Cadmium induces GAPDH- and- MDH mediated delayed cell aging and dysfunction in Candida tropicalis 3Aer.

Eukaryotes employ various mechanisms to survive environmental stress conditions. Multicellular organisms eliminate permanently damaged cells by apoptosis, while unicellular eukaryotes like yeast react by decelerating cell aging. In the present study, transcriptomic and proteomic approaches were employed to elucidate the underlying mechanism of delayed apoptosis. Our findings suggest that Candida tropicalis 3Aer has a set of tightly controlled genes that are activated under Cd⁺² exposition. Acute exposure to Cd⁺² halts the cell cycle at the G₂/M phase checkpoint and activates multiple cytoplasmic proteins that overcome effects of Cd⁺²-induced reactive oxygen species. Prolonged Cd⁺² stress damages DNA and initiates GAPDH amyloid formation. This is the first report that Cd⁺² challenge initiates dynamic redistribution of GAPDH and MDH and alters various metabolic pathways including the pentose phosphate pathway. In conclusion, the intracellular redistribution of GAPDH and MDH induced by prolonged cadmium stress modulates various cellular reactions, which facilitate delayed aging in the yeast cell.

Keywords: Cd+2 stress; Candida tropicalis 3Aer; Non-protein thiols; Cell cycle; Apoptosis

Microorganisms whether prokaryotes or eukaryotes closely maintain their internal environment to optimize activities that depend on the extracellular environment. Fluctuations in the external environment can interfere with cellular physiology and destabilize the cell. The cell reacts to this by activating survival strategies that depend on the manner of the environmental challenges. Yeasts survive acute and prolonged environmental stresses, particularly heavy metal exposure exceptionally well (Gasch [13]; Gibson et al. [14]; Khan et al. [27]). There is no known biological role for cadmium (Chemek et al. [4]), the most toxic of the heavy metals. It inhibits cell growth, ultimately causes cell death (Kim et al. [29]; Rani et al. [45]) and is also highly carcinogenic (Li and Yuan [32]; Kuang et al. [30]). It inhibits DNA repair and the indirectly induces oxidative stress (Jin et al. [24]; Gomes et al. [16]). Initially metal ions inhibit the growth of plants (Bączek-Kwinta et al. [2]) but in the final biomass of the seedlings it was not found due to redirection of photosynthates towards protective mechanisms against toxic effects of metal ions (Bączek-Kwinta et al. [2]).

Intense, prolonged exposure to Cd+2 damages the cell, shutting down all metabolic activity and initiating programmed cell death, apoptosis, which involves the activation of a set of tightly regulated genes and proteins including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), metacaspases and malate dehydrogenase (MDH) (Saunders et al. [48]). Apoptosis is an evolutionarily conserved mechanism, and the underlying cascade is similar in all eukaryotic cells including yeasts. Several orthologs of mammalian apoptotic proteins have been identified in yeasts, and many conserved apoptotic pathways have been described. Yeasts have therefore been considered as model organisms for studying the complex apoptotic cascade to solve unanswered questions about apoptosis in mammalian cells (Madeo et al. [34]; Pozniakovsky et al. [42]; Gourlay and Ayscough [17]; Walter et al. [50]). In addition, one can explore different apoptotic pathways in yeast cells in a way that would not be possible in human cells due to the differential physiological reactions to same types of stress.

Replicative aging and chronological aging are two aging phenomena in yeast. Chronological aging in yeast is reported to be similar to mammalian apoptosis (Longo et al. [33]). Replicative aging is the maximum number of divisions undergone by an individual cell before death (Madeo et al. [35]), while chronological aging is the duration of culture viability during the stationary phase (Falcone and Mazzoni [11]). Both types of aging result in apoptotic cell death (Fabrizio et al. [10]). However, changes in cellular physiology such as overexpression of YAP1 and disruption of YCA1 may delay age-prompted cell death (Herker et al. [19]; Hill et al. [21]).

We previously studied the role of Candida tropicalis 3Aer in bioremediation of Cd+2 contaminated water (Khan et al. [27]). In the current investigation C. tropicalis 3Aer is a model organism for a transcriptomic and proteomic study of the molecular basis of the physiological responses to Cd+2 exposure associated with aging and cell death in eukaryotic cells.

Yeast strain

Candida tropicalis 3Aer was obtained from the First Fungal Culture Bank of Pakistan (FCBP) registered under the accession number FCBP1411. For all experiments yeast potato dextrose (YPD) broth/agar (pH=7.0) was inoculated with the yeast and incubated at 30°C for 36 hours.

Quantification of glutathione and non-protein thiol content

Levels of non-protein thiols, and oxidized (GSSG), and reduced (GSH) glutathione were determined to assess their function in Cd+2 detoxification. Control and Cd+2-treated (1 mM) yeast cell pellets were lysed and the cytoplasmic contents were harvested for spectrophotometric analysis (Rehman and Anjum [46]).

Transcriptomic analysis

The cultured cells were treated with 1 mM Cd+2 for 0, 5, 30, 60, 90, and 120 minutes. Cell cultures without Cd+2 served as controls. Cell pellets were harvested by centrifugation, and total cellular mRNA was extracted using a modified acid guanidinium thiocyanate-phenol-chloroform method. cDNA was synthesized and used as templates in real time PCR to determine the mRNA levels of the studied various genes: G2/M cyclins, cyclin-dependent kinases (Cdks), cytochrome P450, cysteine-γ-lyase and proteasome (Khan et al. [27]). After RNA quantification, using random hexamers complementary DNA was synthesized with the SuperScript™ III RT Kit (Invitrogen, USA), following the manufacturer's instructions.

Protein profiling and peptide identification

Cells were cultivated in YPD broth without Cd+2 (Control), and with 10 mM or 20 mM Cd+2 (Treated). The cell pellets were harvested by centrifugation and washed once with PBS. After processing, the protein in the washed cell pellets was quantified by Bradford assay and subjected to 2D gel electrophoresis. In the first step, the proteins were separated by isoelectric focusing on 7 cm Bio-Rad IPG strips (USA) followed by equilibration to minimize the effect of thiol residues. The proteins on the IPG strips were then resolved in the second dimension on 12 % polyacrylamide gel. Protein spots were visualized by silver staining and identified by Delta 2D software version 3.6. The protein spots displaying variations were excised and digested in the gel. The polypeptides were then eluted from the gel and were characterized by ESI Z-spray harboring Q-TOF Ultima Global (Micromass, Manchester, UK) mass spectrometer (Ramljak et al. [44]; Khan et al. [27],[28]).

Non-protein thiols and glutathione contents

Alterations in the levels of non-protein thiols (10.0 mM/g), GSSG (12.9 mM/g), and GSH (32.5 mM/g) in the presence of metal clearly reflected their involvement in counteracting Cd+2 toxicity. Exposure to Cd+2 doubled the content of non-protein thiols and GSH, while reducing the amount of GSSG (Fig. 1).

Graph: Fig. 1Alterations in the levels of non-protein thiols, oxidized (GSSG) and reduced glutathione (GSH) after Cd +2 exposure.

Transcriptomic analysis

Quantitative PCR showed that Cd+2 exposure up-regulated the expression of cysteine-γ-lyase, cytochrome P450, and proteasome genes, in a time-dependent fashion. After 15 minutes of exposure to Cd+2, Cd+2 exposure down-regulated the expression of G2/M cyclins and cyclin-dependent kinases (Cdks) genes (Fig. 2), which indicate delayed cell division.

Graph: Fig. 2Relative increase of Cdk and G2/M genes monitored by real time PCR over GAPDH as internal control (expressed as (n) fold increase).

Proteomic analysis

Twelve proteins were identified as part of the response to Cd+2 exposure (Table 1). The proteins displaying an altered expression were H2B (histone H2B), H4 (histone H4 type VIII), three different GAPDH isoforms (glyceraldehyde-3-phosphate dehydrogenase), MDH (malate dehydrogenase), FBP aldolase (fructose-bisphosphate aldolase), PGM1 (phosphoglycerate mutase 1), ODCase (orotidine 5'-phosphate decarboxylase), NHase (cobalt-containing nitrile hydratase), UTP-glucose-1-phosphate uridylyltransferase, Grx1 (glutaredoxin-1) and protein kinase domain-containing protein PPK3 (PPK3). Cd+2 exposure induced overexpression of NHase, MDH, ODCase, and GAPDH. These increased two-fold with doubling of the Cd+2 concentration. The levels of FBP aldolase, PPK3 and UTP-glucose-1-phosphate uridylyltransferase did not change markedly. However, even low Cd+2 concentrations altered the levels of PGM1 and H4.

Proteins exhibiting altered expression were identified by mass spectrometry

Heavy metals have cytotoxic effects that cause cell damage and death (Arshad et al. [1]). Various organisms have evolved different mechanisms to counteract these cytotoxic effects (Khan et al. [25], [26]). The present study used C. tropicalis 3Aer as a model organism to decipher the cellular responses to acute and prolonged Cd+2 exposure that results in DNA damage, protein misfolding and cytoplasmic imbalance.

C. tropicalis 3Aer has evolved two different strategies for dealing with and overcoming Cd+2 stress. Acute and mild exposure to Cd+2 activates acute cellular responses with an increase in cysteine contents, activation of protein folding machinery, activation of cellular redox system and cell cycle arrest at G2 (Fig. 3). The second approach is based on GAPDH- and MDH- regulated protein cascades designed to cope with prolonged and higher levels of Cd+2 contact (Fig. 3).

Graph: Fig. 3Acute exposure to Cd +2 directly or indirectly initiates protein misfolding. To monitor protein functionality C. tropicalis activates various members of the protein folding and degradation machinery (4). Cd +2 accumulation induces production of ROS, which aredetoxified by the Grx/GSH system (2), while some Cd +2 forms complexes with GSH and is trapped in vacuoles via the Ycf1 channel (3). In addition, cytochrome P450 in the ER plays a significant role in overcoming Cd +2 toxicity. All events are tightly regulated by the nucleus (1) to combat Cd +2 stress. Finally, C. tropicalis halts the cell cycle at G 2 /M checkpoint to rectify Cd +2 genotoxicity (6).

Cd+2 has been reported to induce production of reactive oxygen species (ROS) by interfering with the electron transport chain. This results in the accumulation of highly unstable semiubiquinones, which reduce molecular oxygen and generate ROS (Wang et al. [51]). Cd+2-induced ROS are the potential cause of the protein denaturation that activates the chaperons and protein folding machinery. Permanently denatured proteins cannot be refolded to their proper shapes and are targeted for degradation by cellular proteasomes (Fig. 3).

GSH plays a significant role by detoxifying ROS and sequestering Cd+2 resulting in the formation of bis-glutathionato-cadmium (Cd[GS]2) complexes (Ilyas and Rehman [22]; Elahi and Rehman [9]).. These are channeled to vacuoles via the YCF1 transmembrane protein (Sastry et al. [47]; Mandal et al. [36]). The presence of the Ycf1 gene and the increased production of GSH after exposure to Cd+2 supports the proposed model of Cd+2 detoxification. GSH is thought to reduce Cd+2 to elemental Cd through its sulfhydryl (-SH) group while being oxidized to GSSG with the help of glutaredoxin (Grx) (Draculic et al. [8]; Ilyas and Rehman [22]). Grx is an important component of the glutaredoxin system together with GSH, glutathione reductase and NADPH (Grant et al. [18]). There are two isoforms of Grx in yeast, Grx1 and Grx2. Grx1 is cytosolic while Grx2 is the mitochondrial form (Pai et al. [40]; Discola et al. [7]). In this study, Grx1 expression increased after Cd+2 exposure in a concentration dependent manner, strongly suggesting the direct involvement of the Grx/GSH system in reducing Cd+2-induced ROS damage (Fig. 3). The non-protein thiol, cysteine is found to be involved in the reduction of oxidative stress by sequestrating Cd+2.

The CYP450 cysteinato-heme enzyme that is localized in reticular membranes, i.e., endoplasmic reticulum also contributed to counteracting ROS (Meunier et al. [38]), as suggested by the massive production of the transcript (Fig. 2a). During the course of Cd+2 exposure, cysteine is actively synthesized to maintain the cellular cysteine reserves. These are required for the biosynthesis of cysteine-rich proteins; the expression of CGL increased by up to eight-fold (Fig. 2a). There is a direct or indirect involvement of ROS in the genotoxic effects that lead to the activation of the DNA repair system and cell cycle arrest (Arshad et al. [1]). The results of the qPCR studies showed that the cell cycle is arrested at the G2/M phase checkpoint. This ensures DNA repair prior to the M phase (Fig. 2b) and prevents entry into mitosis with a defective G2/M phase checkpoint, which would result in cell death after division (Cuddihy and O'connell [6]). Cell survival due to delayed cell division under Cd+2 exposure is comparable to delayed cell aging (Fig. 3).

Prolonged Cd+2 exposure resulted in the accumulation of H2O2, which initiated post-translational modification of GAPDH. GAPDH exists in various isoforms; one of which is involved in glucose metabolism, while the others are involved in various cellular processes at the nuclear level (Galibert et al. [12]; Goffeau et al. [15]). According to the proteomic analysis, the expression levels of the two isozymes increased in a concentration dependent manner, while levels of the third isoform decreased with increasing Cd+2 concentration. H2O2 usually initiates NO-mediated S-nitrosylation or GSH-mediated S-thiolation of GAPDH. S-thiolated GAPDH acts as a switch regulating cellular redox homeostasis, which activates the pentose phosphate pathway and increases cellular NADPH.

Proteomic analysis showed that prolonged Cd+2 exposure resulted in the overexpression of enzymes (FBP aldolase, PGM1 and UTP-glucose-1-phosphate uridylyltransferase) that directly or indirectly regulate glucose metabolism (glycolysis or pentose phosphate pathway). S-thiolation of GAPDH leads to its complexation and aggregate biosynthesis (shown in TEM images Figure 3), which activates the yeast metacaspases. These metacaspases function either as pro-apoptotic or pro-survival activators (Hill and Nystrom [20]). According to the current analysis, inactivated GAPDH stimulates the pro-survival activity of the metacaspases, and stimulates various chaperons and proteasomes to monitor protein folding. In addition, up-regulation of GAPDH supports the generation of Cd+2-induced DNA lesions as seen in Saccharomyces cerevisiae in response to H2O2 (Silva et al. [49]). Up-regulation of NHase suggests a role in S-nitrosylation of GAPDH leading to the generation of DNA stress-monitoring-GAPDH in C. tropicalis. It is first report to demonstrate alterations in histone expression levels associated with Cd+2 exposure in yeast. Generally, non-stoichiometric increases in histone levels are related to abnormal chromosome segregation and gene expression (Meeks-Wagner and Hartwell [37]; Clark-Adams et al. [5]).

GAPDH is actively involved in H2B histone expression during the S -phase of the cell cycle, depicting DNA replication (Zheng et al. [52]). However, modulation of H4 expression is related to DNA mutagenesis, recombination and repair (Prado and Aguilera [43]). The present study demonstrated the generation of Cd+2-mediated DNA lesions, while the modulation of H2B and H4 histone levels represents DNA replication and recombination-mediated repair as a reaction to the applied stress. Moreover, up-regulation of ODCase (pyrimidine synthesizing enzymes) also reflects DNA damage and repair, especially in pyrimidine residues due to prolonged Cd+2 exposure (O'brien et al. [39]). GAPDH is also involved in modifying the cytoplasmic Ca+2 balance, by modulating the ER channel command and control system, which leads to serious alterations in cellular behavior. In summary, GAPDH initiates cascade mechanisms that delay the normal cell cycle in the course of Cd+2 stress (Fig. 4). This strategy resembles delayed cell aging.

Graph: Fig. 4GAPDH and MDH plays predominant roles in delayed apoptosis. Prolonged Cd +2 stress induces formation of amyloid GAPDH, which in turn activates metacaspase. The activated metacaspase functionalizes chaperons and proteasomes to monitor protein folding (1). The S-thiolated GAPDH acts as a cellular switch regulating redox homeostasis and redirects glycolysis to the pentose phosphate pathway to maintain optimum cellular NADPH levels (3). Moreover, GAPDH also undergoes S-nitrosylation during stress and is converted to a nuclear regulator, which induces the expression of histones (H2B and H4) and ODCase, illustrating the genotoxic effects of Cd +2 stress (3). Furthermore, GAPDH also regulates Ca +2 ion mobility from ER channels (5), modulating cellular behavior. Overall, Cd +2 stress results in glucose deprivation leading to MDH accumulation, which in turn modulates the expression of those proteins involved in cell cycle arrest.

The proteomic analysis showed that the yeast cells faced glucose deprivation. Such circumstances generally lead to accumulation of MDH, which stabilizes p53-like elements (Lee et al. [31]). p53 modulates the expression of the proteins involved in cell cycle arrest. Moreover, yeast cells exhibit a cytoplasmic accumulation of MDH, supporting the role of Cd+2 in glucose deprivation. Alterations in PPK3 levels further support the activation of the carbon storage and stress response (Breitkreutz et al. [3]) that result in cell cycle arrest. Activation of PPK3 causes momentary changes in the glycogen metabolism, glycolysis and gluconeogenesis since many enzymes of these pathways are regulated by PK-mediated phosphorylation (Fig. 4).

Up-regulation of PGM1 by 10 mM Cd+2 indicates an accelerated rate of glycolysis and fermentation, synthesis of cellular RNA, and lipid biosynthesis, which ultimately increase the proliferation rate. Recent studies in cancer cells indicate that down-regulation of PGM1 in malignant cells severely targets cellular proliferation (Jiang et al. [23]). Likewise, up-regulation of FBP aldolase (regulator of fructose-1,6-bisphosphate) by 20 mM Cd+2 indicates that the cell is trying to maintain its normal life style and manage the stress. Fructose-1,6-bisphosphate is a key player in the Ras-cAMP-PKA pathway, which has an important role in stress management and cellular proliferation (Peeters et al. [41]).

In conclusion, Candida tropicalis 3Aer possesses two pathways to deal with Cd+2 toxicity. Acute exposure to Cd+2 activated an acute response resulting in cell cycle arrest. Prolonged stress, on the other hand, is met by a cascade controlled by GAPDH and MDH, and leads to delayed cell aging and dysfunction. Current findings open a new horizon to explore the role of previously known metabolic enzymes in the regulation of cell cycle and aging.

The authors express their gratitude to the Pakistan Science Foundation (PSF), Islamabad-Pakistan for support the present research work under project No. PSF/Res/ Envr (97).

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