Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast

FEMS Yeast Research, 15, 2015, fov032 doi: 10.1093/femsyr/fov032 Advance Access Publication Date: 1 June 2015 Research Article RESEARCH ARTICLE Sara Sheibani1,2,† , Natalie K. Jones1,† , Rawan Eid1,3,† , Nada Gharib1 , Nagla T. T. Arab1,3 , Vladimir Titorenko4 , Hojatollah Vali2 , Paul A. Young3 and Michael T. Greenwood1,∗ 1 Department of Chemistry and Chemical Engineering, Royal Military College, Kingston, Ontario, 2 Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, 3 Biology Department, Queen’s University, Kingston, Ontario and 4 Department of Biology, Concordia University, Montreal, Quebec ∗ Corresponding author: Department of Chemistry and Chemical Engineering, Royal Military College (RMC), PO Box 17000, Station Forces, Kingston, Ontario, Canada K7K 7B4. Tel: +001-(613)-641-6000 ext. 3575; Fax: +001-(613)-542-9489; E-mail: † Contributed equally. One sentence summary: The analysis of human genes in yeast serves as a model to study the mechanisms by which lactic acidosis occurs and how it contributes to disease by inducing cell death. Editor: Cristina Mazzoni ABSTRACT We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast. Keywords: apoptosis; programmed cell death; anti-apoptosis; cell survival; lactate; LDHB INTRODUCTION Lactate production is catalyzed in a reversible reaction by the lactate dehydrogenase (LDH) mediated conversion of pyruvate, the end product of glycolysis. Lactate production and function is best understood in the context of anaerobic glycolysis of glucose (Draoui and Feron 2011). In the absence of oxygen, pyruvate that is produced by glycolysis cannot be further catabolized by the Krebs cycle leading to the accumulation of NADH. Given that the NADH cannot be further oxidized due to the anaerobic block in the Krebs cycle, there is depletion of NAD+. This prevents further catabolism of glucose and as a consequence the production of ATP is also halted. The re-routing of pyruvate to lactate by LDH circumvents this by utilizing NADH and regenerating of NAD+ (Diaz-Ruiz, Rigoulet and Devin 2011; Ring et al. 2012). Thus, lactate is critical for maintaining electron balance Received: 17 February 2015; Accepted: 19 May 2015  C FEMS 2015. All rights reserved. For permissions, please e-mail: 1 Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast 2 FEMS Yeast Research, 2015, Vol. 15, No. 0 dent LDH. Such a simple modification that permits the production of L-lactate is consistent with the concept that yeast has an endogenous NAD+ /NADH dependent LDH but that this activity is still poorly characterized (Passarella et al. 2008). A great deal of work looking at yeast responses to lactic acid including genome wide mutant screens suggest that lactic acid, in a lactate specific manner, serves to induce stress-dependent vacuolar cell death as it does in mammalian cells subjected to conditions of lactic acidosis (Abbott et al. 2008, 2009; Suzuki et al. 2012, 2013; Xu et al. 2013). In effect, the fact that catalase overexpression can reduce lactate mediated stress in yeast suggests that a form of programmed cell death is induced and that overexpression of pro-survival genes can prevent this effect (Abbott et al. 2009). In mammalian cells, there are two L-LDH genes, LDHA and LDHB. The LDHA isoform preferentially catalyzes the formation of lactate from pyruvate while the LDHB encoded enzyme preferentially carries out the reverse reaction and produces pyruvate from lactate. Thus, when examined, lactic acidosis has been associated with a preferential increase in LDHA over LDHB (Kay, Zhu and Tsoi 2007; Guo et al. 2011; Zhou, Liotta and Petricoin 2013). Further evidence for the importance of LDHA in mediating the apoptotic effect of lactic acidosis has been provided in studies by gene knock down in CHO cells (Jeong et al. 2001). In contrast evidence suggests a possible pro-survival function for LDHB since it is upregulated in some tumors, and it has been shown to act as a growth promoting gene in some tumors (McCleland et al. 2012; Zhou, Liotta and Petricoin 2013). Here, we have identified a cDNA for human L-lactate dehydrogenase B (LDHB) as a Bax suppressor in yeast. Analysis of this clone suggests that LDHB is a pro-survival sequence that protects yeast from multiple stresses. The ability of LDHB to prevent the lethal effects of exogenous lactate provides further evidence of the importance of yeast as a model for the study of lactate. MATERIALS AND METHODS Yeast strains, plasmid and growth The S. cerevisiae BY4742 (MATα his31 leu20 lys150 ura30) strain was used as the wild-type strain (EUROSCARF). All the mutants used in this study were purchased from Thermo Scientific and are isogenic to BY4742. Plasmids containing the cDNAs for L-LDHB (Bh134) and 14-3-3β/α expressed under the control of the galactose inducible GAL1 promoter in pYES-DEST52 (URA3 selectable marker) were isolated in our previously described Bax screen (Yang et al. 2006). The plasmid with a HIS3 selectable marker expressing activated form of mouse Bax under the regulation of the yeast GAL1 promoter was previously described (Yang et al. 2006). Yeast transformation was performed using the lithium acetate method. The yeast media used in this study consists of yeast nitrogen base (YNB) and 2% glucose as a carbon source supplemented with the required amino acids (leucine, histidine and lysine). When expression of genes under the control of the GAL1 promoter was required, the carbon source was replaced with 1% raffinose and 2% galactose. Spot and viability assays The ability of yeast transformants to grow under different (...truncated)