Proteins with polyQ of different lengths show different characteristics.

<p>A Schematic representation of the constructs used to express polyQ proteins. Each contains a FLAG tag at its N-terminus, followed by huntingtin's exon 1 encoding the first 17 amino acids (Htt), a polyQ tract of different length, and GFP at the C-terminus. Expression of all polyQ proteins is driven by the pGal1 promoter that is induced by galactose and repressed by glucose. B Wild-type cells (W303–1b) expressing the 25Q, 47Q or 103Q were grown logarithmically (0.6–1 A₆₀₀) for 8 hours under galactose induction. Cell lysates were resolved by SDS-PAGE and polyQ proteins were detected by immunoblotting (IB) with a mouse anti-FLAG antibody followed by IRDye 800CW-conjugated goat anti-mouse IgG (right panel) or a rabbit anti-GFP followed by Dylight 680-labeled goat anti-rabbit IgG (left panel, red). A mouse anti-actin followed by IRDye 800CW-conjugated goat anti-mouse IgG was used as a loading control (left panel, green). Blots were visualized by the Odyssey Infrared Imaging System. C Wild-type cells (W303–1b) expressing the 25Q, 47Q, 103Q or an empty plasmid were grown logarithmically in glucose and 10-fold serial dilutions (starting with 7.5×10⁶ cells) were spotted on glucose or galactose plates.</p

Aggregation of PolyQ Proteins Is Increased upon Yeast Aging and Affected by Sir2 and Hsf1: Novel Quantitative Biochemical and Microscopic Assays

<div><p>Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in <i>Saccharomyces cerevisiae</i> aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an “aggregation timeline”. This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, <i>SIR2</i> and <i>HSF1</i>, affect aggregation of the polyQ proteins. In Δ<i>sir2</i> strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.</p></div

Quantitative microscopic assay reveals that aggregation of 47Q increases upon aging.

<p>A Wild-type cells (W303–1b) expressing 25Q, 47Q or 103Q were grown under galactose induction for up to 42 hrs. At each time point hundreds of cells were imaged by fluorescence microscopy. Images of 3 representative time points are presented. B Microscopic images from a similar experiment were analyzed for the presence of aggregates in individual cells as presented by the ratio between the maximal density (y axis) and the density (x axis) and 3 representative time points are presented (for all time points, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone.0044785.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone.0044785.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone.0044785.s003" target="_blank">S3</a>). C The ratio <i>R</i> between the maximal density and the density throughout the experiment plotted over time. At each time point the mean values ± SD are shown and the error bars reflect the variability between individual cells. D The fraction of cells with aggregates (defined as cells with <i>R</i> above a cutoff of 1.5) plotted over time.</p

Quantitative retardation assay reveals that aggregation of 47Q increases upon aging.

<p>Wild-type cells (W303–1b) expressing 25Q, 47Q or 103Q were grown under galactose induction for the indicated time, and at each time point 11.25×10⁵ cells (within the linear range; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone-0044785-g002" target="_blank">Figure 2</a>) were lysed. Early time points (8–16 hrs) were done separately from later time points. Total amounts A and captured aggregates B of polyQ proteins were quantified and Aggregation Index C was calculated as described in and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone-0044785-g002" target="_blank">Figure 2</a>. Data from 3 independent transformants, 3 triplicates each, are presented as mean ± SE. Statistical significance between the different polyQ was calculated by split-plot ANOVA. We found p<0.0001 between 25Q and 47Q; p<0.001 between 47Q and 103Q; and no significance between experiments of each polyQ.</p

Sir2 and Hsf1 are involved in the aging-dependent aggregation of polyQ proteins.

<p>A Δ<i>sir2</i> (RS1717; W303–1b <i>sir2</i>Δ::<i>his5</i>⁺) cells expressing 25Q, 47Q or 103Q were grown under galactose induction for the indicated time, collected, lysed, and Aggregation Index was calculated for each time point, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone-0044785-g003" target="_blank">Figure 3</a>. Data from 3 independent transformants, 3 triplicates each, are presented as mean ± SE. B Wild-type (WT, W303–1b) or Δ<i>sir2</i> cells expressing 47Q were grown for the indicated time under galactose induction. Wild-type cells were also exposed to 10mM nicotinamide (NAM). Cells were, collected and lysed and Aggregation Index for each time point was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone-0044785-g003" target="_blank">Figure 3</a>. C Wild-type cells (W303–1b) expressing the indicated polyQ proteins were grown under galactose induction for 72 hours with the indicated concentration (mM) of NAM. Cells were collected, lysed and Aggregation Index was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone-0044785-g003" target="_blank">Figure 3</a>. D Wild-type (W303–1b) cells expressing the indicated polyQ proteins and harboring either an empty (pRS314) or p<i>HSF1</i> plasmid were grown under galactose induction for the indicated time. Cells were collected, lysed and Aggregation Index was calculated for each time point as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044785#pone-0044785-g003" target="_blank">Figure 3</a>. Data from 3 independent transformants, 3 triplicates each, are presented as mean ± SE.</p

Deteriorated Stress Response in Stationary-Phase Yeast: Sir2 and Yap1 Are Essential for Hsf1 Activation by Heat Shock and Oxidative Stress, Respectively

<div><p>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 <i>Saccharomyces cerevisiae</i> 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 <i>S. cerevisiae</i> 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 Δ<i>yap1</i> mutant, Hsf1 does not respond to oxidative stress, while in Δ<i>sir2</i> 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.</p></div

Hsf1 activation by glucose starvation is maintained in stationary-phase yeast.

<p>Exponential BY4741 cells expressing Btn2-GFP were grown at 30°C in SC medium containing 2% (w/v) glucose. Cells were transferred to fresh media supplemented with the standard 2% or low 0.05% glucose and were either maintained at exponential growth (EG) or allowed to reach stationary-phase (SP) in these media. Prior to heat shock, cells were transferred to fresh media supplemented with the respective 2% or 0.05% glucose and further incubated at 30°C for 3 hrs. Cells were either incubated for 20 min at 30°C (−) or subjected to a 20 min HS at 42°C (+). Hsf1 activity was measured as levels of Btn2-GFP relative to actin (a loading control), as determined by quantified immunoblotting (upper panel). The data are the mean plus standard error of at least 4 independent experiments.</p

Hsf1 response to heat shock is lost in stationary-phase yeast.

<p>BY4741 cells harboring the HSE2-<i>LacZ</i> plasmid (A) or expressing Hsp26-GFP (B) or Btn2-GFP (C) were grown at 30°C to the indicated growth stages ((A, B) indicated as A₆₀₀; (C) EG, exponentially-growing; SP stationary-phase). Cells were either incubated for 20 min at 30°C (blue bars) or subjected to a 20 min heat shock at 42°C (red bars). (A) Hsf1 activity was measured as β-galactosidase specific activity. Mann-Whitney rank sum test indicates that the difference between 30°C and 42°C is statistically significant (p<0.001) up to 1.3 A₆₀₀ and not later (A₆₀₀≥1.8). (B, C) Hsf1 activity was measured as levels of Hsp26-GFP or Btn2-GFP relative to actin as a loading control, determined by SDS-PAGE and immunoblotting (upper panels). Blots were visualized and quantified by the Odyssey Infrared Imaging System (LI-COR Biosciences). The data are the mean plus standard error of 7–15 independent experiments. The fold induction by heat shock (A–C, insets) is the ratio of β-galactosidase specific activity or levels of Hsp26-GFD or Btn2-GFP relative to actin, at 42°C and at 30°C. (D) Wild-type BY4741 cells were grown at 30°C and on the indicated days were spotted on rich agar plates as 10-fold serial dilutions starting with 0.5A₆₀₀. (E) Wild-type <i>CDC48</i> strain and two independent colonies of the <i>cdc48-10</i> temperature-sensitive strain were grown at 30°C either exponentially (EG) or kept in culture for 2 days (SP). Ten-fold serial dilutions starting with 0.5A₆₀₀ were spotted on rich agar plates and incubated for 2 days at either 30°C or 39°C, as indicated.</p

Activation of Hsf1 by heat shock is mimicked by excess Sir2 and improved by the NAD⁺ precursor.

<p>(A) Wild-type BY4741 cells harboring HSE2-<i>LacZ</i> plasmid were transformed with an empty vector (−) or a centromeric p<i>SIR2</i> plasmid (+). Cells grown at 30°C either exponentially (EG) or to stationary-phase (SP) were either incubated for 20 min at 30°C (−) or subjected to a 20 min HS at 42°C (+). (B) Wild-type BY4741 cells harboring HSE2-<i>LacZ</i> plasmid were transformed with an empty vector (−) or a p<i>SIR2</i> plasmid (+). Cells grown at 30°C to the indicated growth phase were incubated for 30 min with (+) or without (−) NR (10 µM) 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 (+). (C) Activity in SP yeast from (B) drawn to a smaller scale. Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.</p

A schematic presentations of the various Hsf1 activation pathways.

<p>The three stresses, heat shock, oxidative stress and sugar starvation, activate the inactive Hsf1 through different mediators, Sir2, Yap1 and Snf1, respectively. Consequently, three distinct types of active Hsf1 are generated, HSF1^HS, Hsf1^OS and Hsf1^SS, respectively. These, in turn, transactivate the transcription of the indicated subsets of target genes.</p