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1.
Food Chem ; 340: 127900, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32871359

RESUMO

The development of innovative and more cost-effective approaches of making beer throughout continuous fermentation process remains a challenging problem, which is worthy of serious exploration. The current work focuses on the application of a commercial brewing yeast (S. cerevisiae Nottingham Ale), entrapped into chitosan-calcium alginate double layer microcapsules, for the production of a Pale Ale beer. During the primary alcoholic fermentation, the consumption rate of fermentable brewing sugars and dissolved O2, estimated by the Gompertz equation, was halved in the beer obtained by encapsulated yeast in comparison with the free cell. The physical-chemical parameters of beer (i.e. pH, alcohol content, color and bitterness) were not remarkably affected by the different yeast-inoculating form. However, the volatile profiles identified by means of HS-SPME-GC-MS analysis, significantly differed in terms of terpenes, esters and alcohols content, thus proving that the yeast-inoculating form may typify the odor and flavor descriptors of the green beer.


Assuntos
Cerveja/análise , Cerveja/microbiologia , Saccharomyces cerevisiae , Compostos Orgânicos Voláteis/análise , Adulto , Álcoois/análise , Encapsulamento de Células , Ésteres/análise , Feminino , Fermentação , Microbiologia de Alimentos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Odorantes/análise , Saccharomyces cerevisiae/química , Paladar
2.
Food Chem ; 338: 128089, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33091980

RESUMO

Antarctic krill oil (KO) was encapsulated into yeast cells (YCs), and the physicochemical, morphological, and conformational characterizations of KO-loaded YCs (KYCs) were investigated. Moreover, the oxidation stability and in vitro release behavior of KYCs were evaluated. Results showed that KYCs provided significantly higher oxidative stability than native KO. The fatty acid profile remained obviously unchanged after encapsulation. Most interestingly, the phospholipid proportion increased from 49.76% ± 1.42% to 59.92% ± 1.39% after encapsulation. Furthermore, there was a slow and prolonged release of KYCs, along with higher bioaccessibility of docosahexaenoic acid and eicosapentaenoic acid than the KO-in-water emulsion (69.62% ± 7.67% and 66.67% ± 4.55% vs 47.44% ± 4.4% and 39.74% ± 3.89%). KO encapsulation in YCs can be considered as an efficient approach for extending the oxidative and in vitro stability of this nutritious oil and facilitating its application in food products.


Assuntos
Cápsulas/química , Euphausiacea/metabolismo , Óleos de Peixe/química , Saccharomyces cerevisiae/química , Animais , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Emulsões/química , Ácidos Graxos/análise , Ácidos Graxos/química , Óleos de Peixe/metabolismo , Oxirredução
3.
Food Chem ; 337: 128006, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32919277

RESUMO

Due to the global warming, more and more often the red-pink grape varieties grown in the Mediterranean basin reveal an insufficient accumulation of anthocyanins and thus a scarce coloration. Nowadays, this is becoming an important technological issue, which may result in the reduction of the fresh market value of table grape. This study aimed at assessing the effect of the pre-harvest treatment by specific inactivated yeasts (YE) on the qualitative parameters and anthocyanin pattern of three red table grape varieties, which typically present poor and/or incomplete coloration during warm years. An increment of anthocyanins level up to almost seven folds corresponding to an improvement of red skin appearance was observed in all the treated table grapes, which was preserved after cold storage, too. While, no significant influence on chemical composition and berry texture and dimensions was found, meaning that YE did not cause appreciable taste changes in grapes.


Assuntos
Antocianinas/análise , Saccharomyces cerevisiae/química , Vitis/química , Cromatografia Líquida de Alta Pressão , Folhas de Planta
4.
Mol Cell ; 80(5): 764-778.e7, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33207182

RESUMO

Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.


Assuntos
Família da Proteína 8 Relacionada à Autofagia , Autofagia , Endocitose , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Microscopia Crioeletrônica , Ligação Proteica , Proteólise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nat Commun ; 11(1): 5952, 2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-33230227

RESUMO

Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein's mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein's stepping cycle and underscores the complexity and crosstalk among the motor's multiple AAA+ domains.


Assuntos
Dineínas do Citoplasma/química , Dineínas do Citoplasma/metabolismo , Domínio AAA , Trifosfato de Adenosina/metabolismo , Dineínas do Citoplasma/genética , Hidrólise , Microtúbulos/metabolismo , Movimento , Mutagênese , Pinças Ópticas , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo
6.
Nat Commun ; 11(1): 5226, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067463

RESUMO

Signs of proteostasis failure often entwine with those of metabolic stress at the cellular level. Here, we study protein sequestration during glucose deprivation-induced ATP decline in Saccharomyces cerevisiae. Using live-cell imaging, we find that sequestration of misfolded proteins and nascent polypeptides into two distinct compartments, stress granules, and Q-bodies, is triggered by the exhaustion of ATP. Both compartments readily dissolve in a PKA-dependent manner within minutes of glucose reintroduction and ATP level restoration. We identify the ATP hydrolase activity of Hsp104 disaggregase as the critical ATP-consuming process determining compartments abundance and size, even in optimal conditions. Sequestration of proteins into distinct compartments during acute metabolic stress and their retrieval during the recovery phase provide a competitive fitness advantage, likely promoting cell survival during stress.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Hidrólise , Agregados Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade
7.
Sci Rep ; 10(1): 17090, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33051497

RESUMO

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.


Assuntos
Adjuvantes Imunológicos/química , Vacinas contra Influenza/imunologia , Triterpenos/química , Animais , Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Citocinas/sangue , Imunoglobulina G/sangue , Vacinas contra Influenza/química , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos
8.
Nucleic Acids Res ; 48(20): 11284-11303, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33080019

RESUMO

The revolution in understanding higher order chromosome dynamics and organization derives from treating the chromosome as a chain polymer and adapting appropriate polymer-based physical principles. Using basic principles, such as entropic fluctuations and timescales of relaxation of Rouse polymer chains, one can recapitulate the dominant features of chromatin motion observed in vivo. An emerging challenge is to relate the mechanical properties of chromatin to more nuanced organizational principles such as ubiquitous DNA loops. Toward this goal, we introduce a real-time numerical simulation model of a long chain polymer in the presence of histones and condensin, encoding physical principles of chromosome dynamics with coupled histone and condensin sources of transient loop generation. An exact experimental correlate of the model was obtained through analysis of a model-matching fluorescently labeled circular chromosome in live yeast cells. We show that experimentally observed chromosome compaction and variance in compaction are reproduced only with tandem interactions between histone and condensin, not from either individually. The hierarchical loop structures that emerge upon incorporation of histone and condensin activities significantly impact the dynamic and structural properties of chromatin. Moreover, simulations reveal that tandem condensin-histone activity is responsible for higher order chromosomal structures, including recently observed Z-loops.


Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Cromatina/metabolismo , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Simulação de Dinâmica Molecular , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Alelos , Cromatina/química , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/química , Biologia Computacional , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/química , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Nucleossomos/química , Nucleossomos/metabolismo , Polímeros/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Nucleic Acids Res ; 48(19): 11172-11184, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32976599

RESUMO

Kinetochores are large multi-subunit complexes that attach centromeric chromatin to microtubules of the mitotic spindle, enabling sister chromatid segregation in mitosis. The inner kinetochore constitutive centromere associated network (CCAN) complex assembles onto the centromere-specific Cenp-A nucleosome (Cenp-ANuc), thereby coupling the centromere to the microtubule-binding outer kinetochore. CCAN is a conserved 14-16 subunit complex composed of discrete modules. Here, we determined the crystal structure of the Saccharomyces cerevisiae Cenp-HIKHead-TW sub-module, revealing how Cenp-HIK and Cenp-TW interact at the conserved Cenp-HIKHead-Cenp-TW interface. A major interface is formed by the C-terminal anti-parallel α-helices of the histone fold extension (HFE) of the Cenp-T histone fold domain (HFD) combining with α-helix H3 of Cenp-K to create a compact three α-helical bundle. We fitted the Cenp-HIKHead-TW sub-module to the previously determined cryo-EM map of the S. cerevisiae CCAN-Cenp-ANuc complex. This showed that the HEAT repeat domain of Cenp-IHead and C-terminal HFD of Cenp-T of the Cenp-HIKHead-TW sub-module interact with the nucleosome DNA gyre at a site close to the Cenp-ANuc dyad axis. Our structure provides a framework for understanding how Cenp-T links centromeric Cenp-ANuc to the outer kinetochore through its HFD and N-terminal Ndc80-binding motif, respectively.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Cinetocoros , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Proteínas de Ligação a DNA/química , Cinetocoros/química , Nucleossomos , Ligação Proteica , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Fuso Acromático
10.
Ceska Slov Farm ; 69(3): 130-136, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32972156

RESUMO

Geranylated flavanone diplacone is a flavanone iso- lated from Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae) with anti-inflammatory and antioxidant properties, nevertheless showing high lipophilicity and low solubility in water. Diplacone was therefore used as a model molecule for incorporation into glucan particles (GPs). GPs are prepared by intensive washing of yeast (Saccharomyces cerevisiae) leading to hollow shells consisting of β-(13)/β-(16) glucan mainly. The aim of this study was to compare anti-inflammatory potential of GPs-diplacone composites with the compound itself, GPs themselves and the physical mixture of GPs and diplacone. The cell line THP1-XBlueTM-MD2-CD14 derived from human leukemic monocytes was stimulated with lipopolysaccharide (LPS) from Escherichia coli to trigger inflammatory reaction. The composites of GPs with diplacone significantly decreased the activity of pro-inflammatory transcription factors nuclear factor κB (NF-κB) and activator protein 1 (AP-1).


Assuntos
Anti-Inflamatórios/farmacologia , Flavanonas/farmacologia , Glucanos/química , Saccharomyces cerevisiae/química , Humanos , NF-kappa B/metabolismo , Células THP-1 , Fator de Transcrição AP-1/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(38): 23539-23547, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907940

RESUMO

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.


Assuntos
RNA Fúngico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Uridina
12.
Biochim Biophys Acta Gen Subj ; 1864(12): 129706, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32805320

RESUMO

BACKGROUND: ScPrx1 is a yeast mitochondrial 1-Cys peroxiredoxins (Prx), a type of Prx enzyme which require thiol-containing reducing agents to resolve its peroxidatic cysteine. ScPrx1 plays important role in protection against oxidative stress. Mitochondrial thioredoxin ScTrx3 and glutathione have been reported to be the physiological electron donor for ScPrx1. However, the mechanism underlying their actions, especially the substrate recognition of ScPrx1 requires additional elucidation. METHODS: The structure of ScPrx1 was obtained through crystallization experiments. The oligomeric state of ScPrx1 was monitored by Blue-Native PAGE. Mutations were generated by the QuikChange PCR-based method. The ScPrx1 activity assay was carried out by measuring the change of 340 nm absorption of the NADPH oxidation. RESULTS: ScPrx1 exist as a homodimer in solution. The structure adopts a typical Prx-fold core which is preceded by an N-terminal ß-hairpin and has a C-terminal extension. Mutations (Glu94Ala, Arg198Ala and Trp126) close to the active site could enhance the catalytic efficiency of ScPrx1 while His83Ala and mutations on α4-ß6 region exhibited reduced activity. The biochemical data also show that the deletion or mutations on ScPrx1 C-terminal have 2-4.56 fold increased activity. CONCLUSION: We inferred that conformational changes of ScPrx1 C-terminal segment were important for its reaction, and the α4-ß6 loop regions around the ScPrx1 active sites were important for the catalytic function of ScPrx1. Collectively, these structural features provides a basis for understanding the diverse reductant species usage in different 1-Cys Prxs.


Assuntos
Peroxidases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Mitocôndrias/química , Mitocôndrias/metabolismo , Modelos Moleculares , Peroxidases/metabolismo , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Tiorredoxinas/metabolismo
13.
Int J Nanomedicine ; 15: 5083-5095, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764938

RESUMO

Background: ß-glucans are chiral polysaccharides with well-defined immunological properties and supramolecular wrapping ability of its chiral feature. However, the exploitation of chiral properties of these nanoparticles in drug delivery systems was seldom conducted. Methods: ß-glucan molecules with different chain lengths were extracted from yeast Saccharomyces cerevisiae and thereafter modified. In a conformation transition process, these ß-glucan molecules were then self-assembled with anti-cancer drug doxorubicin into nanoparticles to construct drug delivery systems. The chiral interactions between the drug and carriers were revealed by circular dichroism spectra, ultraviolet and visible spectrum, fourier transform infrared spectroscopy, dynamic light scattering and transmission electron microscope. The immune-potentiation properties of modified ß-glucan nanoparticles were evaluated by analysis of the mRNA expression in RAW264.7 cell model. Further, the antitumor efficacy of the nanoparticles against the human breast cancer were studied in MCF-7 cell model by cellular uptake and cytotoxicity experiments. Results: ß-glucan nanoparticles can activate macrophages to produce immune enhancing cytokines (IL-1ß, IL-6, TNF-α, IFN-γ). A special chirality of the carriers in diameter of 50~160 nm can also associate with higher drug loading ability of 13.9% ~38.2% and pH-sensitive release with a change of pH from 7.4 to 5.0. Cellular uptake and cytotoxicity experiments also prove that the chiral-active ß-glucan nanoparticles can be used in anti-cancer nanomedicine. Conclusion: This work demonstrates that ß-glucans nanoparticles with special chiral feature which leading to strong immunopotentiation ability and high drug loading efficiency can be developed as a novel type of nanomedicine for anti-cancer treatment.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , beta-Glucanas/imunologia , Adjuvantes Imunológicos/química , Animais , Antineoplásicos/imunologia , Dicroísmo Circular , Portadores de Fármacos/química , Humanos , Células MCF-7 , Camundongos , Nanopartículas/química , Células RAW 264.7 , Saccharomyces cerevisiae/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Estereoisomerismo , beta-Glucanas/química
14.
Appl Environ Microbiol ; 86(22)2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-32769193

RESUMO

Peptides present in growth media are essential for nitrogen nutrition and optimal growth of lactic acid bacteria. In addition, according to their amino acid composition, they can also directly or indirectly play regulatory roles and influence global metabolism. This is especially relevant during the propagation phase to produce high cell counts of active lactic acid bacteria used as starters in the dairy industry. In the present work, we aimed at investigating how the respective compositions of two different yeast extracts, with a specific focus on peptide content, influenced Streptococcus thermophilus metabolism during growth under pH-controlled conditions. In addition to free amino acid quantification, we used a multi-omics approach (peptidomics, proteomics, and transcriptomics) to identify peptides initially present in the two culture media and to follow S. thermophilus gene expression and bacterial protein production during growth. The free amino acid and peptide compositions of the two yeast extracts differed qualitatively and quantitatively. Nevertheless, the two yeast extracts sustained similar levels of growth of S. thermophilus and led to equivalent final biomasses. However, transcriptomics and proteomics showed differential gene expression and protein production in several S. thermophilus metabolic pathways, especially amino acid, citrate, urease, purine, and pyrimidine metabolisms. The probable role of the regulator CodY is discussed in this context. Moreover, we observed significant differences in the production of regulators and of a quorum sensing regulatory system. The possible roles of yeast extract peptides on the modulation of the quorum sensing system expression are evaluated.IMPORTANCE Improving the performance and industrial robustness of bacteria used in fermentations and food industry remains a challenge. We showed here that two Streptococcus thermophilus fermentations, performed with the same strain in media that differ only by their yeast extract compositions and, more especially, their peptide contents, led to similar growth kinetics and final biomasses, but several genes and proteins were differentially expressed/produced. In other words, subtle variations in peptide composition of the growth medium can finely tune the metabolism status of the starter. Our work, therefore, suggests that acting on growth medium components and especially on their peptide content, we could modulate bacterial metabolism and produce bacteria differently programmed for further purposes. This might have applications for preparing active starter cultures.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica , Peptídeos/metabolismo , Saccharomyces cerevisiae/química , Streptococcus thermophilus/metabolismo , Fermentação , Proteínas Fúngicas/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Peptídeos/administração & dosagem , Percepção de Quorum , Streptococcus thermophilus/efeitos dos fármacos
15.
Nucleic Acids Res ; 48(20): 11215-11226, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747934

RESUMO

The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5' to 3' exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between its subunits. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. Applying our approach to collections of ChIP-exo data, we demonstrate that it can recover aspects of regulatory complex spatial organization at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to analyze Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.


Assuntos
Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/metabolismo , Exonucleases/química , RNA de Transferência/genética , Proteínas Ribossômicas/genética , Alinhamento de Sequência/métodos , Fatores de Transcrição/metabolismo , Algoritmos , Animais , Sítios de Ligação , Simulação por Computador , Proteínas de Ligação a DNA/química , Bases de Dados Genéticas , Drosophila/química , Drosophila/genética , Drosophila/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/química , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA/métodos , Fator de Transcrição TFIIIB/química , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição TFIII/química , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Sítio de Iniciação de Transcrição
16.
PLoS Pathog ; 16(8): e1008733, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817694

RESUMO

Staphylococcus aureus (S. aureus) is one of the most common bacterial infections worldwide, and antibiotic resistant strains such as Methicillin-Resistant S. aureus (MRSA) are a major threat and burden to public health. MRSA not only infects immunocompromised patients but also healthy individuals and has rapidly spread from the healthcare setting to the outside community. However, all vaccines tested in clinical trials to date have failed. Immunocompromised individuals such as patients with HIV or decreased levels of CD4+ T cells are highly susceptible to S. aureus infections, and they are also at increased risk of developing fungal infections. We therefore wondered whether stimulation of antifungal immunity might promote the type of immune responses needed for effective host defense against S. aureus. Here we show that vaccination of mice with a fungal ß-glucan particle (GP) loaded with S. aureus antigens provides protective immunity to S. aureus. We generated glucan particles loaded with the four S. aureus proteins ClfA, IsdA, MntC, and SdrE, creating the 4X-SA-GP vaccine. Vaccination of mice with three doses of 4X-SA-GP promoted protection in a systemic model of S. aureus infection with a significant reduction in the bacterial burden in the spleen and kidneys. 4X-SA-GP vaccination induced antigen-specific Th1 and Th17 CD4+ T cell and antibody responses and provided long-term protection. This work suggests that the GP vaccine system has potential as a novel approach to developing vaccines for S. aureus.


Assuntos
Saccharomyces cerevisiae/imunologia , Infecções Estafilocócicas/imunologia , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Coagulase/administração & dosagem , Coagulase/genética , Coagulase/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae/química , Infecções Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/genética , Staphylococcus aureus/genética , Células Th1/imunologia , Células Th17/imunologia , Vacinação , beta-Glucanas/administração & dosagem , beta-Glucanas/imunologia
17.
Nat Commun ; 11(1): 3398, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636384

RESUMO

SWI/SNF remodelers play a key role in regulating chromatin architecture and gene expression. Here, we report the cryo-EM structure of the Saccharomyces cerevisiae Swi/Snf complex in a nucleosome-free state. The structure consists of a stable triangular base module and a flexible Arp module. The conserved subunits Swi1 and Swi3 form the backbone of the complex and closely interact with other components. Snf6, which is specific for yeast Swi/Snf complex, stabilizes the binding of the ATPase-containing subunit Snf2 to the base module. Comparison of the yeast Swi/Snf and RSC complexes reveals conserved structural features that govern the assembly and function of these two subfamilies of chromatin remodelers. Our findings complement those from recent structures of the yeast and human chromatin remodelers and provide further insights into the assembly and function of the SWI/SNF remodelers.


Assuntos
Adenosina Trifosfatases/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Humanos , Nucleossomos , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química
18.
Proc Natl Acad Sci U S A ; 117(31): 18459-18469, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694211

RESUMO

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Associadas a Diversas Atividades Celulares/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Proc Natl Acad Sci U S A ; 117(28): 16383-16390, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601238

RESUMO

Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Transporte Biológico , Canais de Cálcio/química , Canais de Cálcio/genética , Cardiolipinas/genética , Cardiolipinas/metabolismo , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mioblastos/metabolismo , Fosfolipídeos , Estabilidade Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
20.
Nat Struct Mol Biol ; 27(8): 743-751, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661420

RESUMO

Complexes containing a pair of structural maintenance of chromosomes (SMC) family proteins are fundamental for the three-dimensional (3D) organization of genomes in all domains of life. The eukaryotic SMC complexes cohesin and condensin are thought to fold interphase and mitotic chromosomes, respectively, into large loop domains, although the underlying molecular mechanisms have remained unknown. We used cryo-EM to investigate the nucleotide-driven reaction cycle of condensin from the budding yeast Saccharomyces cerevisiae. Our structures of the five-subunit condensin holo complex at different functional stages suggest that ATP binding induces the transition of the SMC coiled coils from a folded-rod conformation into a more open architecture. ATP binding simultaneously triggers the exchange of the two HEAT-repeat subunits bound to the SMC ATPase head domains. We propose that these steps result in the interconversion of DNA-binding sites in the catalytic core of condensin, forming the basis of the DNA translocation and loop-extrusion activities.


Assuntos
Proteínas de Transporte/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
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