Quantifying yeast lipidomics by high-performance thin-layer chromatography (HPTLC) and comparison to mass spectrometry-based shotgun lipidomics.

Lipidomic analysis in diverse biological settings has become a frequent tool to increase our understanding of the processes of life. Cellular lipids play important roles not only as being the main components of cellular membranes, but also in the regulation of cell homeostasis as lipid signaling molecules. Yeast has been harnessed for biomedical research based on its good conservation of genetics and fundamental cell organisation principles and molecular pathways. Further application in so-called humanised yeast models have been developed which take advantage of yeast as providing the basics of a living cell with full control over heterologous expression. Here we present evidence that high-performance thin-layer chromatography (HPTLC) represents an effective alternative to replace cost intensive mass spectrometry-based lipidomic analyses. We provide statistical comparison of identical samples by both methods, which support the use of HPTLC for quantitative analysis of the main yeast lipid classes.

A dynamic actin cytoskeleton is required to prevent constitutive VDAC-dependent MAPK signalling and aberrant lipid homeostasis.

The dynamic nature of the actin cytoskeleton is required to coordinate many cellular processes, and a loss of its plasticity has been linked to accelerated cell aging and attenuation of adaptive response mechanisms. Cofilin is an actin-binding protein that controls actin dynamics and has been linked to mitochondrial signaling pathways that control drug resistance and cell death. Here we show that cofilin-driven chronic depolarization of the actin cytoskeleton activates cell wall integrity mitogen-activated protein kinase (MAPK) signalling and disrupts lipid homeostasis in a voltage-dependent anion channel (VDAC)-dependent manner. Expression of the cof1-5 mutation, which reduces the dynamic nature of actin, triggers loss of cell wall integrity, vacuole fragmentation, disruption of lipid homeostasis, lipid droplet (LD) accumulation, and the promotion of cell death. The integrity of the actin cytoskeleton is therefore essential to maintain the fidelity of MAPK signaling, lipid homeostasis, and cell health in S. cerevisiae.

The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity.

Amyloid beta 42 (Abeta42) is the principal trigger of neurodegeneration during Alzheimer's disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, we here identify the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42-mediated cell death. We demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co-purification of Abeta42 with mitochondria and prevents Abeta42-induced mitochondria-dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.

Bacteria Are New Targets for Inhibitors of Human Farnesyltransferase.

Farnesyltransferase inhibitors (FTIs) are focus for the treatment of several diseases, particularly in the field of cancer therapy. Their potential, however, goes even further, as a number of studies have evaluated FTIs for the treatment of infectious diseases such as malaria, African sleeping sickness, leishmaniosis, and hepatitis D virus infection. Little is known about protein prenylation mechanisms in human pathogens. However, disruption of IspA, a gene encoding the geranyltranstransferase of Staphylococcus aureus (S. aureus) leads to reprogramming of cellular behavior as well as impaired growth and decreased resistance to cell wall-targeting antibiotics. We used an agar well diffusion assay and a time kill assay and determined the minimum inhibitory concentrations of the FTIs lonafarnib and tipifarnib. Additionally, we conducted cell viability assays. We aimed to characterize the effect of these FTIs on S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), Escherichia coli (E. coli), Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and Streptococcus pneumoniae (S. pneumoniae). Both the FTIs lonafarnib and tipifarnib were capable of inhibiting the growth of the Gram-positive bacteria S. aureus, MRSA, S. epidermidis, and S. pneumoniae, whereas no effect was observed on Gram-negative bacteria. The analysis of the impact of lonafarnib and tipifarnib on common human pathogens might lead to novel insights into their defense mechanisms and therefore provide new therapeutic targets for antibiotic-resistant bacterial infections.

Porin 1 Modulates Autophagy in Yeast.

Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

Lipotoxicty in yeast: a focus on plasma membrane signalling and membrane contact sites.

Lipotoxicity is a pathophysiological process triggered by lipid overload. In metazoans, lipotoxicity is characterised by the ectopic deposition of lipids on organs other than adipose tissue. This leads to organ dysfunction, cell death, and is intimately linked to lipid-associated diseases such as cardiac dysfunction, atherosclerosis, stroke, hepatosteatosis, cancer and the metabolic syndrome. The molecules involved in eliciting lipotoxicity include FAs and their acyl-CoA derivatives, triacylglycerol (TG), diacylglycerol (DG), ceramides, acyl-carnitines and phospholipids. However, the cellular transport of toxic lipids through membrane contact sites (MCS) and vesicular mechanisms as well as lipid metabolism that progress lipotoxicity to the onset of disease are not entirely understood. Yeast has proven a useful model organism to study the molecular mechanisms of lipotoxicity. Recently, the Rim101 pathway, which senses alkaline pH and the lipid status at the plasmamembrane, has been connected to lipotoxicity. In this review article, we summarise recent research advances on the Rim101 pathway and MCS in the context of lipotoxicity in yeast and present a perspective for future research directions.

Trans-Fats Inhibit Autophagy Induced by Saturated Fatty Acids.

Depending on the length of their carbon backbone and their saturation status, natural fatty acids have rather distinct biological effects. Thus, longevity of model organisms is increased by extra supply of the most abundant natural cis-unsaturated fatty acid, oleic acid, but not by that of the most abundant saturated fatty acid, palmitic acid. Here, we systematically compared the capacity of different saturated, cis-unsaturated and alien (industrial or ruminant) trans-unsaturated fatty acids to provoke cellular stress in vitro, on cultured human cells expressing a battery of distinct biosensors that detect signs of autophagy, Golgi stress and the unfolded protein response. In contrast to cis-unsaturated fatty acids, trans-unsaturated fatty acids failed to stimulate signs of autophagy including the formation of GFP-LC3B-positive puncta, production of phosphatidylinositol-3-phosphate, and activation of the transcription factor TFEB. When combined effects were assessed, several trans-unsaturated fatty acids including elaidic acid (the trans-isomer of oleate), linoelaidic acid, trans-vaccenic acid and palmitelaidic acid, were highly efficient in suppressing autophagy and endoplasmic reticulum stress induced by palmitic, but not by oleic acid. Elaidic acid also inhibited autophagy induction by palmitic acid in vivo, in mouse livers and hearts. We conclude that the well-established, though mechanistically enigmatic toxicity of trans-unsaturated fatty acids may reside in their capacity to abolish cytoprotective stress responses induced by saturated fatty acids.

Diacylglycerol triggers Rim101 pathway-dependent necrosis in yeast: a model for lipotoxicity.

The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21-dependent sensing complex and the activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity, i.e., high-fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.

Mitochondrial energy metabolism is required for lifespan extension by the spastic paraplegia-associated protein spartin.

Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. Here we show that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. We demonstrate that spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26) and is sufficient to complement for PFK26-deficiency at least in early aging. We conclude that mitochondria-related energy metabolism is crucial for spartin's vital function during aging and uncover a network of specific interactors required for this function.

Cardioprotection and lifespan extension by the natural polyamine spermidine.

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.

Ethanolamine: A novel anti-aging agent.

Ethanolamine (Etn) is a naturally occurring aminoalcohol necessary for synthesis of the phospholipid phosphatidylethanolamine (PE), a major component of biological membranes. We recently reported that Etn treatment increases cellular PE levels, thereby inducing cytoprotective autophagy and protecting against aging across species.

Unsaturated fatty acids induce non-canonical autophagy.

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1(+/-) mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.

Spermidine feeding decreases age-related locomotor activity loss and induces changes in lipid composition.

Spermidine is a natural polyamine involved in many important cellular functions, whose supplementation in food or water increases life span and stress resistance in several model organisms. In this work, we expand spermidine's range of age-related beneficial effects by demonstrating that it is also able to improve locomotor performance in aged flies. Spermidine's mechanism of action on aging has been primarily related to general protein hypoacetylation that subsequently induces autophagy. Here, we suggest that the molecular targets of spermidine also include lipid metabolism: Spermidine-fed flies contain more triglycerides and show altered fatty acid and phospholipid profiles. We further determine that most of these metabolic changes are regulated through autophagy. Collectively, our data suggests an additional and novel lipid-mediated mechanism of action for spermidine-induced autophagy.

Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Ceramide triggers metacaspase-independent mitochondrial cell death in yeast.

The activation of ceramide-generating enzymes, the blockade of ceramide degradation, or the addition of ceramide analogues can trigger apoptosis or necrosis in human cancer cells. Moreover, endogenous ceramide plays a decisive role in the killing of neoplastic cells by conventional anticancer chemotherapeutics. Here, we explored the possibility that membrane-permeable C2-ceramide might kill budding yeast (Saccharomyces cerevisiae) cells under fermentative conditions, where they exhibit rapid proliferation and a Warburg-like metabolism that is reminiscent of cancer cells. C2-ceramide efficiently induced the generation of reactive oxygen species (ROS), as well as apoptotic and necrotic cell death, and this effect was not influenced by deletion of the sole yeast metacaspase. However, C2-ceramide largely failed to cause ROS hypergeneration and cell death upon deletion of the mitochondrial genome. Thus, mitochondrial function is strictly required for C2-ceramide-induced yeast lethality. Accordingly, mitochondria from C2-ceramide-treated yeast cells exhibited major morphological alterations including organelle fragmentation and aggregation. Altogether, our results point to a pivotal role of mitochondria in ceramide-induced yeast cell death.

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis.

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.

Fatty acids trigger mitochondrion-dependent necrosis.

Obesity is characterised by lipid accumulation in non-adipose tissues, leading to organ degeneration and a wide range of diseases, including diabetes, heart attack and liver cirrhosis. Free fatty acids (FFA) are believed to be the principal toxic triggers mediating the adverse cellular effects of lipids. Here, we show that various cooking oils used in human nutrition cause cell death in yeast in the presence of a triacylglycerol lipase, mimicking the physiological microenvironment of the small intestine. Combining genetic and cell death assays, we demonstrate that elevated FFA concentrations lead to necrotic cell death, as evidenced by loss of membrane integrity and release of nuclear HMGB1. FFA-mediated necrosis depends on functional mitochondria and leads to the accumulation of reactive oxygen species. We conclude that lipotoxicity is executed via a mitochondrial necrotic pathway, challenging the dogma that the adverse effects of lipid stress are exclusively apoptotic.

Ageing and eating.

Epidemiological studies propose that extension of the human lifespan or the reduction of age associated diseases may be achieved by physical exercise, caloric restriction, and by consumption of certain substances such as resveratrol, selenium, flavonoids, zinc, omega 3 unsaturated fatty acids, vitamins E and C, Ginkgobiloba extracts, aspirin, green tea catechins, antioxidants in general, and even by light caffeine or alcohol consumption. Though intriguing, these studies only show correlative (not causative) effects between the application of the particular substance and longevity. On the other hand, obesity is yet a strong menace to the western society and it will emerge even more so throughout the next decades according to the prediction of the WHO. Although obesity is considered a severe problem, very little is known about the molecular mechanisms causing the associated degeneration of organs and finally death. Nutrient related adverse consequences for health and thus ageing may be due to a high sugar or high fat diet, excessive alcohol consumption and cigarette smoke amongst others. In this article we examine the interdependencies of eating and ageing and suggest yeast, one of the most successful ageing models, as an easy tool to elucidate the molecular pathways from eating to ageing. The conservation of most ageing pathways in yeast and their easy genetic tractability may provide a chance to discriminate between the correlative and causative effects of nutrition on ageing.

Apoptotic death of ageing yeast.

Yeast has been a valuable model to study replicative and chronological ageing processes. Replicative ageing is defined by the number of daughter cells a mother can give birth to and hence reflects the ageing situation in proliferating cells, whereas chronological ageing is widely accepted as a model for postmitotic tissue ageing. Since both ageing forms end in yeast programmed death (necrotic and apoptotic), and abrogation of cell death by deletion of the apoptotic machinery or diminishment of oxidative radicals leads to longevity, apoptosis and ageing seem closely connected. This review focuses on ageing as a physiological way to induce yeast apoptosis, which unexpectedly defines apoptosis as a pro- and not an anti-ageing mechanism.