A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response inSchizosaccharomyces pombe

Abstract

Using an integrated approach incorporating proteomics, metabolomics and published mRNA data, we have investigated the effects of hydrogen peroxide on wild type and a Sty1p-deletion mutant of the fission yeast Schizosaccharomyces pombe. Differential protein expression analysis based on the modification of proteins with matched fluorescent labelling reagents (2-D-DIGE) is the foundation of the quantitative proteomics approach. This study identifies 260 differentially expressed protein isoforms from 2-D-DIGE gels using MALDI MS and reveals the complexity of the cellular response to oxidative stress and the dependency on the Sty1p stress-activated protein kinase. We show the relationship between these protein changes and mRNA expression levels identified in a parallel whole genome study, and discuss the regulatory mechanisms involved in protecting cells against hydrogen peroxide and the involvement of Sty1p-dependent stress-activated protein kinase signalling. Metabolomic profiling of 29 intermediates using 1 H NMR was also conducted alongside the protein analysis using the same sample sets, allowing examination of how the protein changes might affect the metabolic pathways and biological processes involved in the oxidative stress response. This combined analysis identifies a number of interlinked metabolic pathways that exhibit stress-and Sty1-dependent patterns of regulation.

Saccharomyces Cerevisiae as a Tool to Evaluate the Effects of Herbicides on Eukaryotic Life

“…Additionally, the comparative proteomic analysis showed very clearly the under-expression of several enzymes belonging to the carbohydrate metabolism group in herbicide-treated cells (Braconi et al, 2009). Our data were, again, in accordance with works studying cellular responses to oxidative stress (Godon et al, 1998;Magherini et al, 2007;Perzov et al, 2000;Sales et al 2000;Weeks et al, 2006) and underlined the susceptibility of glycolysis to oxidative insults. A decrease in the activity of glycolytic enzymes is thought to be beneficial during exposure to oxidants (Costa et al, 2002;Shanmuganathan et al, 2004).…”

Oxidative stress response pathways: Fission yeast as archetype

“…Another mechanism employed by Sty1 to alter translation initiation and affect protein expression in response to oxidative stimuli involves the inhibition of the proteolytic cleavage of the Pabp polyA-binding protein, which is involved in shortening the polyA tail of cellular transcripts (Weeks et al, 2006).…”

Dynamic cofilin phosphorylation in the control of lamellipodial actin homeostasis

“…In-gel tryptic digestion of excised gel pieces was performed as previously described (Weeks et al, 2006). For peptide mass fingerprinting, 0.5 l of tryptic digest was mixed with 1 l of matrix -saturated aqueous 2,5-dihydroxybenzoic acid (DHB) -and spotted onto a sample target and dried.…”

“…2D difference gel electrophoresis (2D-DIGE) was carried out in a dedicated clean-room essentially as described (Weeks et al, 2006). For this, 120 g of protein extract from each triplicate sample was labelled with NHS-Cy3 or NHS-Cy5 at 4 pmol dye/g protein on ice in the dark for 30 minutes.…”