Genome wide analysis of small heat shock proteins involved in yeast ageing

Ageing is a phenomenon common to almost all living organisms and is characterised by the accumulation of changes with time that are associated with the ever-increasing susceptibility to disease and inevitably death. The rate of ageing is species-specific, indicating a strong genetic component. It is now widely accepted that genes involved in basic cellular processes such as stress resistance, metabolic regulation and genomic stability determine longevity in divergent organisms from yeast to mammals. This suggests that there may be conserved universal mechanisms involved in ageing. Furthermore, lifespan can be increased in all these model organisms by reducing the nutrients consumed - a phenomenon known as dietary restriction (DR). Saccharomyces cerevisiae is a useful model organism in which to study ageing and has been at the forefront of recent pioneering work on the molecular mechanisms underlying lifespan extension by DR. DR can be studied by reducing the glucose concentration from the standard 2% down to 0.5% or below, or by using genetic mimics. However, the exact mechanisms of lifespan extension by dietary restriction remain unclear and highly controversial. Research in our laboratory has identified two proteins in yeast that are induced in response to DR and thus correlate with longevity. Both proteins, Hsp12 and Hsp26, belong to the small heat shock protein (sHsp) family. Previous work by the Morgan laboratory has found that Hsp12 is essential for the longevity effect of DR and have solved the structure of the protein by NMR. Further studies have revealed a genetic interaction between HSP12 and HSP26, as hsp12/hsp26Δ double knockouts show a strongly reduced mean and maximum replicative lifespan. Despite this, the hsp12/hsp26∆ double knockout is not defective in various processes associated with yeast ageing, including stress resistance, rDNA silencing or protein aggregation. To shed light on the mechanisms by which Hsp12 and Hsp26 affect longevity, we employed unbiased approaches of synthetic genetic array (SGA) and quantitative fitness analysis (QFA) to identify genetic interactions of HSP12 and HSP26 on a genome-wide scale. This involved the generation of thousands of double mutant strains and analysis of their growth under various conditions, including DR. Results from the SGA analysis have revealed genetic interactions between HSP12 and HSP26 with the regulation of transcription from RNA polymerase II and processes associated with the mitochondria and vacuoles. QFA data is still to be analysed but ultimately we hope that QFA will re-confirm the genetic interactions identified by SGA analysis. We hope both SGA analysis and QFA data will provide insight into the cellular functions of Hsp12 and Hsp26 and how these proteins affect ageing, in particular lifespan extension by DR

Proteomic analysis of dietary restriction in yeast reveals a role for Hsp26 in lifespan extension

Dietary restriction (DR) has been shown to increase lifespan in organisms ranging from yeast to mammals. This suggests that the underlying mechanisms may be evolutionarily conserved. Indeed, upstream signalling pathways, such as TOR, are strongly linked to DR-induced longevity in various organisms. However, the downstream effector proteins that ultimately mediate lifespan extension are less clear. To shed light on this, we used a proteomic approach on budding yeast. Our reasoning was that analysis of proteome-wide changes in response to DR might enable the identification of proteins that mediate its physiological effects, including replicative lifespan extension. Of over 2500 proteins we identified by liquid chromatography-mass spectrometry, 183 were significantly altered in expression by at least 3-fold in response to DR. Most of these proteins were mitochondrial and/or had clear links to respiration and metabolism. Indeed, direct analysis of oxygen consumption confirmed that mitochondrial respiration was increased several-fold in response to DR. In addition, several key proteins involved in mating, including Ste2 and Ste6, were downregulated by DR. Consistent with this, shmoo formation in response to α-factor pheromone was reduced by DR, thus confirming the inhibitory effect of DR on yeast mating. Finally, we found that Hsp26, a member of the conserved small heat shock protein (sHSP) family, was upregulated by DR and that overexpression of Hsp26 extended yeast replicative lifespan. As overexpression of sHSPs in Caenorhabditis elegans and Drosophila has previously been shown to extend lifespan, our data on yeast Hsp26 suggest that sHSPs may be universally conserved effectors of longevity

Hsp12 is unstructured in solution, but folds in the presence of SDS.

<p>(A) ¹H-¹⁵N HSQC spectrum of Hsp12 in aqueous solution at 298 K. The spectrum shows only sharp peaks with random coil shifts indicating the absence of any structured regions. (B) ¹H-¹⁵N HSQC spectrum of Hsp12 at 303 K in the presence of increasing concentrations of SDS (0, 1, 2, 5, 8 mM Red -> Blue). SDS causes a considerable increase in the amount of chemical shift dispersion implying increased levels of folded material/regions. (C) Assigned ¹H-¹⁵N HSQC spectrum of Hsp12 at 318 K in the presence of 100 mM SDS.</p

Ensemble of structures calculated for micelle-bound Hsp12 overlaid on each of the four helices.

<p>Ensemble of twenty structures overlaid on helices I (A), II (B), III (C) and IV (D). No long-range interactions were detected and so the helices appear free to move independently with no overall fold being evident.</p

DR induces expression of a relatively small number of proteins.

<p>Wild type BY4741 yeast cells were grown in standard (2% glucose) and DR (0.5% glucose) conditions before lysis and separation of proteins by 2-D electrophoresis. Wide-range (pH 3–10) gels revealed no obvious reproducible differences in protein expression, as illustrated by representative gels shown in panel (A). Narrow pH range gels (pH 3–5.6 and 5.3–6.5) revealed changes in protein spots, which were identified by mass spectrometry. Selected identified proteins are indicated by arrows in panels (B) and (C).</p

Backbone dynamics and chemical shift-based secondary structure of Hsp12.

<p><i>T</i>₁, <i>T</i>₂ and <i>T</i>₁/<i>T</i>₂ relaxation values are shown for Hsp12 in the presence (A,C,E) and absence (B,D,F) of 100 mM SDS at 318 K. <i>T</i>₁ and <i>T</i>₂ relaxation times for micelle-bound (A,C) Hsp12 show significant variation; contrasting with the similar relaxation values observed for free Hsp12 (B,D). Micelle-bound Hsp12 (E) shows grouped variations in the <i>T</i>₁/<i>T</i>₂ values ranging from approximately 1.5 to 14, indicating a wide range of mobility and a clear differentiation of secondary structure elements; whereas the free form (F) shows consistent values of around 2, indicating a completely unstructured protein. (G) The assigned chemical shifts at 318 K in 100 mM SDS expressed as deviation from random coil are shown aligned with the primary sequence and the positions of the α-helices.</p

Helical properties of micelle-bound Hsp12.

<p>(A) The four α-helices are represented as ribbons and colour coded from the N-terminus (blue) to the C-terminus (red) in a representative structure. (B,C) Analysis of charge distribution with hydrophobic residues labelled green and charged residues labelled red in both ribbon (B) and surface (C) representation, illustrating the amphipathic nature of Hsp12. Structures were generated using Chimera.</p