Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance.

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.

Anticholinergic Exposure in Elderly Complex Chronic Patients: A Cross-Sectional Study.

BACKGROUND: Elderly patients with multiple chronic conditions are closely linked to polymedication, a condition that is also highly associated with the presence of adverse effects, such as those observed by anticholinergic activity. Anticholinergic burden is defined in a very variable way and is described inconsistently using different scores and providing different interpretations of the risk of suffering from anticholinergic adverse effects. OBJECTIVE: The objective is to analyse the anticholinergic risk exposure in elderly complex chronic patients. METHODS: A observational multicentre study was performed for a cohort of complex chronic patients over 65 years who received treatment with at least one drug with anticholinergic activity. Anticholinergic exposure was assessed using ten scales included in the Anticholinergic Burden Calculator. RESULTS: 473 patients were recruited, being 67.7% with excessive polypharmacy. 80 was the total number of anticholinergic drugs with any scale, with a median of 2 drugs with anticholinergic activity per patient (IQR=2). Three scales evaluated more than 70% of the patients (Chew: 79.1%; Drug Burden Index (DBI): 77.8%; Anticholinergic Cognitive Burden (ACB): 75.9%). The percentage of different drugs with anticholinergic properties evaluated ranged from 13.8% (Anticholinergic Burden Classification (ABC)) to 57.5% (DBI) and anticholinergic drugs prescriptions oscillated from 14% (Anticholinergic Risk Scale (ARS)) to 53.3% (DBI). 71.1% of patients were at risk (moderate and high risk) according to DBI vs. 9.7% by ARS at the opposite side. Important differences of anticholinergic risk in patients with excessive polypharmacy were in ACB, ABC and DBI scales. CONCLUSION: This study has highlighted clear differences between the scales used. DBI seems to be the scale that identifies a higher number of elderly chronic complex patients at risk of developing anticholinergic adverse effects.

The N-Terminal Region of Yeast Protein Phosphatase Ppz1 Is a Determinant for Its Toxicity.

The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. We show here that, in spite of their conserved catalytic domain, Ppz2 was not toxic when tested under the same conditions as Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1, even if its expression level was comparable to that of Ppz2. Similar amounts of yeast PP1c (Glc7) produced an intermediate effect on growth. Mutation of the Ppz1 myristoylable Gly2 to Ala avoided the localization of the phosphatase at the cell periphery but only slightly attenuated its toxicity. Therefore, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. This region is predicted to be intrinsically disordered and contains several putative folding-upon-binding regions which are absent in Ppz2 and might be relevant for toxicity.

Yeast Ppz1 protein phosphatase toxicity involves the alteration of multiple cellular targets.

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

Overexpression of budding yeast protein phosphatase Ppz1 impairs translation.

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.

Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study.

Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase.

Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation.

BACKGROUND: Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. RESULTS: We used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 °C. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1 (Thr108)) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. CONCLUSIONS: In this study we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.

Global phenotypic and genomic comparison of two Saccharomyces cerevisiae wine strains reveals a novel role of the sulfur assimilation pathway in adaptation at low temperature fermentations.

BACKGROUND: The wine industry needs better-adapted yeasts to grow at low temperature because it is interested in fermenting at low temperature to improve wine aroma. Elucidating the response to cold in Saccharomyces cerevisiae is of paramount importance for the selection or genetic improvement of wine strains. RESULTS: We followed a global approach by comparing transcriptomic, proteomic and genomic changes in two commercial wine strains, which showed clear differences in their growth and fermentation capacity at low temperature. These strains were selected according to the maximum growth rate in a synthetic grape must during miniaturized batch cultures at different temperatures. The fitness differences of the selected strains were corroborated by directly competing during fermentations at optimum and low temperatures. The up-regulation of the genes of the sulfur assimilation pathway and glutathione biosynthesis suggested a crucial role in better performance at low temperature. The presence of some metabolites of these pathways, such as S-Adenosilmethionine (SAM) and glutathione, counteracted the differences in growth rate at low temperature in both strains. Generally, the proteomic and genomic changes observed in both strains also supported the importance of these metabolic pathways in adaptation at low temperature. CONCLUSIONS: This work reveals a novel role of the sulfur assimilation pathway in adaptation at low temperature. We propose that a greater activation of this metabolic route enhances the synthesis of key metabolites, such as glutathione, whose protective effects can contribute to improve the fermentation process.

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast.

Low-temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low-temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature.

Functional analysis of lipid metabolism genes in wine yeasts during alcoholic fermentation at low temperature.

Wine produced by low-temperature fermentation is mostly considered to have improved sensory qualities. However few commercial wine strains available on the market are well-adapted to ferment at low temperature (10 - 15°C). The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. One strategy to modify lipid composition is to alter transcriptional activity by deleting or overexpressing the key genes of lipid metabolism. In a previous study, we identified the genes of the phospholipid, sterol and sphingolipid pathways, which impacted on growth capacity at low temperature. In the present study, we aimed to determine the influence of these genes on fermentation performance and growth during low-temperature wine fermentations. We analyzed the phenotype during fermentation at the low and optimal temperature of the lipid mutant and overexpressing strains in the background of a derivative commercial wine strain. The increase in the gene dosage of some of these lipid genes, e.g., PSD1, LCB3, DPL1 and OLE1, improved fermentation activity during low-temperature fermentations, thus confirming their positive role during wine yeast adaptation to cold. Genes whose overexpression improved fermentation activity at 12°C were overexpressed by chromosomal integration into commercial wine yeast QA23. Fermentations in synthetic and natural grape must were carried out by this new set of overexpressing strains. The strains overexpressing OLE1 and DPL1 were able to finish fermentation before commercial wine yeast QA23. Only the OLE1 gene overexpression produced a specific aroma profile in the wines produced with natural grape must.

Metabolomic comparison of Saccharomyces cerevisiae and the cryotolerant species S. bayanus var. uvarum and S. kudriavzevii during wine fermentation at low temperature.

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12 °C and 28 °C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12 °C and 28 °C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD(+) synthesis.

Phenotypic analysis of mutant and overexpressing strains of lipid metabolism genes in Saccharomyces cerevisiae: implication in growth at low temperatures.

The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10-15°C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition.

Analysis of low temperature-induced genes (LTIG) in wine yeast during alcoholic fermentation.

Fermentations carried out at low temperatures, that is, 10-15 °C, not only enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. In this study, we determined the transcriptional activity of 10 genes that were previously reported as induced by low temperatures and involved in cold adaptation, during fermentation with the commercial wine yeast strain QA23. Mutant and overexpressing strains of these genes were constructed in a haploid derivative of this strain to determine the importance of these genes in growth and fermentation at low temperature. In general, the deletion and overexpression of these genes did affect fermentation performance at low temperature. Most of the mutants were unable to complete fermentation, while overexpression of CSF1, HSP104, and TIR2 decreased the lag phase, increased the fermentation rate, and reached higher populations than that of the control strain. Another set of overexpressing strains were constructed by integrating copies of these genes in the delta regions of the commercial wine strain QA23. These new stable overexpressing strains again showed improved fermentation performance at low temperature, especially during the lag and exponential phases. Our results demonstrate the convenience of carrying out functional analysis in commercial strains and in an experimental set-up close to industrial conditions.