Sip18 hydrophilin prevents yeast cell death during desiccation stress.

AIMS: For this study, we performed a genetic screen of S. cerevisiae's deletion library for mutants sensitive to dehydration stress, with which we aimed to discover cell dehydration-tolerant genes. METHODS AND RESULTS: We used a yeast gene deletion set (YGDS) of 4850 viable mutant haploid strains to perform a genome-wide screen for the identification of desiccation stress modifiers. SIP18 is among the genes identified as essential for cells surviving to drying/rehydration process. Deletion of SIP18 promotes the accumulation of reactive oxygen species and enhances apoptotic and necrotic cell death in response to dehydration/rehydration process. CONCLUSIONS: SIP18p acts as an inhibitor of apoptosis in yeast under dehydration stress, as suggested by its antioxidative capacity through the ROS accumulation reduction after an H(2) O(2) attack. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first systematic screen for the identification of putative genes essential to overcoming cell dehydration process. A broad range of identified genes could help to understand why some strains of high biotechnological interest cannot cope with the drying and rehydration treatments. Dehydration sensitivity makes these strains not suitable to be commercialized by yeast manufactures.

No death without life: vital functions of apoptotic effectors.

As a result of the genetic experiments performed in Caenorhabditis elegans, it has been tacitly assumed that the core proteins of the 'apoptotic machinery' (CED-3, -4, -9 and EGL-1) would be solely involved in cell death regulation/execution and would not exert any functions outside of the cell death realm. However, multiple studies indicate that the mammalian orthologs of these C. elegans proteins (i.e. caspases, Apaf-1 and multidomain proteins of the Bcl-2 family) participate in cell death-unrelated processes. Similarly, loss-of-function mutations of ced-4 compromise the mitotic arrest of DNA-damaged germline cells from adult nematodes, even in a context in which the apoptotic machinery is inoperative (for instance due to mutations of egl-1 or ced-3). Moreover, EGL-1 is required for the activation of autophagy in starved nematodes. Finally, the depletion of caspase-independent death effectors, such as apoptosis-inducing factor (AIF) and endonuclease G, provokes cell death-independent consequences, both in mammals and in yeast (Saccharomyces cerevisiae). These results corroborate the conjecture that any kind of protein that has previously been specifically implicated in apoptosis might have a phylogenetically conserved apoptosis-unrelated function, most likely as part of an adaptive response to cellular stress.

The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death.

The exposure of calreticulin (CRT) on the plasma membrane can precede anthracycline-induced apoptosis and is required for cell death to be perceived as immunogenic. Mass spectroscopy, immunofluorescence and immunoprecipitation experiments revealed that CRT co-translocates to the surface with another endoplasmic reticulum-sessile protein, the disulfide isomerase ERp57. The knockout and knockdown of CRT or ERp57 inhibited the anthracycline-induced translocation of ERp57 or CRT, respectively. CRT point mutants that fail to interact with ERp57 were unable to restore ERp57 translocation upon transfection into crt(-/-) cells, underscoring that a direct interaction between CRT and ERp57 is strictly required for their co-translocation to the surface. ERp57(low) tumor cells generated by retroviral introduction of an ERp57-specific shRNA exhibited a normal apoptotic response to anthracyclines in vitro, yet were resistant to anthracycline treatment in vivo. Moreover, ERp57(low) cancer cells (which failed to expose CRT) treated with anthracyclines were unable to elicit an anti-tumor response in conditions in which control cells were highly immunogenic. The failure of ERp57(low) cells to elicit immune responses and to respond to chemotherapy could be overcome by exogenous supply of recombinant CRT protein. These results indicate that tumors that possess an intrinsic defect in the CRT-translocating machinery become resistant to anthracycline chemotherapy due to their incapacity to elicit an anti-cancer immune response.

A yeast mutant showing diagnostic markers of early and late apoptosis.

A Saccharomyces cerevisiae mutant in cell division cycle gene CDC48 shows typical markers of apoptosis: membrane staining with annexin V, indicating an exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane; intense staining, using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method, indicating DNA fragmentation; and chromatin condensation and fragmentation. The coordinate occurrence of these events at different locations in the cell, which have no obvious connection except their relation to apoptosis, implies the presence of the molecular machinery performing the basic steps of apoptosis already in yeast. Saccharomyces cerevisiae may prove a suitable model to trace the roots of apoptosis.

Oxygen stress: a regulator of apoptosis in yeast.

Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.

Physical interaction of apoptosis-inducing factor with DNA and RNA.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which upon apoptosis induction translocates to the nucleus where it interacts with DNA by virtue of positive charges clustered on the AIF surface. Here we show that the AIF interactome, as determined by mass spectroscopy, contains a large panel of ribonucleoproteins, which apparently bind to AIF through the RNA moiety. However, AIF is devoid of any detectable RNAse activity both in vitro and in vivo. Recombinant AIF can directly bind to DNA as well as to RNA. This binding can be visualized by electron microscopy, revealing that AIF can condense DNA, showing a preferential binding to single-stranded over double-stranded DNA. AIF also binds and aggregates single-stranded and structured RNA in vitro. Single-stranded poly A, poly G and poly C, as well double-stranded A/T and G/C RNA competed with DNA for AIF binding with a similar efficiency, thus corroborating a computer-calculated molecular model in which the binding site within AIF is the same for distinct nucleic acid species, without a clear sequence specificity. Among the preferred electron donors and acceptors of AIF, nicotine adenine dinucleotide phosphate (NADP) was particularly efficient in enhancing the generation of higher-order AIF/DNA and AIF/RNA complexes. Altogether, these data support a model in which a direct interaction of AIF contributes to the compaction of nucleic acids within apoptotic cells.

Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p.

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homologue VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycle-dependent phosphorylation of a tyrosine residue near the carboxy terminus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues.

The proteasomal substrate Stm1 participates in apoptosis-like cell death in yeast.

We have identified the yeast gene STM1 in an overexpression screen for new proteasomal substrates. Stm1 is unstable in wild-type cells and stabilized in cells with defective proteasomal activity and thus a bona fide substrate of the proteasome. It is localized in the perinuclear region and is required for growth in the presence of mutagens. Overexpression in cells with impaired proteasomal degradation leads to cell death accompanied with cytological markers of apoptosis: loss of plasma membrane asymmetry, chromatin condensation, and DNA cleavage. Cells lacking Stm1 display deficiency in the apoptosis-like cell death process induced by treatment with low concentrations of H(2)O(2). We suggest that Stm1 is involved in the control of the apoptosis-like cell death in yeast. Survival is increased when Stm1 is completely missing from the cells or when inhibition of Stm1 synthesis permits proteasomal degradation to decrease its amount in the cell. Conversely, Stm1 accumulation induces cell death. In addition we identified five other genes whose overexpression in proteasomal mutants caused similar apoptotic phenotypes.

Methods to Assess Autophagy and Chronological Aging in Yeast.

Autophagy is a catabolic process that is crucial for cellular homeostasis and adaptive response to changing environments. Importantly, autophagy has been shown to be induced in many longevity-associated scenarios and to be required to maintain lifespan extension. Notably, autophagy is a highly conserved cellular process among eukaryotes, and the yeast Saccharomyces cerevisiae has become a universal model system for unraveling the molecular machinery underlying autophagic mechanisms. Here, we discuss different protocols to monitor survival and autophagy of yeast cells upon chronological aging. These include the use of propidium iodide to assess the loss of cell membrane integrity, as well as clonogenic assays to directly determine survival rates. Additionally, we describe methods to quantify autophagic flux, including the alkaline phosphatase activity or the GFP liberation assays, which measure the delivery of autophagosomal cargo to the vacuole. In sum, we have recapped established protocols used to evaluate a link between lifespan extension and autophagy in yeast.

Prostaglandin D2 induces programmed cell death in Trypanosoma brucei bloodstream form.

African trypanosomes produce some prostanoids, especially PGD2, PGE2 and PGF2alpha (Kubata et al. 2000, J. Exp. Med. 192: 1327-1338), probably to interfere with the host's physiological response. However, addition of prostaglandin D2 (but not PGE2 or PGF2alpha) to cultured bloodstream form trypanosomes led also to a significant inhibition of cell growth. Based on morphological alterations and specific staining methods using vital dyes, necrosis and autophagy were excluded. Here, we report that in bloodstream form trypanosomes PGD2 induces an apoptosis-like programmed cell death, which includes maintenance of plasma membrane integrity, phosphatidylserine exposure, loss of mitochondrial membrane potential, nuclear chromatin condensation and DNA degradation. The use of caspase inhibitors cannot prevent the cell death, indicating that the process is caspase-independent. Based on these results, we suggest that PGD2-induced programmed cell death is part of the population density regulation as observed in infected animals.

Phosphatidylethanolamine positively regulates autophagy and longevity.

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.

Spermidine induces autophagy by inhibiting the acetyltransferase EP300.

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.

Identification of the regions of porcine VCP preventing its function in Saccharomyces cerevisiae.

Cdc48p is essential for homotypic endoplasmic reticular fusion in Saccharomyces cerevisiae. It is localized at the endoplasmic reticulum during most of the cell division cycle but concentrates in the nucleus at the G1/S-transition. Its mammalian homologue VCP alternates between the endoplasmic reticulum and the centrosome in dependence of the cell cycle. Though Cdc48p and porcine VCP show a high sequence conservation--almost 70% of their amino acid residues are identical the VCP gene fails to complement a disruption of CDC48. Complementation studies with CDC48 and VCP gene hybrids show that an exchange of the central Cdc48p domain for the central VCP domain prevents a complementation of a CDC48 disruption, although this is the best conserved region between the two proteins. Protein chimeras containing the N-terminal part of VCP only complement a disruption of CDC48 when expressed at high levels. The respective yeast strain shows a nucleus devoid of Cdc48p. In contrast to VCP, Cdc48p contains an almost perfect nuclear targeting sequence in this region. Exchange of the C-terminal Cdc48p domain for the C-terminus of VCP leads to normal viability of the cell, even at low expression levels.

Apoptosis in yeast--a monocellular organism exhibits altruistic behaviour.

Apoptosis is a highly regulated form of programmed cell death crucial for life and health in metazoan animals. Apoptosis is defined by a set of cytological alterations. The recent discovery of these markers in yeast indicates the presence of the basic mechanisms of apoptosis already in unicellular eukaryotes. Oxygen radicals regulate both mammalian and yeast apoptosis. We suggest that apoptosis originated in unicellular organisms as an altruistic response to severe oxidative damage. Later, cells developed mechanisms to purposely produce reactive oxygen species as a regulator of apoptosis. Yeast may become an important model to investigate the conserved steps of apoptosis.

Mammalian Bax triggers apoptotic changes in yeast.

Apoptosis is co-regulated by the conserved family of Bcl-2-related proteins, which includes both its agonists (Bax) and antagonists (Bcl-X(L)). A mutant strain of the yeast Saccharomyces cerevisiae has been shown to express all morphological signs of apoptosis. Overexpression of Bax is lethal in S. cerevisiae, whereas simultaneous overexpression of Bcl-X(L) rescues the cells. We report that overexpression of mammalian Bax in a S. cerevisiae wild type strain triggers morphological changes similar to those of apoptotic metazoan cells: the loss of asymmetric distribution of plasma membrane phosphatidylserine, plasma membrane blebbing, chromatin condensation and margination, and DNA fragmentation. Simultaneous overexpression of Bcl-X(L) prevents these changes. We demonstrate that Bax triggers phenotypic alterations in yeast strongly resembling those it causes in metazoan apoptotic cells.

Apoptosis in yeast: a new model for aging research.

Apoptosis is a form of programmed cell death with a central role in development and homeostasis of metazoan organisms. Recent research indicates the presence of an apoptotic cell death program in unicellular eukaryotes. Yeast can be killed by expression of mammalian proapoptotic genes or in response to oxygen stress, which is an inducer of mammalian apoptosis. The dying yeast cells show morphological alterations typical for apoptosis. Yeast provides a simple model for cellular aging. The observation that old yeast cells produce oxygen radicals and die apoptotically may provide clues to a similar sequence of events in mammalian aging.

Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8.

The exposure of calreticulin (CRT) on the surface of stressed and dying cancer cells facilitates their uptake by dendritic cells and the subsequent presentation of tumor-associated antigens to T lymphocytes, hence stimulating an anticancer immune response. The chemotherapeutic agent mitoxantrone (MTX) can stimulate the peripheral relocation of CRT in both human and yeast cells, suggesting that the CRT exposure pathway is phylogenetically conserved. Here, we show that pheromones can act as physiological inducers of CRT exposure in yeast cells, thereby facilitating the formation of mating conjugates, and that a large-spectrum inhibitor of G protein-coupled receptors (which resemble the yeast pheromone receptor) prevents CRT exposure in human cancer cells exposed to MTX. An RNA interference screen as well as transcriptome analyses revealed that chemokines, in particular human CXCL8 (best known as interleukin-8) and its mouse ortholog Cxcl2, are involved in the immunogenic translocation of CRT to the outer leaflet of the plasma membrane. MTX stimulated the production of CXCL8 by human cancer cells in vitro and that of Cxcl2 by murine tumors in vivo. The knockdown of CXCL8/Cxcl2 receptors (CXCR1/Cxcr1 and Cxcr2) reduced MTX-induced CRT exposure in both human and murine cancer cells, as well as the capacity of the latter-on exposure to MTX-to elicit an anticancer immune response in vivo. Conversely, the addition of exogenous Cxcl2 increased the immunogenicity of dying cells in a CRT-dependent manner. Altogether, these results identify autocrine and paracrine chemokine signaling circuitries that modulate CRT exposure and the immunogenicity of cell death.

The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca(2+) levels to α-synuclein toxicity in Parkinson's disease models.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein α-synuclein (αSyn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca(2+) fluxes, arguing for an involvement of deregulated Ca(2+) homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca(2+)/Mn(2+) ATPase PMR1 (plasma membrane-related Ca(2+)-ATPase 1) as a phylogenetically conserved mediator of αSyn-driven changes in Ca(2+) homeostasis and cytotoxicity. Expression of αSyn in yeast resulted in elevated cytosolic Ca(2+) levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented αSyn-induced loss of dopaminergic neurons in nematodes and flies. In addition, αSyn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the αSyn-driven rise of cytosolic Ca(2+) levels is pivotal for its cytotoxicity and requires PMR1.

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways.

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.

C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages.

Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca(2+) from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl-/- and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl-/- macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl-/- macrophages, FB1 treatment rescued Atgl-/- macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl-/- macrophages by activation of the mitochondrial apoptosis pathway.