Effect of cadmium on essential metals and their impact on lipid metabolism in Saccharomyces cerevisiae

Abstract

Cadmium (Cd) is a toxic heavy metal that induces irregularity in numerous lipid metabolic pathways. Saccharomyces cerevisiae, a model to study lipid metabolism, has been used to establish the molecular basis of cellular responses to Cd toxicity in relation to essential minerals and lipid homeostasis. Multiple pathways sense these environmental stresses and trigger the mineral imbalances specifically calcium (Ca) and zinc (Zn). This review is aimed to elucidate the role of Cd toxicity in yeast, in three different perspectives: (1) elucidate stress response and its adaptation to Cd, (2) understand the physiological role of a macromolecule such as lipids, and (3) study the stress rescue mechanism. Here, we explored the impact of Cd interference on the essential minerals such as Zn and Ca and their influence on endoplasmic reticulum stress and lipid metabolism. Cd toxicity contributes to lipid droplet synthesis by activating OLE1 that is essential to alleviate lipotoxicity. In this review, we expanded our current findings about the effect of Cd on lipid metabolism of budding yeast.

Introduction

Cadmium (Cd) is a toxic heavy metal, hazardous for both humans and animals and has been declared by the International Agency for Research on Cancer as a carcinogen (IARC 1993). Cd possesses a biological half-life of 30 years in humans, because of its high retention and low excretion rate. Exposure to Cd occurs via plant-derived foods, seafood, tobacco smoking, and inhalation of industrially emitted air (Järup and Åkesson 2009). Chronic exposure to Cd causes damage in the cardiovascular, immune, and reproductive systems (Nordberg 2009), and epidemiological data indicate an increase in the risk of prostate, genitourinary, breast, lung, and colon cancers and hepatocellular carcinoma (Luevano and Damodaran 2014). Recently, Cd was shown as a possible etiological factor for human neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease (Chin-Chan et al. 2015).

Cd induces reactive oxygen species (ROS) generation and thereby results in oxidative stress, mitochondrial dysfunction and altered homeostasis of certain essential metals (Nair et al. 2013; Choong et al. 2014; Pereira et al. 2014; Al Kaddissi et al. 2014). The generated ROS targets biomolecules, such as DNA, lipids, and proteins, and eventually induces cell death. Cd is involved in the inhibition of DNA repair enzymes, deregulation of cell proliferation, interference with the apoptosis, disruption of cell adhesion, signal transduction cascades, autophagy, and tumour suppressor functions (Beyersmann and Hartwig 2008; Wysocki and Tamás 2010; Muthukumar et al. 2011; Xie et al. 2016). Interestingly, the mechanisms involved in metal toxicity detoxification are conserved across all eukaryotic organisms (Huh et al. 2003; Bleackley and MacGillivray 2011).

Cell biology has greatly benefited with yeast as a model system whose genetics and metabolism are well studied. Yeast, due to its protein homology with higher eukaryotes, has provided numerous insights into the genetics and biochemistry of several disorders including lipid-related diseases, signalling pathways, and regulatory networks (Buschini et al. 2003; Koch et al. 2014). Yeast has been extensively exploited using, gene disruptions, protein localization, protein-protein interactions, and functional analysis by genetic interactions, to understand diverse cellular processes (Tong et al. 2004; Henry et al. 2012). In the past few decades, Saccharomyces cerevisiae has been used as a tool to study the mechanism of biological response against numerous toxic metals, including Cd (Jin et al. 2008; Hosiner et al. 2014). The genome-wide screening of gene and protein expression underlying Cd toxicity analysed by microarray (Vido et al. 2001), and proteomics arrays/assays (Brzóska and Moniuszko-Jakoniuk 1998; Fauchon et al. 2002), respectively, revealed Cd interference in several vital metabolisms.

Cd is a toxic metal that interrupts numerous signalling pathways resulting in toxicity. Although, increasing consideration is given to interactions between toxic metals and bio-elements such as zinc (Zn), copper, iron, selenium, and calcium (Ca) (Gardarin et al. 2010), the molecular insight into Cd-induced biological effects is uncertain (Jomova and Valko 2011). Cd has a high affinity for sulphhydryl, carboxyl, and phosphate groups and thereby inhibits enzymes and disrupts various metabolic processes, including lipid metabolism (Rogalska et al. 2009). Understanding the lipid homeostasis is critical because, the imbalance in lipid metabolism leads to cardiovascular diseases, fatty liver, and obesity (Wenk 2005). Despite the importance of lipid metabolism, only limited studies have reported the involvement of lipid pathways in Cd-associated resistance mechanisms.

Lipids rank among the main components present in cell membranes, and Cd exposure alters the lipid levels (Muthukumar et al. 2011; Rajakumar et al. 2016a; Rajakumar et al. 2016b). Recent studies explored the molecular effects of Cd on lipid pathways such as lipid transport and energy metabolism in crabs Sinopotamonhenanense (Liu et al. 2013). The role of Cd in oxidative and endoplasmic reticulum (ER) stress, autophagy mechanisms, lipid metabolism, essential metal interference, and numerous signalling pathways has been assessed with various concentrations of Cd in yeast model systems (Table 1). This review focuses on the effect of Cd on lipids and aims to provide the possible mechanism behind stress-associated lipid accumulation using S. cerevisiae as a model organism.

Table 1.

Effects of various concentrations of Cd on the different metabolic pathways in yeast cell

S. No.	Metal	Concentration	Method	Metabolism	Reference
1.	Cadmium sulphate (CdSO4)	50 μM	Spot test	Phospholipids, lipid droplets, zinc and calcium passage, autophagy, Hog1 pathway, functional profiling screens	Muthukumar et al. 2011 Muthukumar and Nachiappan 2013 Rajakumar et al. 2016a Rajakumar et al., 2016b Fang et al., 2016 Guo et al. 2016 Rajakumar and Nachiappan 2017 Chang et al. 2018
		3.5 μM	Barcode sequencing (Bar-seq)	Functional profiling screens	Guo et al. 2016
2.	Cadmium chloride (CdCl2)	10 μM	Spot test	Transcription profiling on target pathways	Hosiner et al. 2014
		100 and 150 μM	Spot test	CWI MAP kinase pathway	Xiong et al. 2015

Cadmium uptake and transports in yeast

All living organisms require processes such as detoxification and homeostatic acquisition of metal ions. Cd is one such metal when in excess is detrimental to cells. Since dead cells are not capable of removing cadmium from the medium, the influx of Cd into yeast cells is dependent on active metabolism. Previous reports have suggested that bivalent cation transporters could play a role in Cd influx (Table 2) into the cells (Gomes et al. 2002), among which the zinc transport system is likely to be the most potent candidate. Studies have shown that the high-affinity zinc transporter, Zrt1, is responsible for Cd uptake (Gitan et al. 1998 and Rajakumar et al. 2016b). Additionally, the level of the Cd–GSH complex controls the transport of Cd though the activation of Yap1 and Ycf1 facilitates Cd uptake through Zrt1 (Gomes et al. 2002). Although metal efflux systems have begun to be characterized, metallothionine (MT) and GSH-mediated sequestration aid in neutralizing toxic metals, in eukaryotes (Gomes et al. 2002). Phytochelatin, a GSH polymer synthesized in plants and Schizosaccharomyces pombe, detoxifies heavy metals (Cobbett and Goldsbrough 2002). A few other examples of transporters include Ycf1, a vacuolar membrane ATP-binding cassette (ABC) transporter and P1B-type heavy metal-transporting ATPase (PCA1) (Eraso et al., 2004; Gomes et al., 2002; Adle et al. 2006). Glutathione and/or other cytosolic Cd carriers interact with the PCA1 metal-binding domain to transfer Cd (Eraso et al., 1997 and Gomes et al., 2002; Cobbett and Goldsbrough 2002; Adle et al. 2007). Similarly, organisms have evolved certain defence mechanisms to negotiate the toxic effects of heavy metals. The P1B-type ATPase family and some essential metal transporters extrude toxic metal ions such as Fe, Cu, Zn, Mn, Ca, and Cd from the cell (Adle et al. 2007 and Wysocki and Tamás 2010).

Table 2.

Intracellular or uptake of Cd on yeast cells

Yeast strains	Concentration of Cd	Incubation (h)	Intracellular/uptake Cd levels		Methods	References
			μg/g	nmol/OD
BY4741	50 μM CdSO4	12	0.76 ± 0.92		ICP-OES	Muthukumar et al. 2011
BY4741	50 μM CdSO4	12	1.34 ± 1.52		ICP-OES	Rajakumar et al. 2016a
3031a	50 μM CdSO4	12	0.64 ± 0.89		ICP-OES	Rajakumar et al. 2016b
BY4741	50 μM CdSO4	48		4.48 ± 3.7	ICP-OES	Abbà et al. 2011
BY4741	25 μM CdCl2	06		3.64 ± 0.02	ICP-AES	Mazzola et al. 2015
BY4742	25 μM CdCl2	06		3.20 ± 0.00	ICP-AES	Caetano et al. 2015

Impact of cadmium interference on zinc transporters

The trace element Zn plays a vital role in growth, development, and cell functioning (Singh et al. 2016) and serves as a cofactor for various essential proteins such as lipid metabolism proteins, alcohol dehydrogenase, Cu/Zn superoxide dismutase, chaperones, DNA or RNA polymerases, and ribosomal proteins (Regalla and Lyons 2005). Cd exhibits similar physical and chemical properties with Zn. Exposure to and interference by Cd leads to Zn deficiency and subsequently serious health concerns (Schrey et al. 2000). The displacement of Zn with Cd occurs in some biological processes decreasing intracellular Zn levels and affecting Zn homeostasis (Rajakumar et al. 2016b). Disturbances in Zn balance alter the function and metabolism of lipids.

Since the accumulation or depletion of Zn is toxic, the cytosolic Zn level is stringently maintained by influx and efflux mechanisms. The Zn transcription factor ZAP1 activates the Zn transporters; Zrt1p (high affinity) and Zrt2p (low affinity) present in the plasma membrane (Zhao and Eide 1996a; Zhao and Eide 1996b; Ehrensberger and Bird 2011). Further, the vacuolar Zn transporter Zrt3p and the plasma membrane transporter Fet4p also assist in maintaining the cytosolic Zn levels (Eide 2009). Zrt1p is important for Cd uptake in Zn-limited cells, and Zrt1p inactivation averts Cd overload (Gitan et al. 2003). Cd accumulation and Cd-mediated toxicity decrease Zn influx (Brzóska and Moniuszko-Jakoniuk 2001). Zn homeostasis is closely linked to lipid metabolism, and Zn deficiency activates Zap1 and causes lipid accumulation (Han et al. 2001; Singh et al. 2016). Cd exposure to wild-type (WT) yeast cells significantly upregulated Zn transporters, ZRT1 and ZRT2, but reduced the intracellular Zn levels. Intriguingly, the ZRT1 mutant strains were resistant to Cd and prevented Cd overload compared to the WT cells and ZRT2 mutant strains (Gitan et al. 2003; Rajakumar et al. 2016b). During Zn depletion, phospholipid metabolism is regulated by ZAP1 (Zhao and Eide 1996b; Koch et al. 2014), and membrane phospholipids, especially phosphatidylethanolamine (PE) and phosphatidylinositol (PI) are altered in S. cerevisiae (Carman and Han 2007; Singh et al. 2016). Cd exposure significantly increased the phospholipids in yeast cells (Muthukumar et al. 2011; Rajakumar et al. 2016a), but decreased phosphatidylcholine (PC), PE, and phosphatidylserine levels in ZRT1 mutant cells.

Concerning Zn homeostasis and phospholipids, the role of Zap1p has been well explored; nevertheless, minimal information is available on the regulation of triacyl glycerol (TAG) metabolism in yeast. Phosphatidic acid phosphohydrolase 1 (PAH1) and dipeptidyl peptidase 1 (DPP1) are highly affected under Zn-limiting conditions and regulated at the transcription level by Zap1p (Carman and Han 2007; Koch et al. 2014). The upregulation of the lipid precursor phosphatidic acid (PA) contributes to TAG formation through PAH1. During Zn deficiency, PAH1 is highly activated through ZAP1 (Soto-Cardalda et al. 2012). Zap1 regulates mitochondrial fatty acid (FA) and TAG. The deletion of FA gene ETR1 causes the accumulation of TAG, and the expression of ETR1 in zap1∆ strain restores the TAG level. These results showed that the conceded mitochondrial FA biosynthesis caused a reduction in lipoic acid and loss of mitochondrial function in zap1∆ cells. Additionally, the deletion of ZAP1 increased TAG and resulted in lipid droplet (LD) accumulation in S. cerevisiae. Cd exposure in WT and zrt2∆ cells increased TAG and sterol esters (SE) levels and resulted in LDs with a larger size and increased number (Rajakumar et al. 2016b). Furthermore, Zn-limiting conditions led to ROS accumulation in budding yeast (Wu et al. 2009). ROS generation in WT and zrt2∆ cells increased on Cd exposure; however, zrt1∆ behaved differently. An apparent decrease in intracellular levels of Cd and TAG was seen in zrt1∆ cells. Cd exposure reduced the Zn levels and served as a risk factor for alteration in lipid homeostasis and ROS levels in S. cerevisiae (Rajakumar et al. 2016a). Thus, the plasma membrane transporter Zrt1 appears to be dependent on Zap1 and protects the cells against Cd toxicity, lipid accumulation, and oxidative damage in S. cerevisiae (Fig. 1).

Fig. 1.

A hypothetical pathway elucidating the uptake of Cd via Zn transporter systems Zrt1p and Zrt2p. Zrt1 aids in the uptake the Cd compared to Zrt2 and subsequently ROS and lipid accumulation in S. cerevisiae

Cadmium-induced apoptosis and autophagy in yeast

S. cerevisiae is used as a model organism for understanding the process of cell killing and its mechanisms (Carmona-Gutierrez et al. 2010). Both exogenous and intrinsic stresses can cause cell death in yeast (Sharon et al. 2009). Usually toxic metal-induced yeast cell killing mechanism is associated with increased intracellular ROS generation, oxidative stress, and loss in Ca²⁺ homeostasis (Gomes et al. 2008; Wu et al. 2013).

Cd-induced apoptosis is a result of cellular GSH: GSSG imbalance and heightened levels of intracellular ROS, particularly in yca1 and gsh1 mutants (Nargund et al. 2008). In response to Cd toxicity, the alterations in cellular sulphhydryls may be the major determining factor for cell death (Kim et al. 2003). Transcription factors such as Yap1p, Skn7p, Msn2p, and Msn4p collectively coordinate appropriate responses to different oxidative stressors, by either repressing or upregulating the transcription of specific genes (Lee et al. 1999; Gasch et al. 2000; Temple et al. 2005) and many of these transcription factors are linked to the antioxidant defence mechanism. Yap1p and Skn7p mainly regulate the yeast cell response to Cd toxicity by upregulating transcription of yeast Cd factor YCF1 (Wemmie et al. 1994). ROS induces mitochondrial dysfunction, release of cytochrome c to the cytoplasm, activation of caspase-9, and hydrolysis of specific cellular proteins. Although elevated ROS levels may play a chief role in inducing apoptosis, additional factors such as Ca²⁺ homeostasis are present (Ruta et al. 2014; Wang et al. 2014); however, the precise pathway remains uncertain.

Besides apoptosis, autophagy plays a pivotal role in Cd-mediated toxicity. Autophagy maintains cellular homeostasis via the removal of damaged organelles and toxic macromolecules and thus prevents the organism from damage and disease (Zhang et al. 2007; Thevenod, 2009; Kroemer et al. 2010; Thorburn 2018). Cd induces ER stress and is associated with degradation of misfolded or damaged proteins. In yeast, Cd toxicity triggers several vacuolar and autophagic responsive genes such as PRB1, LAP4, and PEP4. Another gene, SEC17, which encodes for a membrane protein required for vesicular transport and autophagy is induced in when the cells are exposed to Cd (Hosiner et al. 2014). Additionally, phospholipids, particularly PE, play a major role in autophagosome formation and are reliable markers for autophagy (Nakatogawa et al. 2007). Phospholipids are involved in lipidation of Atg8p. PE depletion causes in the membrane extension around the autophagosomes to lose its integrity (Nebauer et al. 2007). PE accumulation in Cd toxicity was validated by understating the involvement of phosphatidylethanolamine decarboxylase (PSD2) (Muthukumar and Nachiappan, 2013). Under Cd toxicity, PE obtained from PSD2 alone is used for Atg8 lipid conjugation (Atg8–PE). These results suggest that cells were able to tolerate the Cd stress through organelle-specific PE synthesis, and the importance of PE in the assembly of autophagosomes is evident (Muthukumar and Nachiappan, 2013). Thus, xenobiotic metals, like cadmium, interfere with protein folding and degradative mechanisms in living cells, with autophagy and apoptosis targets (Fig. 2). A better understanding of these mechanisms may provide important insights into the contribution of metals and metalloids to protein-misfolding diseases.

Fig. 2.

The Cd-induced apoptosis and autophagy pathways in S. cerevisiae

Calcium impairment causes lipid aberrancy

The maintenance of Ca levels in the ER is essential for protein production (Schröder and Kaufman 2005). The ER provides a platform for the cross-talk of many cellular signalling pathways and metabolic regulatory events through Ca fluxes and metabolism (Verkhratsky 2005). Cd interacts with Ca transport in intracellular stores, such as interference in hepatic Ca sequestration in the microsomes (Zhang et al. 1990), or inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) (Hechtenberg and Beyersmann 1991). The Ca-dependent proteins in the ER have an important role in the maintenance of cellular integrity (Groenendyk et al. 2010). The function of Ca transporters and channels is also modulated by membrane lipid composition, protein-protein interaction, and a range of post-translational modifications (Fu et al. 2011). An alternative theory of ER stress postulates a key role for the downregulation of SERCA2 as a result of ER luminal Ca depletion (Fu et al. 2012). The enhanced ratio of PC/PE in the ER impairs SERCA2 and stimulates ER stress (Fu et al. 2011) indicating a link between lipotoxicity and Ca imbalance. The absence of SERCA in S. cerevisiae led to the discovery of another P-type ATPase, Cod1/Spf1p, that controls the Hmg2p degradation through Ca in the ER (Cronin et al. 2002). These studies indicate a close relationship between the Ca and lipid homeostasis in the ER. The alteration of intracellular Ca and unfolded protein response (UPR) activation are reported in several models of metabolic disease and lipid disequilibrium (Ozcan et al. 2004; Fu et al. 2011; Abhishek et al. 2017). Lipid remodelling occurs as an adaptive stress response and acclimatization during ER stress, but the mechanistic link between lipid aberrancy and Ca imbalance is yet to be elucidated.

The surge in intracellular Ca leads to alteration of phospholipids, especially PC levels. The silencing of phosphatidylethanolamine N-methyltransferase accelerated the SERCA activity and reduced ER stress (Jacobs et al. 2010). On exposure of WT yeast cells to Cd, the Kennedy pathway (CKI1, EKI1, and CPT1) and the methylation pathway (CHO2 and OPI3) genes were upregulated for PC synthesis (Rajakumar et al. 2016a). In yeast, the Biological General Repository for Interaction Datasets (BioGRIDs) showed a negative genetic interaction of yeast phosphatidylethanolamine N-methyltransferase homologue OPI3 with SPF1 and PMR1 (Schuldiner et al. 2005; Jonikas et al. 2009; Surma et al. 2013).

The spf1Δ activated the Ca-responsive genes and significantly increased intracellular Ca levels (Cronin et al. 2002). Some experimental evidence suggests the negative genetic interaction between Opi3 with Spf1 with a high confidence score. Remarkably, SPF1 and PMR1 are downregulated in opi3Δ cells (Rajakumar et al. 2016a). The defects of Pmr1 and Spf1 have implications in protein glycosylation, influence ER stress, and stimulate LD formation in S. cerevisiae (Fei et al. 2009; Cohen et al. 2013). Increase in TAG and LD levels were observed in cho2Δ and opi3Δ cells during Cd exposure (Rajakumar et al. 2016a). In yeast, Cd toxicity modified Ca homeostasis and PC content (lipid alteration) and had an impact on Ca-ATPase in ER (Fig. 3).

Fig. 3.

Cd downregulates the SPF1 (ER Ca²⁺ATPase) and thereby alters phospholipid methylation disrupting lipid homeostasis in S. cerevisiae

The defect in lipid metabolism activated the UPR, suggestive of a compensatory role (Thibault et al. 2012; Surma et al. 2013). Alteration in the ratio of membrane PC/PE resulted in ER stress and Ca imbalance in mice hepatocytes (Fu et al. 2011). The elevation in Kar2p (ER chaperone) expression in opi3Δ cells with or without Cd treatment can be attributed to Ca imbalance or SPF1 downregulation in S. cerevisiae (Rajakumar et al. 2016a). SPF1 is part of the UPR program and regulates Hmg2p degradation in the ER. Mutants lacking SPF1 exhibit defects in membrane protein orientation (Krumpe et al. 2012). The abundance of intracellular Ca-induced lipid aberrancy has an impact on membrane proliferation in S. cerevisiae. Chemical stress induces upregulation of genes involved in phospholipid biosynthesis, cell membrane, and cell wall organization (Murata et al. 2003). Together, these findings demonstrated a relationship between Ca and lipid homeostasis during Cd toxicity.

Mitochondrial dysfunctions are accompanied by PC impairment during Cd stress (Rajakumar et al. (2016a). The physiological and functional interaction of ER with mitochondria accounts for Ca transport and lipid synthesis in the cell (Mitsuhashi et al. 2011; Malhotra and Kaufman 2011). Cd treatment resulted in the fragmentation of mitochondrial structure in opi3Δ and WT strains. PC is essential for Cd tolerance, intracellular membrane maintenance, and preserving mitochondrial morphology (Rajakumar et al. 2016a). The reciprocal relationship with the phospholipids and Ca provides another facet of a stress-regulated mechanism in S. cerevisiae.

Effect of cadmium on fatty acid desaturase—OLE1

Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme responsible for the biosynthesis of monounsaturated fatty acids (MUFA) in rats and human (Kim and Ntambi 1999). In S. cerevisiae, OLE1 encodes for the sole delta-9 FA desaturase, with a sequence like that of human SCD1. Ole1 is an ER membrane protein essential for the synthesis of MUFA. The primary products of Ole1p are palmitoleic (16:1) and oleic (18:1) FAs, formed from palmitoyl (16:0) and stearoyl (18:0) CoA, respectively (Martin et al. 2007). This enzyme (Ole1p) mainly contributes towards cell viability and membrane fluidity by regulating the equilibrium of saturated fatty acid (SFA)and unsaturated fatty acid (UFA). In S. cerevisiae, Ole1 is the target of ER-bound transcription factors Spt23p and Mga2p. In response to stimuli, both Spt23p and Mga2p are activated by ubiquitin-dependent processing into their soluble forms and then targeted to the nucleus (Chellappa et al. 2001; Aguilar and De Mendoza 2006). At the transcriptional and mRNA levels, OLE1 is highly regulated and responds to some different stimuli, including a carbon source, nutrient FAs, metal ions, and oxygen levels (Aguilar and De Mendoza 2006 and Martin et al. 2007). Additionally, impairment of lipid storage and membrane damage is an important aspect of Cd stress (Pierron et al. 2007; Rahoui et al. 2010). Alterations in the lipid composition can tolerate Cd stress in S. cerevisiae (Muthukumar et al. 2011; Rajakumar et al. 2016a; Rajakumar and Nachiappan 2017). Hence, lipid-associated pathways may be involved in the mechanisms of Cd damage and resistance.

A recent investigation of Cd stress and the adaptive mechanisms of yeast were done by screening a genome-wide essential gene overexpression. OLE1 was associated with high Cd stress (Fang et al. 2016). The WT strains bearing the OLE1 plasmid showed strong Cd resistance as well as more accumulation of intracellular Cd (Fang et al. 2016). On exposure to Cd, OLE1 mRNA levels were significantly increased in the WT (Rajakumar and Nachiappan, 2017), mga2Δ, and spt23Δ strains (Fang et al. 2016). Particularly, the spt23Δ strain showed more tolerance compared to mga2Δ revealing a positive correlation between the OLE1 expression level and Cd resistance. Either with and without OLE1 overexpression, WT yeast cells exposed to Cd stress resulted in a significant increase in MUFA (C16:1 and C18:1) (Fang et al. 2016; Rajakumar and Nachiappan 2017).

The effects of oleic acid on membrane integrity decreased in WT and OLE1 overexpressed strains treated with Cd revealed a relationship between MUFA and Cd stress. Interestingly, Cd induced a high percentage of dead cells, nearly 60% higher than that observed in the mga2Δ control that recovered on supplementation of oleic acid or by overexpression of OLE1 in mga2Δ under Cd stress (Fang et al. 2016).

Cd-induced lipid peroxidation enhanced lipoxygenase activity that is involved in catalysing lipid peroxidation by using membrane lipid components, especially unsaturated FAs, as substrates (Thompson et al. 1998). Compared to saturated FAs, unsaturated FAs contain double bonds that are vulnerable to damage from Cd-induced free radicals, resulting in unsaturated FA peroxidation (Upchurch 2008; Tsaluchidu et al. 2008). OLE1, an oxygen-sensing gene, could be upregulated in response to oxidative stress (Kwast et al. 1999). On Cd treatment, the lipid peroxidation levels were significantly increased in both WT yeast BY4741 (Muthukumar et al. 2011) and mga2Δ strain, and there was a decrease following the overexpression of OLE1 or the addition of oleic acid (Fang et al. 2016). This regulation of OLE1 may help yeast cells to counter lipid peroxidation and membrane damage caused during Cd-induced stress. This finding suggested that regulation of OLE1 in response to Cd stress was associated with oxidative stress and independent of Mga2p and Spt23p (Fang et al. 2016). Delta-9 desaturase present in the rat liver, encoded by SCD1, is a target of Cd and is significantly inhibited by Cd (Kudo et al. 1991). The stress response regulation of OLE1 is an important adaptive response of yeast to counter Cd stress (Fig. 4).

Fig. 4.

OLE1-facilitated TAG/LD accumulation alleviates the Cd-induced cell death apoptosis in yeast

Lipid droplets alleviate Cd-induced cytotoxicity

Lipotoxicity, the phenomenon of cell death due to lipid imbalance, is a common consequence of the morbidities associated with metabolic syndromes such as obesity, fatty liver, and cardiovascular diseases. The saturated and unsaturated FAs are pro-apoptotic, but cytotoxicity is achieved through different pathways (Garbarino et al. 2009). Cd is associated with a sequence of deteriorating disorders in humans that also cause programmed cell death (Shivapurkar et al. 2003). Programmed cell death is a valuable tool for the elucidation of several key apoptotic regulators in S. cerevisiae (Ligr et al. 1998; Manon 2004). However, the mechanism for Cd-induced lipotoxicity is yet to be defined.

Cd induces oxidative stress that generates ROS and increased lipid accumulation. In oxidative stress, polyunsaturated Fas and other FAs are redistributed from membrane phospholipids to LD and TAGs. Unlike cell membranes, the LD core provides a protective environment that minimizes poly unsaturated FAs peroxidation chain reactions and limits ROS levels (Bailey et al. 2015).

LDs mediate cellular functions other than fat storage and mobilization relevant for energy homeostasis. For example, LDs can participate in protein degradation, histone storage, viral replication, and antibacterial defence (Walther and Farese 2012; Anand et al. 2012). LDs are currently considered to be metabolically active organelles, and during oxidative stress, lipid metabolism is impaired (Cutler et al. 2004; Furukawa et al. 2004). LD biogenesis, by recycling of structural phospholipids into energy-generating substrates, may serve as a survival strategy during stress. The biology of LD has received increasing interest, due to the link between excess lipid storage in certain environmental disturbances of cells.

Avoidance of excess saturated FAs is important in membrane phospholipids and the scavenging of lipotoxicity (Kamphorst et al. 2013). Lipotoxicity resulting from the accumulation of long chain FAs seems to be caused predominantly by saturated FAs (Listenberger et al. 2001). Conversely, oleic acid supplementation that is well-tolerated by the cells leads to triglyceride accumulation and protects the cells against saturated FA-induced lipotoxicity (Listenberger et al. 2003).

In our earlier studies, Cd exposure in S. cerevisiae increased the phospholipids primarily, PC and PE, and induced ER stress (Muthukumar et al. 2011; Muthukumar and Nachiappan 2013; Rajakumar et al. 2016a). There is a functional link between phospholipid metabolism and TAG formation in S. cerevisiae during the various phases of growth. The precursors are channelled towards phospholipid synthesis during membrane formation and towards lipid droplets during storage (Horvath et al. 2011).

Furthermore, FA sensitivity is significantly enhanced in yeast cells that were unable to form LDs (Petschnigg et al. 2009). FAs significantly contributed to the lipid composition. Cd exposure increased the ratio of UFA in WT cells. Interestingly under Cd exposure, the mRNA expression of FA synthesizing genes ACC1 (acetyl-CoA carboxylase1), FAS1 (FA synthase1), and desaturase were upregulated in S. cerevisiae (Rajakumar and Nachiappan 2017).

Upon Cd exposure, there was an insignificant change in the sterol levels while significant alterations were observed in the TAG as well as DAG (Rajakumar and Nachiappan 2017). The TAG is efficiently packed into LDs (Wang 2015). The overload of TAG and SEs contributes to the increased number of LDs in Cd-treated cells. The transcriptional response of genes related to TAG synthesis (LRO1 and DGA1) were upregulated in Cd-treated WT cells (Rajakumar and Nachiappan 2017). FA esterification and storage into LDs protected the cells against free fatty acid (FFA)-induced lipotoxicity. This result is supported by others (Holland et al. 2007).

Defects in LD formation render FFAs toxic (Listenberger et al. 2001). During Cd exposure, a knockout of dga1Δorlro1Δ showed a reduction in the TAG level and accumulated FFA, whereas TAG was accumulated in are1Δ and are2Δ strains. A similar trend in TAG levels was observed in double knockout strains during Cd toxicity (Rajakumar and Nachiappan 2017).

Schizosaccharomyces pombe and S. cerevisiae deficient in a TAG synthesis undergo lipo-apoptosis upon entry into the stationary phase. These cells show promises of apoptotic cell death, including ROS accumulation (Garbarino and Sturley, 2005; Low et al. 2005). The impaired LD synthesis in cells with the dga1Δ or lro1Δ increased ROS generation during Cd exposure. Significant ROS accumulation was observed in single and double mutants of dga1Δ and lro1Δ strains (Rajakumar and Nachiappan 2017). ROS is not only associated with cell death but also plays essential roles in signalling and adaptation to cellular stress (Carmona-Gutierrez et al. 2010).

Excessive accumulation of FFA and its incorporation into phospholipids in various cell membranes caused severe cytotoxicity and lipo-necrosis, whereas neutral lipid storage shielded the cells and alleviated FFA toxicity (Sheibani et al. 2014). Yeast strains that lack all four acyltransferase genes are viable but completely lack neutral lipids and thus present a genetically malleable system to investigate FA-induced cytotoxicity (Garbarino et al. 2009). The TAG mutants (single and double deletion of dga1Δlro1Δ) resulted in both early and late apoptosis during Cd toxicity, whereas the WT and SE mutant cells showed normal physiology (Rajakumar and Nachiappan 2017).

Apoptosis mediated by the overload of FFA and ROS was confirmed in the TAG-deficient cells. This was confirmed with the overexpression of TAG synthesizing genes (DGA1 and LRO1) in LD quadruple mutant strains. When TAG synthesizing genes were overexpressed, TAG levels were increased, FFA levels were ameliorated, and ROS levels decreased (Rajakumar and Nachiappan 2017).Tgl3p, Tgl4p, and Tgl5p are the major TAG lipases of the S. cerevisiae for the degradation of TAG that is stored in LDs (Athenstaedt and Daum 2005). The blockage or defect in TAG hydrolysis rescued the lipotoxicity due to the accumulation of the LDs. Interestingly; Cd toxicity caused the increase in LD number that accumulated TAG in TGL mutants. The TAG accumulation in the double and quintuple knockouts (tgl3Δtgl4Δ, tgl4Δtgl5Δ, and tg13Δtgl4Δtgl5Δare1Δare2Δ) made the cells tolerant to Cd and increased cell growth (Rajakumar and Nachiappan 2017). These data collectively demonstrate that TAG accumulated in the LD buffered the excessive FFA and increased the cell survival during Cd-induced lipotoxicity and cytotoxicity in S. cerevisiae (Fig. 4).

Upregulation of sulphur assimilation pathway and UPR

Since Cd cannot be degraded or modified to less toxic forms, it persists in cells and alters cellular homeostasis by provoking oxidative stress and stress response pathways (Ballatori 2002). Apart from these effects, Cd mainly targets the sulphur assimilation pathway and methyl cycle in yeast cells (Mendoza-Cózatl et al. 2005; Sohn et al. 2014). In S. cerevisiae, extracellular sulphate is taken up by sulphate transporters and through the assimilation pathway is reduced to sulphide. The assimilated sulphur will either be incorporated into the sulphur-containing amino acids or into the low-molecular-weight thiol molecules S-adenosylmethionine (SAM) and GSH (Wysocki and Tamás 2010). Transcription of the genes encoding the incorporation of assimilated sulphur is regulated by the transcriptional activator Met4p. When cells were subjected to Cd toxicity, MET transcriptional factors like MET4, MET28, MET31, and MET32 were upregulated (Hosiner et al. 2014), indicative of significant links to the sulphur and methyl cycle genes (Tables 3 and 4). SAM regulated de novo PC synthesis genes such as CHO2 and OPI3 that controlled the major membrane phospholipids, PC and PE, in both yeast and mammals (Visram et al. 2018). Deletion of CHO2 and OPI3 induced lipid alterations including TAG, LD accumulation, increased FA content, and altered FA profiles in yeast (Rajakumar et al. 2016a). Additionally, upregulation of phospholipid methyl transferase-encoding genes CHO2 and OPI3 were observed in Cd toxicity. Therefore, using the STRING bioinformatics tool (Jensen et al. 2009), we constructed a protein interaction network in yeast S. cerevisiae. Through applying a cut off on the confidence score from STRING, we used only genes/protein upregulated in Cd stress from different cellular processes such as sulphur pathway, lipid, and FA biogenesis and UPR/ER stress mechanism. The results showed that Acc1, Fas1, and Cho2 directly interact with Cys3, Sam2, and Met17 and also controlled activity of S-adenosyl homocysteine hydrolase (Sah1) (Fig. 5). This data strongly supports the recent findings of S-adenosyl homocysteine as the key trigger of lipid deregulation (Visram et al. 2018). As a response to Cd stress, the host cell’s defence mechanism activates UPR transcription factor and UPR genes and proteins (Table 2) (Gardarin et al. 2010; Wysocki and Tamás 2010; Rajakumar et al. 2016a). Interestingly, the ability of UPR transducers to sense lipid homeostasis disequilibrium is conserved in eukaryotes. Lipid perturbations in yeast induced alterations in protein quality control, with minimal changes to lipid metabolism (Thibault and Ng 2012). Several protein degradation genes control lipid biosynthetic proteins like Rpn4, Ubx2 control Lpl1, and Ole1 activity in yeast (Surma et al. 2013; Weisshaar et al. 2017). The core interaction network contains HAC1 and kar2 interactions between proteins that share with phospholipid synthetic genes OPI3, CKI1, CPT1, and FA enzyme ACC1 (Table 4 and Fig. 5). These results collectively emphasize how Cd-induced cellular processes are interlinked with lipid metabolism.

Table 3.

Significant GO terms associated to the Cd metal stress conditions

S. No.	GO terms	Proteins	Genes	Transcription factors (TFs)	Reference
1.	Sulphur compound metabolism: transsulphuration pathway and methyl cycle		CYS3, SAM2, MET2, MET3, MET5, MET6, MET14, MET16, MET17	Met4, Met32, Met28	Hosiner et al. 2014
2.	Protein folding and proteasomal degradation/autophagy	Kar2, Atg8, Ape1, Pca1	PDI1, ERO1, FKB2, MCD1, MPD1, JEM1, KAR2, EUG1, MPD1, LHS1, ATG17	Hac1, Ire1, Rpn4	Gardarin et al. 2010; Muthukumar and Nachiappan 2013; Hosiner et al. 2014; Smith et al. 2016
3.	Lipid and fatty acid metabolism		CHO2, OPI3, CPT1, EKI1, CKI1, ARE1, ARE2, DGA1, LRO1, ACC1, FAS1, OLE1		Fang et al. 2016; Rajakumar and Nachiappan 2017

Table 4.

Protein-protein interaction were analysed with major lipid/fatty acid metabolic related proteins

Connecting proteins	Phospholipid biosynthesis	Score	Lipid droplet biosynthesis	Score	Fatty acid biosynthesis	Score	Sulphur pathway	Score	UPR	Score
ACC1 Acetyl-CoA carboxylase 1	OPI3	0.524	DGA1	0.786	FAS1	0.999	CYS4	0.901	KAR2	0.630
			ARE2	0.624	FAS2	0.999
			ARE1	0.526	OLE1	0.940
FAS1 Fatty acid synthase 1	OPI3	0.561	LRO1	0.564	ACC1	0.999
					FAS2	0.999
					OLE1	0.854
FAS2 Fatty acid synthase 2					ACC1	0.999	CYS4	0.691
					FAS1	0.99	SAM2	0.519
					OLE1	0.829
OLE1 Fatty acid desaturase	OPI3	0.535	DGA1	0.693	ACC1	0.940
			ARE2	0.611	FAS1	0.854
					FAS2	0.829
OPI3 PL methyl transferase	CPT1	0.997	LRO1	0.591	FAS1	0.561			HAC1	0.607
	CHO2	0.983			OLE1	0.535			EUG1	0.518
	CKI1	0.794			ACC1	0.524
	EKI1	0.687
CHO2 PE methyl transferase	OPI3	0.997	LRO1	0.574			MET17	0.721
	CKI1	0.782	DGA1	0.524
	CPT1	0.763
	EKI1	0.637
CKI1 Choline kinase 1	EKI1	0.987							HAC1	0.529
	CPT1	0.895
	CHO2	0.892
	OPI3	0.794
CPT1 Choline phosphor transferase 1	OPI3	0.983	LRO1	0.629					HAC1	0.774
	CKI1	0.895
	CHO2	0.782
	EKI1	0.735
DGA1 Diacylglycerol acyltransferase 1	CHO2	0.524	LRO1	0.998	ACC1	0.786
			ARE2	0.982	OLE1	0.693
			ARE1	0.939
LRO1 Lecithin cholesterol acyl transferase 1	CPT1	0.629	DGA1	0.998	FAS1	0.564
	CHO2	0.574	ARE2	0.982
	OPI3	0.591	ARE1	0.940

Fig. 5.

Network illustrating functional trends of protein participating in dynamic complexes, such as lipid, fatty acid homeostasis, UPR regulations, and transsulphuration pathway

.

Conclusions and future perspectives

Several epidemiologic studies offer the evidence of Cd exposure with adverse effects that induce oxidative stress and is associated with obesity, hyperglycaemia, and type II diabetes (Han et al. 2015; Pizzino et al. 2017). This approach could be useful in endotyping lipid-related disorders, improving exposure assessment, identifying biochemical and transport mechanisms for Cd exposures in S. cerevisiae, and for clarifying the role of the stress scavenging proteins in yeast microbiology.

The ion transport system is essential for Cd influx, accumulation, and toxicity of the cells. This review discusses the involvement of vital metals like Zn and Ca and their transport and homeostatic mechanisms during Cd toxicity. Cd competes for the binding sites of essential metal absorptive and enzymatic proteins (Bridges and Zalups 2005). The replacement of Zn by Cd leads to oxidative stress. The Zn transporters efficiently play a role in LD accumulation during Cd exposure (Rajakumar et al. 2016b). Furthermore, cross-talk regulations of Ca and lipids were observed in yeast cells during Cd stress. Particularly, ER Ca-ATPase SPF1 was downregulated in opi3 deletion strain (Rajakumar et al. 2016a). This result correlated with TAG accumulation during spf1 deletion (Unpublished data) and the LDs effectively rescued the ROS-mediated programmed cell death. This observation showed that LDs play a role in heavy metal-induced oxidative stress. The lipid overload during Cd stress in yeast alleviates the ROS induction and the essential metal imbalances.

The Ca transporters are modulated by membrane lipid composition, protein-protein interaction, and a range of post-translational modifications (Traaseth et al. 2008; Fu et al. 2011). ER Ca-ATPases play a vital role in cellular Ca homeostasis as well as lipid regulation in the cell (Arruda and Hotamisligil, 2015). Thus, understanding the cross-talk mechanism of Ca and lipid homeostasis in yeast can provide a key factor for targeting strategy for the development of therapeutic drugs to combat human diseases such as metabolic syndromes. Moreover, advance studies are required to elucidate the role of metal transport proteins and lipid under Cd toxicity to alleviate the pollutant in the current eco-system.

In the future, studies need to be focused on the role of Zn and Ca and their transport system on lipid homeostasis. Zn limitation has an impact on signalling with the adverse effect on lipid homeostasis (Wei et al. 2018). The identification of target genes that are involved in Cd transport with impact on lipid homeostasis will provide pharmacotherapy progress to treat diseases caused by zinc and Ca alterations during Cd exposure.