesol biosynthesis was inhibited to decrease the consumption of FPP. Glucose was used to balance the trade-off between the competitive squalene and patchoulol pathways. The patchoulol production was 59.2 ± 0.7 mg/L in a shaken flask with a final production of 466.8 ± 12.3 mg/L (20.5 ± 0.5 mg/g dry cell weight) combined with fermentation optimization, which was 7.8-fold higher than the reported maximum production. The work significantly promoted the industrialization process of patchoulol production using biobased microbial platforms.


Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo , Fermentação , Ácido Mevalônico/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esqualeno/metabolismo
5.
J Agric Food Chem ; 67(32): 8986-8993, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31347835

RESUMO

Trehalose plays a crucial role in response to freezing stress in baker's yeast. MAL62, a gene involved in the adenosine diphosphoglucose-dependent trehalose synthesis pathway, can increase trehalose content. However, the difference between MAL62-related trehalose synthesis and traditional uridine diphosphoglucose-dependent trehalose synthesis is not well-understood. MAL62 overexpression showed less effect in enhancing intracellular trehalose compared to TPS1 overexpression. However, MAL62 overexpression elicited trehalose synthesis before fermentation with enhanced maltose metabolism and had a similar effect on cell viability after freezing. Furthermore, MAL62 and TPS1 overexpression in the NTH1 deletion background further strengthened freezing tolerance and improved leavening ability. Our results suggest that the enhancement in freezing tolerance by MAL62 overexpression may involve multiple pathways rather than simply enhancing trehalose synthesis. The results reveal valuable insights into the relationship between maltose metabolism and freezing tolerance and may help to develop better yeast strains for enhancing fermentation characteristics of frozen dough.


Assuntos
Glucosiltransferases/metabolismo , Maltose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , alfa-Glucosidases/metabolismo , Farinha/análise , Farinha/microbiologia , Congelamento , Regulação Fúngica da Expressão Gênica , Glucosiltransferases/genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Trealase/genética , Trealase/metabolismo , Trealose/metabolismo , alfa-Glucosidases/genética
6.
Nature ; 571(7765): 366-370, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31243363

RESUMO

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.


Assuntos
ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Microscopia Crioeletrônica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Transporte Biológico , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/ultraestrutura , Ativação Enzimática , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/ultraestrutura
7.
Biochimie ; 163: 101-107, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31175919

RESUMO

Saccharomyces cerevisiae has high level of inorganic polyphosphate and a multicomponent system of its metabolism, including polyphosphatases Ppx1, Ppn1, Ddp1, and Ppn2. The aim of the study was to construct the yeast strain overexpressing Ppn2 and to compare the properties of Ppn2, Ppx1, Ppn1, and Ddp1 purified from overexpressing strains of S. cerevisiae. We overexpressed Ppn2 in S. cerevisiae under a strong constitutive promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase-encoding gene and suggested biochemical criteria for distinguishing among yeast polyphosphatases, which is important for their identification and understanding of their functions. Ppn2, Ppn1, and Ddp1 had endopolyphosphatase activities, whereas Ppx1 did not. Ppx1 and Ppn1 exhibited high and Ddp1 and Ppn2 low exopolyphosphatase activity: 240, 500, 0.05 and 0.1 U/mg protein, respectively. The enzymes had distinct patterns of exopolyphosphatase activities stimulation by divalent metal ions. Ppn2, Ppn1 and Ddp1 displayed endopolyphosphatase activity in the presence of 1 mM Mg2+. The endopolyphosphatase activities of Ppn2 and Ppn1 were induced by 0.01 mM of Co2+ or Zn2+, whereas that of Ddp1 required 0.1 mM of these cations. The endopolyphosphatase activity of Ppn1 was inhibited by 0.01 mg mL-1 of heparin, while endopolphosphatase activity of Ppn2 was weakly sensitive to 0.25 mg mL-1 of heparin. The Ppx1 and Ppn1 activity with guanosine tetraphosphate was nearly 80% of activity with long-chain polyphosphates. The Ppn1 hydrolyzed dATP, while Ppx1 did not. The differences in the mode of polyphosphate hydrolysis, substrate specificity, metal ion dependence and cell localization suggest distinct roles of these enzymes in yeast.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Polifosfatos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Hidrolases Anidrido Ácido/genética , Cátions Bivalentes/metabolismo , Hidrólise , Microrganismos Geneticamente Modificados , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
8.
Eur J Med Chem ; 178: 752-766, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31229877

RESUMO

This work describes the synthesis of hybrid tetrahydro-1,5-naphthyridine and 1,5-naphthyridine derivatives fused with heterocycles such as chromenes and chromen-2-ones or coumarins, which were synthesized in good to high general yields. The synthetic route involves an intramolecular [4 + 2]-cycloaddition reaction of functionalized aldimines obtained by the condensation of 3-aminopyridine and aldehydes containing a double or triple carbon-carbon bond in orto position and allows the selective generation of three stereogenic centers in a short, efficient and reliable synthesis. The subsequent dehydrogenation of the fused tetrahydrochromeno[4,3-b][1,5]naphthyridines and/or tetrahydrochromeno[4,3-b][1,5]naphthyridin-6-ones leads to the formation of the corresponding tetracyclic chromeno[4,3-b][1,5]naphthyridine derivatives and/or chromeno[4,3-b][1,5]naphthyridin-6-ones in quantitative yields. Some of the prepared products showed activity as inhibitors of Topoisomerase I (TopI). Additionally, the cytotoxic behavior of these compounds has been studied in cell lines derived from human lung adenocarcinoma (A549) and human ovarian carcinoma (SKOV03), and on non-cancerous lung fibroblasts cell line (MRC5) where, on the last ones, the absence of cytotoxicity was observed. 7-Phenyl-6H-6a,7,12,12a-tetrahydrochromeno[4,3-b][1,5]naphthyridine 5a showed excellent cytotoxic activity with a IC50 value of 1.03 ±â€¯0.30 µM against the A549 cell line and a IC50 value of 1.75 ±â€¯0.20 µM against the SKOV03 cell line. The obtained results point to these compounds as good antiproliferative candidates.


Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Naftiridinas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/isolamento & purificação , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Naftiridinas/síntese química , Naftiridinas/química , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/química , Células Tumorais Cultivadas
9.
J Basic Microbiol ; 59(7): 744-753, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31087563

RESUMO

The recalcitrance of lignocellulosic biomass is a major factor limiting its conversion into biofuels. Therefore, in this study, we pretreated corn stalk with 2% Na2 CO3 and 2% H2 O2 for time 70 min at 130°C using a corn stalk to liquid ratio of 1:10. The fermentation broth from multicopy Saccharomyces cerevisiae strains engineered for lignocellulase synthesis was used to enzymatically hydrolyze the pretreated corn stalk. The highest monosaccharide yield (102 mmol/L) was obtained using 15 IU endocellulase, 10 IU exocellulase, and 15 IU ß-glucosidase per gram of cellulose and 20 IU xylanase per gram of hemicellulose. Subsequently, the high-efficiency ethanol-producing S. cerevisiae strain WXY12, which can produce ethanol from glucose and xylose simultaneously, was added to the fermentation system. The entire process involved ethanol production through simultaneous saccharification and fermentation (SSF). Optimization of the fermentation conditions (yeast powder as a nitrogen source, temperature of 30°C, MgSO4 as a metal ion inducer, rotation speed of 180 rpm, solid-liquid ratio of 1:8, and inoculation amount of 2%) resulted in an ethanol output of 46.87 g/L and a theoretical conversion rate of 27.4%. The results of this study improved corn stalk utilization, reduced hydrolysis costs, and generated high ethanol yields.


Assuntos
Biocombustíveis , Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/metabolismo , Zea mays/química , Zea mays/metabolismo , Bioengenharia , Biomassa , Hidrólise , Lignina/química , Lignina/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Talanta ; 200: 279-287, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036185

RESUMO

Plants are well-recognized sources of inhibitors for α-glucosidase - a key target enzyme for management of type 2 diabetes. Recently, two advanced bioactivity-profiling techniques, i.e., ligand fishing and high-resolution inhibition profiling, have shown great promises for accelerating identification of α-glucosidase inhibitors from complex plant extracts. Non-specific affinities and non-specific inhibitions are major sources of false positive hits from ligand fishing and high-resolution inhibition profiling, respectively. In an attempt to minimize such false positive hits, we describe a new screening approach based on ligand fishing and high-resolution inhibition profiling for detection of high-affinity ligands and assessment of inhibitory activity, respectively. The complementary nature of ligand fishing and high-resolution inhibition profiling was explored to identify α-glucosidase inhibitory ligands from a complex mixture, and proof-of-concept was demonstrated with crude ethyl acetate extract of Ginkgo biloba. In addition to magnetic beads with a 3-carbon aliphatic linker, α-glucosidase was immobilized on magnetic beads with a 21-carbon aliphatic linker; and the two different types of magnetic beads were compared for their hydrolytic activity and fishing efficiency.


Assuntos
Biflavonoides/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Extratos Vegetais/farmacologia , alfa-Glucosidases/metabolismo , Biflavonoides/química , Biflavonoides/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos , Ginkgo biloba/química , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Ligantes , Fenômenos Magnéticos , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade
11.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 392-396, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045569

RESUMO

Grx1, a cytosolic thiol-disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates (reduced, GSH, and oxidized, GSSG). Here, the crystallization of reduced Grx1 from the yeast Saccharomyces cerevisiae (yGrx1) in space group P212121 and its structure solution and refinement to 1.22 Šresolution are reported. To study the structure-function relationship of yeast Grx1, the crystal structure of reduced yGrx1 was compared with the existing structures of the oxidized and glutathionylated forms. These comparisons revealed structural differences in the conformations of residues neighbouring the Cys27-Cys30 active site which accompany alterations in the redox status of the protein.


Assuntos
Cisteína/química , Glutarredoxinas/química , Glutationa/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
12.
Comput Biol Chem ; 80: 168-176, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30965174

RESUMO

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Quitina Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Antifúngicos/metabolismo , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Quitina Sintase/química , Quadruplex G , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Técnica de Seleção de Aptâmeros , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência
13.
Nat Methods ; 16(5): 437-445, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988468

RESUMO

Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed candidate enzymes from different organisms. The rNTase activities of 22 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a cytidine-adding enzyme that is likely to be part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, Caenorhabditis elegans MUT-2, that adds alternating uridine and guanosine nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutants of the enzyme that result in defective silencing did not add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Nucleotidiltransferases/genética , Interferência de RNA , RNA Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Animais , Caenorhabditis elegans/enzimologia , Mutação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética
14.
Biomolecules ; 9(4)2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934776

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient's brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.


Assuntos
Técnicas de Cocultura , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Doença de Parkinson/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Linhagem Celular Tumoral , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Bioelectrochemistry ; 128: 148-154, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31003053

RESUMO

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.


Assuntos
Apoptose/efeitos dos fármacos , Candida/efeitos dos fármacos , Caspases/metabolismo , Eletroporação/métodos , Formiatos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Candida/classificação , Candida/citologia , Candida/enzimologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Especificidade da Espécie
16.
Genes Dev ; 33(9-10): 578-589, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30846429

RESUMO

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.


Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Fatores de Alongamento de Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , RNA Polimerase II/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
17.
Proc Natl Acad Sci U S A ; 116(15): 7333-7342, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30918129

RESUMO

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.


Assuntos
Nucleotídeos de Adenina/química , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Nucleotídeos de Adenina/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Espectrometria de Massas , Domínios Proteicos , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
18.
DNA Repair (Amst) ; 76: 40-49, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30818168

RESUMO

DNA polymerases influence genome stability through their involvement in DNA replication, response to DNA damage, and DNA repair processes. Saccharomyces cerevisiae possess four non-essential DNA polymerases, Pol λ, Pol η, Pol ζ, and Rev1, which have varying roles in genome stability. In order to assess the contribution of the non-essential DNA polymerases in genome stability, we analyzed the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant in microhomology mediated repair, due to recent studies linking some of these DNA polymerases to this repair pathway. Our results suggest that the length and quality of microhomology influence both the overall efficiency of repair and the involvement of DNA polymerases. Furthermore, the non-essential DNA polymerases demonstrate overlapping and redundant functions when repairing double-strand breaks using short microhomologies containing mismatches. Then, we examined genome-wide mutation accumulation in the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant compared to wild type cells. We found a significant decrease in the overall rate of mutation accumulation in the quadruple mutant cells compared to wildtype, but an increase in frameshift mutations and a shift towards transversion base-substitution with a preference for G:C to T:A or C:G. Thus, the non-essential DNA polymerases have an impact on the nature of the mutational spectrum. The sequence and functional homology shared between human and S. cerevisiae non-essential DNA polymerases suggest these DNA polymerases may have a similar role in human cells.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Mutação
19.
Molecules ; 24(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889828

RESUMO

The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal-a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.


Assuntos
Aldeídos/metabolismo , Engenharia Metabólica/métodos , Monoterpenos/metabolismo , NADPH Desidrogenase/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Modelos Moleculares , Mutagênese/genética , Proteínas Mutantes/metabolismo , NADPH Desidrogenase/metabolismo , Oxirredução , Estereoisomerismo
20.
Appl Biochem Biotechnol ; 187(4): 1131-1142, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30903384

RESUMO

Escherichia coli isocitrate dehydrogenase (IDH) is regulated by reversible phosphorylation on Ser113. Latest phosphoproteomic studies revealed that eukaryotic IDHs can also be phosphorylated on the analogous Ser site. So as to understand the possible phosphorylation mechanism, the equivalent Ser of NADP-IDHs from yeast Saccharomyces cerevisiae (ScIDH) and Yarrowia lipolytica(YlIDH) were investigated by site-directed mutagenesis. ScIDH Ser110 and YlIDH Ser103 were replaced by Asp or Glu to mimic a continuous phosphorylation state. Meanwhile, the effects of another four amino acids (Thr, Tyr, Gly, Ala) with various side chain on IDH activity were determined as well. Enzymatic analysis showed that replacement of Ser with Asp or Glu nearly inactivated ScIDH and YlIDH. Four other mutant enzymes of ScIDH, S110T, S110G, S110A, and S110Y, retained 38.07%, 3.24%, 2.65%, and 0.01% of its original activity, and four other mutant enzymes of YlIDH, S103T, S103G, S103A, and S103Y retained 44.26%, 27.99%, 16.29%, and 0.01% of its original activity, respectively. These results suggested that phosphorylation on eukaryotic IDHs has identical consequence to that on the bacterial IDHs. We thus presume that phosphorylation on the substrate-binding Ser shall be a common regulatory mechanism among IDHs.


Assuntos
Isocitrato Desidrogenase/metabolismo , Saccharomyces cerevisiae/enzimologia , Yarrowia/enzimologia , Sequência de Aminoácidos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Cinética , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Alinhamento de Sequência , Especificidade por Substrato
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