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Deteriorated stress response in stationary-phase yeast: Sir2 and Yap1 are essential for Hsf1 activation by heat shock and oxidative stress, respectively.

Nussbaum I, Weindling E, Jubran R, Cohen A, Bar-Nun S - PLoS ONE (2014)

Bottom Line: However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging.This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses.Rather, factors that participate in Hsf1 activation appear to be compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.

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Precursors of NAD+ affect Hsf1 activity.BY4741 cells expressing Hsp26-GFP (A,B) or Btn2-GFP (C,D) grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with either NR (10 µM; A,C (+)) or NAM (10 mM; B,D (+)) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as levels of Hsp26-GFP (A,B) or Btn2-GFP (C,D) relative to actin (a loading control), as determined by quantified immunoblotting. Insets in C, D, levels of proteins in SP yeast drawn to a smaller scale. The data shown are mean plus standard error of at least 10 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (A) indicates a statistically significant difference (P = 0.001) between untreated cells and cells exposed to either HS or to HS plus NR, and between 30°C and 42°C in cells exposed to NR. Paired t-test applied to data of SP cells in (A) indicates a statistically significant difference (p = 0.07) between untreated cells and cells exposed to NR (*), and between 30°C or 42°C only in cells exposed to NR (**). There is no statistically significant difference between 30°C and 42°C in cells not exposed to NR (***).
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pone-0111505-g005: Precursors of NAD+ affect Hsf1 activity.BY4741 cells expressing Hsp26-GFP (A,B) or Btn2-GFP (C,D) grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with either NR (10 µM; A,C (+)) or NAM (10 mM; B,D (+)) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as levels of Hsp26-GFP (A,B) or Btn2-GFP (C,D) relative to actin (a loading control), as determined by quantified immunoblotting. Insets in C, D, levels of proteins in SP yeast drawn to a smaller scale. The data shown are mean plus standard error of at least 10 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (A) indicates a statistically significant difference (P = 0.001) between untreated cells and cells exposed to either HS or to HS plus NR, and between 30°C and 42°C in cells exposed to NR. Paired t-test applied to data of SP cells in (A) indicates a statistically significant difference (p = 0.07) between untreated cells and cells exposed to NR (*), and between 30°C or 42°C only in cells exposed to NR (**). There is no statistically significant difference between 30°C and 42°C in cells not exposed to NR (***).

Mentions: To investigate the possible contribution of NAD+ to Hsf1 activation, we supplied yeast with NR or nicotinamide (NAM) in order to increase NAD+ levels [69], [70]. Addition of NR or NAM to exponentially-growing cells had no significant effect on the basal activity or the heat shock response of Hsf1, as manifested by the levels of Hsp26-GFP or Btn2-GFP (Figure 5). In stationary-phase yeast, NR or NAM also exerted similar responses. On one hand, they attenuated the basal activity of Hsf1, as reflected by the levels of Hsp26-GFP (Figure 5A,B) or Btn2-GFP (Figure 5C,D, insets). More importantly, both NAD+ precursors inverted the effect of heat shock. Instead of its inhibitory effect in untreated stationary-phase yeast, supplementing either NR or NAM allowed a slight activation of Hsf1 by heat shock (Figure 5). These marginal effects are statistically significant, showing a difference in the basal Hsf1 activity in stationary-phase yeast between untreated cells and cells supplemented with either NAD+ precursor, as well as between 30°C and 42°C only in cells exposed to NAD+ precursor, but not in cells not supplemented with NAD+ (Figure 5).


Deteriorated stress response in stationary-phase yeast: Sir2 and Yap1 are essential for Hsf1 activation by heat shock and oxidative stress, respectively.

Nussbaum I, Weindling E, Jubran R, Cohen A, Bar-Nun S - PLoS ONE (2014)

Precursors of NAD+ affect Hsf1 activity.BY4741 cells expressing Hsp26-GFP (A,B) or Btn2-GFP (C,D) grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with either NR (10 µM; A,C (+)) or NAM (10 mM; B,D (+)) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as levels of Hsp26-GFP (A,B) or Btn2-GFP (C,D) relative to actin (a loading control), as determined by quantified immunoblotting. Insets in C, D, levels of proteins in SP yeast drawn to a smaller scale. The data shown are mean plus standard error of at least 10 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (A) indicates a statistically significant difference (P = 0.001) between untreated cells and cells exposed to either HS or to HS plus NR, and between 30°C and 42°C in cells exposed to NR. Paired t-test applied to data of SP cells in (A) indicates a statistically significant difference (p = 0.07) between untreated cells and cells exposed to NR (*), and between 30°C or 42°C only in cells exposed to NR (**). There is no statistically significant difference between 30°C and 42°C in cells not exposed to NR (***).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214751&req=5

pone-0111505-g005: Precursors of NAD+ affect Hsf1 activity.BY4741 cells expressing Hsp26-GFP (A,B) or Btn2-GFP (C,D) grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with either NR (10 µM; A,C (+)) or NAM (10 mM; B,D (+)) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as levels of Hsp26-GFP (A,B) or Btn2-GFP (C,D) relative to actin (a loading control), as determined by quantified immunoblotting. Insets in C, D, levels of proteins in SP yeast drawn to a smaller scale. The data shown are mean plus standard error of at least 10 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (A) indicates a statistically significant difference (P = 0.001) between untreated cells and cells exposed to either HS or to HS plus NR, and between 30°C and 42°C in cells exposed to NR. Paired t-test applied to data of SP cells in (A) indicates a statistically significant difference (p = 0.07) between untreated cells and cells exposed to NR (*), and between 30°C or 42°C only in cells exposed to NR (**). There is no statistically significant difference between 30°C and 42°C in cells not exposed to NR (***).
Mentions: To investigate the possible contribution of NAD+ to Hsf1 activation, we supplied yeast with NR or nicotinamide (NAM) in order to increase NAD+ levels [69], [70]. Addition of NR or NAM to exponentially-growing cells had no significant effect on the basal activity or the heat shock response of Hsf1, as manifested by the levels of Hsp26-GFP or Btn2-GFP (Figure 5). In stationary-phase yeast, NR or NAM also exerted similar responses. On one hand, they attenuated the basal activity of Hsf1, as reflected by the levels of Hsp26-GFP (Figure 5A,B) or Btn2-GFP (Figure 5C,D, insets). More importantly, both NAD+ precursors inverted the effect of heat shock. Instead of its inhibitory effect in untreated stationary-phase yeast, supplementing either NR or NAM allowed a slight activation of Hsf1 by heat shock (Figure 5). These marginal effects are statistically significant, showing a difference in the basal Hsf1 activity in stationary-phase yeast between untreated cells and cells supplemented with either NAD+ precursor, as well as between 30°C and 42°C only in cells exposed to NAD+ precursor, but not in cells not supplemented with NAD+ (Figure 5).

Bottom Line: However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging.This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses.Rather, factors that participate in Hsf1 activation appear to be compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.

Show MeSH
Related in: MedlinePlus