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1.
Methods Mol Biol ; 2564: 213-222, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107344

RESUMO

Fluorescent proteins within fluorescent fusions have been reported to affect cellular growth fitness via altering native protein function and intracellular localization. Here we report in detail a procedure to analyze the growth characteristics of yeast cells expressing such fusions in comparison to unmodified parental strain. This approach can serve as an initial step in fluorescent protein characterization in vivo.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Corantes/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
2.
Methods Mol Biol ; 2564: 269-286, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36107348

RESUMO

The expression of plant cysteine oxidase (PCO) enzyme in Saccharomyces cerevisiae enables the Arg/Cys N-degron pathway (Cys-NDP) for selective protein degradation that, in plants, functions as direct oxygen perception mechanism. A synthetic construct based on the plant Cys-NDP substrate related to apetala 2.12 (RAP2.12), the dual luciferase oxygen reporter (DLOR), exploits the N-terminal Cys of RAP2.12, and its oxygen-dependent degradation through the Cys-NDP. The luminescent output of DLOR can be used as a proxy for intracellular oxygen dynamics in budding yeast. Replacement of the luciferase reporter of the DLOR with fluorescent proteins would furthermore facilitate the imaging of reporter dynamics in living cells. In this chapter, we describe the methods for delivering the DLOR synthetic construct to yeast and calibrating its output by means of oxygen quantification in the culture with a physical oxygen sensor. We explain the setup needed to carry out hypoxic treatments with several colonies as replicates. We also describe the method to measure oxygen concentration in the culture, the closest indication of intracellular oxygen levels, as a way that would serve to calibrate the DLOR output. Finally, we propose a strategy to replace the luminescent reporters in the DLOR with fluorescent proteins to visualize oxygen dynamics in vivo.


Assuntos
Cisteína Dioxigenase , Saccharomyces cerevisiae , Cisteína/metabolismo , Cisteína Dioxigenase/metabolismo , Luciferases/metabolismo , Oxigênio/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
3.
Microb Cell Fact ; 21(1): 180, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064410

RESUMO

BACKGROUND: Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly. RESULTS: We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein. CONCLUSIONS: Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.


Assuntos
COVID-19 , Humanos , Fator de Acasalamento , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Saccharomyces cerevisiae/metabolismo , Saccharomycetales
4.
Cell Rep ; 40(10): 111316, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36070694

RESUMO

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Microscopia Crioeletrônica , Poli T , RNA Polimerase III/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões Terminadoras Genéticas
5.
Am J Hum Genet ; 109(9): 1692-1712, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36055214

RESUMO

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.


Assuntos
Proteínas de Ligação ao Cálcio , Doenças Mitocondriais , Proteínas de Ligação ao Cálcio/genética , Homeostase/genética , Humanos , Proteínas de Membrana/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Sistema Nervoso/metabolismo , Saccharomyces cerevisiae/metabolismo
6.
Zhongguo Zhong Yao Za Zhi ; 47(15): 4066-4073, 2022 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-36046896

RESUMO

CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.


Assuntos
Produtos Biológicos , Saccharomyces cerevisiae , Sistemas CRISPR-Cas , Carotenoides/metabolismo , Edição de Genes/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
7.
Nat Commun ; 13(1): 5273, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36071116

RESUMO

Binding to binding site clusters has yet to be characterized in depth, and the functional relevance of low-affinity clusters remains uncertain. We characterized transcription factor binding to low-affinity clusters in vitro and found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, small clusters with binding sites an order of magnitude lower in affinity give rise to high mean occupancies at physiologically-relevant transcription factor concentrations. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters in a synthetic and native gene regulatory network in S. cerevisiae. In both systems, clusters of low-affinity binding sites generated transcriptional output comparable to single or even multiple consensus sites. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.


Assuntos
Saccharomyces cerevisiae , Fatores de Transcrição , Sequência de Bases , Sítios de Ligação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076954

RESUMO

Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.


Assuntos
Autofagossomos , Proteínas de Saccharomyces cerevisiae , Animais , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Endopeptidases/metabolismo , Mamíferos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
9.
Molecules ; 27(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36080396

RESUMO

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c.


Assuntos
Citocromos c , Dimetil Sulfóxido , Citocromos c/metabolismo , Dimetil Sulfóxido/química , Cinética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Solventes , Termodinâmica , Água
10.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077205

RESUMO

Ripened oriental melon (Cucumis melo) with orange-colored flesh is rich in ß-carotene. Lycopene ß-cyclase (LCYB) is the synthetic enzyme that directly controls the massive accumulation of ß-carotene. However, the regulatory mechanism underlying the CmLCYB-mediated ß-carotene accumulation in oriental melon is fairly unknown. Here, we screened and identified a transcription factor, CmNAC34, by combining bioinformatics analysis and yeast one-hybrid screen with CmLCYB promoter. CmNAC34 was located in the nucleus and acted as a transcriptional activator. The expression profile of CmNAC34 was consistent with that of CmLCYB during the fruit ripening. Additionally, the transient overexpression of CmNAC34 in oriental melon fruit promoted the expression of CmLCYB and enhanced ß-carotene concentration, while transient silence of CmNAC34 in fruit was an opposite trend, which indicated CmNAC34 could modulate CmLCYB-mediated ß-carotene biosynthesis in oriental melon. Finally, the yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), ß-glucuronidase (GUS) analysis assay, and luciferase reporter (LUC) assay indicated that CmNAC34 could bind to the promoter of CmLCYB and positively regulated the CmLCYB transcription level. These findings suggested that CmNAC34 acted as an activator to regulate ß-carotene accumulation by directly binding the promoter of CmLCYB, which provides new insight into the regulatory mechanism of carotenoid metabolism during the development and ripening of oriental melon.


Assuntos
Cucumis melo , Cucumis melo/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta Caroteno/metabolismo
11.
PLoS One ; 17(9): e0272878, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36048821

RESUMO

Ribosomal DNA (rDNA) is the genetic loci that encodes rRNA in eukaryotes. It is typically arranged as tandem repeats that vary in copy number within the same species. We have recently shown that rDNA repeats copy number in the yeast Saccharomyces cerevisiae is controlled by cell volume via a feedback circuit that senses cell volume by means of the concentration of the free upstream activator factor (UAF). The UAF strongly binds the rDNA gene promoter, but is also able to repress SIR2 deacetylase gene transcription that, in turn, represses rDNA amplification. In this way, the cells with a smaller DNA copy number than what is optimal evolve to increase that copy number until they reach a number that sequestrates free UAF and provokes SIR2 derepression that, in turn, blocks rDNA amplification. Here we propose a mathematical model to show that this evolutionary process can amplify rDNA repeats independently of the selective advantage of yeast cells having bigger or smaller rDNA copy numbers. We test several variants of this process and show that it can explain the observed experimental results independently of natural selection. These results predict that an autoregulated feedback circuit may, in some instances, drive to non Darwinian deterministic evolution for a limited time period.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Variações do Número de Cópias de DNA , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Retroalimentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Seleção Genética , Fatores de Transcrição/metabolismo
12.
PLoS Genet ; 18(9): e1010056, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36054210

RESUMO

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3' end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt-the latter being the size of the in vivo templates-we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3' end. Mismatch incorporation depends on the 3'→ 5' proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3' end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ "chews back" the 3' end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3' end of the donor.


Assuntos
Proteínas de Saccharomyces cerevisiae , DNA Polimerase III/genética , Reparo do DNA/genética , Replicação do DNA/genética , Exonucleases/genética , Proteína 2 Homóloga a MutS/genética , Nucleotídeos/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Nat Commun ; 13(1): 5152, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36056028

RESUMO

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Recombinação Genética , Proteína de Replicação A/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
14.
Plant Signal Behav ; 17(1): 2115747, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36093942

RESUMO

The protein kinase GCN2 (General Control Nonderepressible2) and its phosphorylation target, the eukaryotic translation initiation factor (eIF)2α represent the core module of the plant's integrated stress response, a signaling pathway widely conserved in eukaryotes that can rapidly regulate translation in response to stressful conditions. Recent findings indicate that the Arabidopsis thaliana GCN2 protein operates under the command of reactive oxygen species (ROS) emanating from the chloroplast under a variety of abiotic stresses such as excess light. To get deeper insights into the mechanism of GCN2 activation under excess light, we assessed the role of amino acids in view of the classic function of GCN2 as a sensor of amino acid status. Additionally, given that osmoprotectants can counteract ROS-related stresses, we tested their ability to mitigate GCN2 activity. Our results demonstrate that certain amino acids and osmoprotectants attenuate eIF2α-phosphorylation under excess light stress to some degree. Future investigations into the biochemical mechanisms of these natural compounds on GCN2 signaling activity will provide better insights into the GCN2-eIF2α regulation.


Assuntos
Proteínas de Saccharomyces cerevisiae , Aminoácidos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas Serina-Treonina Quinases , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Fungal Biol ; 126(10): 658-673, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36116898

RESUMO

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.


Assuntos
Saccharomyces cerevisiae , Saccharum , Etanol/metabolismo , Genótipo , Microbiologia Industrial , Saccharomyces cerevisiae/metabolismo , Açúcares
16.
J Cell Biol ; 221(10)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36069810

RESUMO

The chaperone-mediated sequestration of misfolded proteins into inclusions is a pivotal cellular strategy to maintain proteostasis in Saccharomyces cerevisiae, executed by small heat shock proteins (sHsps) Hsp42 and Btn2. Direct homologs of Hsp42 and Btn2 are absent in other organisms, questioning whether sequestration represents a conserved proteostasis strategy and, if so, which factors are involved. We examined sHsps from Escherchia coli, Caenorhabditis elegans, and humans for their ability to complement the defects of yeast sequestrase mutants. We show that sequestration of misfolded proteins is an original and widespread activity among sHsps executed by specific family members. Sequestrase positive C. elegans' sHsps harbor specific sequence features, including a high content of aromatic and methionine residues in disordered N-terminal extensions. Those sHsps buffer limitations in Hsp70 capacity in C. elegans WT animals and are upregulated in long-lived daf-2 mutants, contributing to lifespan extension. Cellular protection by sequestration of misfolded proteins is, therefore, an evolutionarily conserved activity of the sHsp family.


Assuntos
Evolução Molecular , Proteínas de Choque Térmico Pequenas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/metabolismo , Humanos , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
J Agric Food Chem ; 70(36): 11336-11343, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36047715

RESUMO

Longifolene as an important sesquiterpene had enormous biological benefits. However, the low productivity of longifolene relying on chemical catalysis and plant extraction limited its wide application. Herein, the longifolene biosynthetic pathway was introduced into Saccharomyces cerevisiae, and multiple genetic strategies were applied to debottleneck the synthesis of longifolene, including the regulation of the rate-limiting enzymes, the elimination of the competitive pathways, the screening of the molecular chaperone to improve synthase activity, and the enhancement of the precursor supply. After combinationally applying these optimum strategies, the production of longifolene reached 27.30 mg/L in shake flasks and 1249 mg/L in fed-batch fermentation, respectively, which was the highest yield of longifolene reported thus far. It was demonstrated that the strategies applied in our work were effective in promoting the biosynthesis of longifolene, which not only laid a significant foundation for its industrial production but also provided a platform for the synthesis of other terpenoids.


Assuntos
Proteínas de Saccharomyces cerevisiae , Sesquiterpenos , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo
18.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077401

RESUMO

Ca2+ is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca2+ indicators allows real-time monitoring of cytosolic Ca2+ levels to study such responses. Here we explored the possibility of using the ratiometric Ca2+ indicator GEM-GECO for monitoring cytosolic Ca2+ concentration ([Ca2+]cyt) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca2+ ATPase Pmc1, which is involved in [Ca2+]cyt control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca2+ depletion, but to excessive Ca2+ signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca2+-free to Ca2+-bound state minutes after external addition of CaCl2. This was followed by gradual return of GEM-GECO to a Ca2+-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca2+]cyt monitoring in budding yeast.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
19.
Cells ; 11(17)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-36078056

RESUMO

Proteins are vital for the significant cellular activities of living organisms. However, not all of them are essential. Identifying essential proteins through different biological experiments is relatively more laborious and time-consuming than the computational approaches used in recent times. However, practical implementation of conventional scientific methods sometimes becomes challenging due to poor performance impact in specific scenarios. Thus, more developed and efficient computational prediction models are required for essential protein identification. An effective methodology is proposed in this research, capable of predicting essential proteins in a refined yeast protein-protein interaction network (PPIN). The rule-based refinement is done using protein complex and local interaction density information derived from the neighborhood properties of proteins in the network. Identification and pruning of non-essential proteins are equally crucial here. In the initial phase, careful assessment is performed by applying node and edge weights to identify and discard the non-essential proteins from the interaction network. Three cut-off levels are considered for each node and edge weight for pruning the non-essential proteins. Once the PPIN has been filtered out, the second phase starts with two centralities-based approaches: (1) local interaction density (LID) and (2) local interaction density with protein complex (LIDC), which are successively implemented to identify the essential proteins in the yeast PPIN. Our proposed methodology achieves better performance in comparison to the existing state-of-the-art techniques.


Assuntos
Mapas de Interação de Proteínas , Saccharomyces cerevisiae , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo
20.
Cells ; 11(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36078161

RESUMO

Ageing is accompanied by dramatic changes in chromatin structure organization and genome function. Two essential components of chromatin, the linker histone Hho1p and actin-related protein 4 (Arp4p), have been shown to physically interact in Saccharomyces cerevisiae cells, thus maintaining chromatin dynamics and function, as well as genome stability and cellular morphology. Disrupting this interaction has been proven to influence the stability of the yeast genome and the way cells respond to stress during chronological ageing. It has also been proven that the abrogated interaction between these two chromatin proteins elicited premature ageing phenotypes. Alterations in chromatin compaction have also been associated with replicative ageing, though the main players are not well recognized. Based on this knowledge, here, we examine how the interaction between Hho1p and Arp4p impacts the ageing of mitotically active yeast cells. For this purpose, two sets of strains were used-haploids (WT(n), arp4, hho1Δ and arp4 hho1Δ) and their heterozygous diploid counterparts (WT(2n), ARP4/arp4, HHO1/hho1Δ and ARP4 HHO1/arp4 hho1Δ)-for the performance of extensive morphological and physiological analyses during replicative ageing. These analyses included a comparative examination of the yeast cells' chromatin structure, proliferative and reproductive potential, and resilience to stress, as well as polysome profiles and chemical composition. The results demonstrated that the haploid chromatin mutants arp4 and arp4 hho1Δ demonstrated a significant reduction in replicative and total lifespan. These findings lead to the conclusion that the importance of a healthy interaction between Arp4p and Hho1p in replicative ageing is significant. This is proof of the concomitant importance of Hho1p and Arp4p in chronological and replicative ageing.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Actinas/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
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