Acquired Resistance to Severe Ethanol Stress in Saccharomyces cerevisiae Protein Quality Control.

Acute severe ethanol stress (10% [vol/vol]) damages proteins and causes the intracellular accumulation of insoluble proteins in Saccharomyces cerevisiae On the other hand, a pretreatment with mild stress increases tolerance to subsequent severe stress, which is called acquired stress resistance. It currently remains unclear whether the accumulation of insoluble proteins under severe ethanol stress may be mitigated by increasing protein quality control (PQC) activity in cells pretreated with mild stress. In the present study, we examined the induction of resistance to severe ethanol stress in PQC and confirmed that a pretreatment with 6% (vol/vol) ethanol or mild thermal stress at 37°C significantly reduced insoluble protein levels and the aggregation of Lsg1, which is prone to denaturation and aggregation by stress, in yeast cells under 10% (vol/vol) ethanol stress. The induction of this stress resistance required the new synthesis of proteins; the expression of proteins comprising the bichaperone system (Hsp104, Ssa3, and Fes1), Sis1, and Hsp42 was upregulated during the pretreatment and maintained under subsequent severe ethanol stress. Since the pretreated cells of deficient mutants in the bichaperone system (fes1Δ hsp104Δ and ssa2Δ ssa3Δ ssa4Δ) failed to sufficiently reduce insoluble protein levels and Lsg1 aggregation, the enhanced activity of the bichaperone system appears to be important for the induction of adequate stress resistance. In contrast, the importance of proteasomes and aggregases (Btn2 and Hsp42) in the induction of stress resistance has not been confirmed. These results provide further insights into the PQC activity of yeast cells under severe ethanol stress, including the brewing process.IMPORTANCE Although the budding yeast S. cerevisiae, which is used in the production of alcoholic beverages and bioethanol, is highly tolerant of ethanol, high concentrations of ethanol are also stressful to the yeast and cause various adverse effects, including protein denaturation. A pretreatment with mild stress improves the ethanol tolerance of yeast cells; however, it currently remains unclear whether it increases PQC activity and reduces the levels of denatured proteins. In the present study, we found that a pretreatment with mild ethanol upregulated the expression of proteins involved in PQC and mitigated the accumulation of insoluble proteins, even under severe ethanol stress. These results provide novel insights into ethanol tolerance and the adaptive capacity of yeast. They may also contribute to research on the physiology of yeast cells during the brewing process, in which the concentration of ethanol gradually increases.

Btn2 is involved in the clearance of denatured proteins caused by severe ethanol stress in Saccharomyces cerevisiae.

Saccharomyces cerevisiae shows similar responses to heat shock and ethanol stress. Cells treated with severe ethanol stress activate the transcription of HSP genes and cause the aggregation of Hsp104-GFP, implying that severe ethanol stress as well as heat shock causes the accumulation of denatured proteins in yeast cells. However, there is currently no concrete evidence to show that severe ethanol stress causes protein denaturation in living yeast cells. In the present study, we investigated whether severe ethanol stress causes protein denaturation, and confirmed that a treatment with 10% (v/v) ethanol stress resulted in the accumulation of insoluble proteins and ubiquitinated proteins in yeast cells. We also found that increased denatured protein levels were efficiently reduced by the ubiquitin-proteasome system after the elimination of ethanol. Since our previous findings demonstrated that the expression of Btn2 was induced by severe ethanol stress, we herein examined the importance of Btn2 in protein quality control in cells treated with severe ethanol stress. btn2∆ cells showed a significant delay in the clearance of denatured proteins during the recovery process. These results provide further insights into the effects of severe ethanol stress on yeast proteostasis and the contribution of Btn2 to the efficient clearance of denatured proteins.

The VFH1 (YLL056C) promoter is vanillin-inducible and enables mRNA translation despite pronounced translation repression caused by severe vanillin stress in Saccharomyces cerevisiae.

Vanillin, furfural and 5-hydroxymethylfurfural (HMF) are representative fermentation inhibitors generated during the pretreatment process of lignocellulosic biomass in bioethanol production. These biomass conversion inhibitors, particularly vanillin, are known to repress translation activity in Saccharomyces cerevisiae. We have reported that the mRNAs of ADH7 and BDH2 were efficiently translated under severe vanillin stress despite marked repression of overall protein synthesis. In this study, we found that expression of VFH1 (YLL056C) was also significantly induced at the protein level by severe vanillin stress. Additionally, we demonstrated that the VFH1 promoter enabled the protein synthesis of other genes including GFP and ALD6 under severe vanillin stress. It is known that transcriptional activation of VFH1 is induced by furfural and HMF, and we verified that Vfh1 protein synthesis was also induced by furfural and HMF. The null mutant of VFH1 delayed growth in the presence of vanillin, furfural and HMF, indicating the importance of Vfh1 for sufficient tolerance against these inhibitors. The protein levels of Vfh1 induced by the inhibitors tested were markedly higher than those of Adh7 and Bdh2, suggesting the superior utility of the VFH1 promoter over the ADH7 or BDH2 promoter for breeding optimized yeast strains for bioethanol production from lignocellulosic biomass.

Protein synthesis of Btn2 under pronounced translation repression during the process of alcoholic fermentation and wine-making in yeast.

Acute high-concentration ethanol (> 9% v/v) has adverse effects on Saccharomyces cerevisiae, including the remarkable repression of bulk mRNA translation. Therefore, increased mRNA levels do not necessarily lead to an increase in the corresponding protein levels in yeast cells under severe ethanol stress. We previously identified that synthesis of Btn2 protein was efficiently induced even under the pronounced translation repression caused by acute severe ethanol stress under laboratory conditions. However, it remains to be clarified whether the translational activity is also repressed and whether the synthesis of Btn2 protein is induced during the process of alcoholic fermentation, in which the ethanol concentration increases gradually to reach high levels. In this study, we revealed that the pronounced translation repression and the translation of BTN2 are induced by high ethanol concentrations that form gradually during alcoholic fermentation using a wine yeast strain EC1118. Furthermore, we confirmed the induced expression of non-native genes driven by the BTN2 promoter during the later stage of the wine-making process. Our findings provide new information on the translation activity in yeast cells during alcoholic fermentation and suggest the utility of the BTN2 promoter for sustaining the fermentation efficiency and quality modification of alcoholic beverages.

Increased expression of 5-HT3 and NK 1 receptors in 5-fluorouracil-induced mucositis in mouse jejunum.

BACKGROUND AND OBJECTIVE: Although 5-fluorouracil (5-FU) is a widely used as chemotherapy agent, severe mucositis develops in approximately 80% of patients. 5-FU-induced small intestinal mucositis can cause nausea and vomiting. The current study was designed to investigate peripheral alterations due to the 5-FU-induced mucositis of neuronal and non-neuronal 5-HT3 and NK1 receptor expression by immunohistochemical analysis. METHODS: 5-FU was administered by i.p. injection to C57BL/6 mice. After 4 days, segments of the jejunum were removed. The specimens were analyzed by immunohistochemistry, real-time PCR, and enzyme immunoassay. RESULTS: The numbers of 5-HT3 receptor immunopositive cells and nerve fibers in mucosa were increased by 5-FU treatment. The 5-HT3 receptor immunopositive cell bodies were found only in jejunal submucosa and myenteric plexus in the 5-FU-treated mice. The numbers of NK1 receptor cells in mucosa and immunopositive expression of NK1 receptors in deep muscular plexus were dramatically increased in 5-FU-treated mice. Real-time PCR demonstrated that 5-FU treatment significantly increased mRNA levels of 5-HT3A, 5-HT3B, and NK1 receptors. The amounts of 5-HT and substance P increased after 5-FU treatment. The 5-HT3 or NK1 receptor immunopositive cells colocalized with both 5-HT and substance P. Furthermore, 5-HT3 and NK1 receptors colocalized with CD11b. CONCLUSIONS: The 5-HT3 and NK1 immunopositive macrophages and mucosal mast cells in lamina propria release 5-HT and substance P, which in turn activate their corresponding receptors on mucosal cells in autocrine and paracrine manners. It is assumed to result in the release of 5-HT and substance P in mucosa.

A reinforcement learning model with choice traces for a progressive ratio schedule.

The progressive ratio (PR) lever-press task serves as a benchmark for assessing goal-oriented motivation. However, a well-recognized limitation of the PR task is that only a single data point, known as the breakpoint, is obtained from an entire session as a barometer of motivation. Because the breakpoint is defined as the final ratio of responses achieved in a PR session, variations in choice behavior during the PR task cannot be captured. We addressed this limitation by constructing four reinforcement learning models: a simple Q-learning model, an asymmetric model with two learning rates, a perseverance model with choice traces, and a perseverance model without learning. These models incorporated three behavioral choices: reinforced and non-reinforced lever presses and void magazine nosepokes, because we noticed that male mice performed frequent magazine nosepokes during PR tasks. The best model was the perseverance model, which predicted a gradual reduction in amplitudes of reward prediction errors (RPEs) upon void magazine nosepokes. We confirmed the prediction experimentally with fiber photometry of extracellular dopamine (DA) dynamics in the ventral striatum of male mice using a fluorescent protein (genetically encoded GPCR activation-based DA sensor: GRABDA2m). We verified application of the model by acute intraperitoneal injection of low-dose methamphetamine (METH) before a PR task, which increased the frequency of magazine nosepokes during the PR session without changing the breakpoint. The perseverance model captured behavioral modulation as a result of increased initial action values, which are customarily set to zero and disregarded in reinforcement learning analysis. Our findings suggest that the perseverance model reveals the effects of psychoactive drugs on choice behaviors during PR tasks.

Trans 18-carbon monoenoic fatty acid has distinct effects from its isomeric cis fatty acid on lipotoxicity and gene expression in Saccharomyces cerevisiae.

Epidemiological studies have suggested that an excess intake of trans-unsaturated fatty acids increases the risk of coronary heart disease. However, the mechanisms of action of trans-unsaturated fatty acids in eukaryotic cells remain unclear. Since the budding yeast Saccharomyces cerevisiae can grow using fatty acids as the sole carbon source, it is a simple and suitable model organism for understanding the effects of trans-unsaturated fatty acids at the molecular and cellular levels. In this study, we compared the physiological effects of Δ9 cis and trans 18-carbon monoenoic fatty acids (oleic acid and elaidic acid) in yeast cells. The results obtained revealed that the two types have distinct effects on the expression of OLE1, which encodes Δ9 desaturase, and lipotoxicity in are1Δare2Δdga1Δlro1Δ and gat1Δ cells. Our results suggest that cis and trans 18-carbon monoenoic fatty acids exert different physiological effects in the regulation of gene expression and processing of excess fatty acids in yeast.