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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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Sir2 is required for Hsf1 response to heat shock but not to oxidative stress.Wild-type and Δsir2 BY4741 cells (A), or wild-type and Δsir2 W303-1b cells (B), harboring HSE2-LacZ plasmid, were grown at 30°C either exponentially (EG) or to stationary-phase (SP). Cells were either incubated for 20 min at 30°C (blue bars) or subjected to 20 min heat shock at 42°C (red bars). (C) Exponentially growing wild-type and Δsir2 BY4741 cells harboring HSE2-LacZ plasmid were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.
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pone-0111505-g006: Sir2 is required for Hsf1 response to heat shock but not to oxidative stress.Wild-type and Δsir2 BY4741 cells (A), or wild-type and Δsir2 W303-1b cells (B), harboring HSE2-LacZ plasmid, were grown at 30°C either exponentially (EG) or to stationary-phase (SP). Cells were either incubated for 20 min at 30°C (blue bars) or subjected to 20 min heat shock at 42°C (red bars). (C) Exponentially growing wild-type and Δsir2 BY4741 cells harboring HSE2-LacZ plasmid were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.

Mentions: A role for NAD+ in aging makes sense in light of the involvement of sirtuins in lifespan determination. These class III protein deacetylases that consume NAD+ are implicated in lifespan extension in many model organisms and in particular in mediating the beneficial effects of dietary restriction [39]–[41]. Sir2, the founding member of the sirtuins family, exerts opposite effects on S. cerevisiae aging, depending on the yeast aging model system. While RLS is extended by excess SIR2 and shortened upon SIR2 deletion, CLS is prolonged in Δsir2 mutant under dietary restriction [4], [6], [43], [44]. Despite the enigmatic contribution of Sir2 to yeast aging, we examined whether activation of Hsf1 was modified in mutants lacking the SIR2 gene. Following β-galactosidase activity, we found that although exponentially-growing Δsir2 cells (two different strains, BY4741 (Figure 6A) and W303-1b (Figure 6B)) exhibited somewhat higher basal Hsf1 activity than their wild-type counterparts, these mutants totally failed to respond to heat shock. In stationary-phase yeast, SIR2 deletion had no effect on the residual basal or heat shock-induced activities of Hsf1 (Figure 6A).


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)

Sir2 is required for Hsf1 response to heat shock but not to oxidative stress.Wild-type and Δsir2 BY4741 cells (A), or wild-type and Δsir2 W303-1b cells (B), harboring HSE2-LacZ plasmid, were grown at 30°C either exponentially (EG) or to stationary-phase (SP). Cells were either incubated for 20 min at 30°C (blue bars) or subjected to 20 min heat shock at 42°C (red bars). (C) Exponentially growing wild-type and Δsir2 BY4741 cells harboring HSE2-LacZ plasmid were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.
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Related In: Results  -  Collection

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pone-0111505-g006: Sir2 is required for Hsf1 response to heat shock but not to oxidative stress.Wild-type and Δsir2 BY4741 cells (A), or wild-type and Δsir2 W303-1b cells (B), harboring HSE2-LacZ plasmid, were grown at 30°C either exponentially (EG) or to stationary-phase (SP). Cells were either incubated for 20 min at 30°C (blue bars) or subjected to 20 min heat shock at 42°C (red bars). (C) Exponentially growing wild-type and Δsir2 BY4741 cells harboring HSE2-LacZ plasmid were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.
Mentions: A role for NAD+ in aging makes sense in light of the involvement of sirtuins in lifespan determination. These class III protein deacetylases that consume NAD+ are implicated in lifespan extension in many model organisms and in particular in mediating the beneficial effects of dietary restriction [39]–[41]. Sir2, the founding member of the sirtuins family, exerts opposite effects on S. cerevisiae aging, depending on the yeast aging model system. While RLS is extended by excess SIR2 and shortened upon SIR2 deletion, CLS is prolonged in Δsir2 mutant under dietary restriction [4], [6], [43], [44]. Despite the enigmatic contribution of Sir2 to yeast aging, we examined whether activation of Hsf1 was modified in mutants lacking the SIR2 gene. Following β-galactosidase activity, we found that although exponentially-growing Δsir2 cells (two different strains, BY4741 (Figure 6A) and W303-1b (Figure 6B)) exhibited somewhat higher basal Hsf1 activity than their wild-type counterparts, these mutants totally failed to respond to heat shock. In stationary-phase yeast, SIR2 deletion had no effect on the residual basal or heat shock-induced activities of Hsf1 (Figure 6A).

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