Involvement of Yeast HSP90 Isoforms in Response to Stress and Cell Death Induced by Acetic Acid

et al. (2013) Involvement of Yeast HSP90 Isoforms in Response to Stress and Cell Death Induced by Acetic Acid. PLoS ONE 8(8): e71294. doi:10.1371/journal.pone.0071294 Involvement of Yeast HSP90 Isoforms in Response to Stress and Cell Death Induced by Acetic Acid Alexandra Silva 0 Bele m Sampaio-Marques 0 A ngela Fernandes 0 Laura Carreto 0 Fernando Rodrigues 0 Martin Holcik 0 Manuel A. S. Santos 0 Paula Ludovico 0 Irina V. Lebedeva, Enzo Life Sciences, Inc., United States of America 0 1 Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal, 2 Life and Health Sciences Research Institute/3B's - PT Government Associate Laboratory, Braga/Guimara es, Portugal, 3 Department of Biology and Centre d'Enseignement de la Statistique Applique e a` la Me decine, University of Aveiro , Aveiro , Portugal , 4 Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute , Ottawa, Ontario , Canada Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2a by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress. - Funding: This work was supported by Fundacao para a Ciencia e Tecnologia and COMPETE/QREN/EU (PTDC/BIA-MIC/114116/2009), and by the Canadian Institute for Health Research (MOP 89737 to MH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Yeast cells respond to stress in multiple ways ranging from activation of pathways that promote survival to those eliciting programmed cell death (PCD). The cells initial response to an imposed stress is driven towards survival and the defense against and recovery from the insult. Nonetheless, the persistency of the noxious unresolved stimulus eventually activates death signaling pathways. Hydrogen peroxide (H2O2) and acetic acid are among the stress agents whose yeast cellular responses are best characterized [13]. Our previous work has shown that acetic acidinduced apoptosis in yeast cells is characterized by the impairment of general protein synthesis, yet it is paradoxically associated with the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) [4]. However, the biological relevance of these alterations in regulating stress recovery or the progression to PCD and the molecular mechanism driving HSP90 induction are still unknown. Mammalian HSP90 chaperones are a family of highly conserved proteins with crucial functions in various cellular processes including signal transduction, protein folding and degradation and protein trafficking between sub-cellular compartments [58]. HSP90 proteins play important anti-apoptotic functions [9]; however, recent reports also suggested their involvement in necrotic mechanisms [813] since the HSP90 inhibitors prevent necrosis induced by Fas- and tumor-necrosis factor receptor 1 [10,11]. Most likely, the different HSP90 isoforms, their selective post-translational modifications and client proteins could explain the dual role of HSP90 in cell survival and death. In yeast, two genes, HSC82 and HSP82, encode Hsp90 proteins, which are 97% identical [14], but are apparently differently regulated [15]. In physiological conditions, Hsc82p is present at 20-fold greater levels than Hsp82p; however, upon heat shock HSP82 expression is induced more than 20-fold [16]. Among the different HSP90 functions, these chaperones are also involved in yeast translational control [17,18] and necrotic cell death [12,19]. Despite this, the translation control of HSP90 mRNAs and their impact on yeast cell death has not yet been addressed. Translation control allows cells to reduce protein synthesis and prevent continued gene expression under potentially error prone conditions, save cellular energy and allow the reprogramming of existing mRNAs and proteins, conferring on cells the plasticity needed to deal with stress. Thus, translational control has been identified as an important biological determinant of cell fate through its tight regulation of stress responses and PCD [2023]. Nonetheless, translational control of yeast PCD is still poorly understood. The deletion of the LSM4 gene, involved in yeast mRNA decay, was shown to inhibit mRNA decapping leading to an increase in mRNA stability and ultimately triggering a caspasedependent apoptotic process [2426]. Furthermore, an extensive degradation of ribosomal RNAs has been described during H2O2and acetic acid-induced apoptosis [27]. In the presence of H2O2, the degradation of ribosomal RNAs is correlated with decreased global translation mediated by the phosphorylation of eIF2a by Gcn2p kinase (the amino acid control kinase) [28]. Acetic acid treatment also decreased the levels of translation factors eIF4A, eEF1A, eEF2 and eEF3A, a condition known to induce a severe amino acid-starvation response [4]. In this study, we demonstrate the existence of a translational reprogramming of the HSP90 isoforms, particularly of HSP82 during the progression of acetic acid-induced cell death. In addition, we show that HSP90 isoforms could play antagonistic roles in the cell death process. Materials and Methods Strains, Media and Treatments Saccharomyces cerevisiae BY4742 (MATa his3D1 leu2D0 lys2D0 ura3D0) strain and its isogenic derivatives Dgcn2, Dhsc82 and Dhsp82 (EUROSCARF) were used. For acetic acid treatment, S. cerevisiae cells were grown until the middle exponential phase in liquid YPD medium containing glucose (2%, w/v), yeast extract (0.5%, w/v) and peptone (1%, w/v). Cells were harvested, resuspended in fresh medium (pH 3.0) and incubated at 26uC for 1 h. Then cells were resuspended (108 cells/mL) in YPD fresh medium (pH 3.0) followed by the addition of 160, 180, 195 or 210 mM acetic acid and incubation for 15, 30, 60, 12 (...truncated)