Search

Significant interest in the development of high-affinity ligands for Siglecs exists due to the various therapeutically relevant functions of these proteins. Here, we report a new strategy to develop and design Siglec ligands as disialyl-oligosaccharide mimetics exemplified on Siglec-2 (CD22). We report insights into development of dimeric ligands with high affinity and avidity to cell surface-expressed CD22, assay development, tool compounds, structure activity relationships, and biological data on calcium flux regulation in B-cells. The binding modes of selected ligands have been modeled based on state-of-the-art molecular dynamics simulations on the microsecond timescale, providing detailed views on ligand binding and opening a new perspective on drug design efforts for Siglecs. High-avidity dimeric ligands containing a linker opening the way towards bispecifics are presented as well.

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.

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.

A cell's reaction to any change in the endogenous or exogenous conditions often involves a complex response that eventually either leads to cell adaptation and survival or to the initiation and execution of (programmed) cell death. The molecular decision whether to live or die, while depending on a cell's genome, is fundamentally influenced by its actual metabolic status. Thus, the collection of all metabolites present in a biological system at a certain time point (the so-called metabolome) defines its physiological, developmental and pathological state and determines its fate during changing and stressful conditions. The budding yeast Saccharomyces cerevisiae is a unicellular organism that allows to easily modify and monitor conditions affecting the cell's metabolome, for instance through a simple change of the nutrition source. Such changes can be used to mimic and study (patho)physiological scenarios, including caloric restriction and longevity, the Warburg effect in cancer cells or changes in mitochondrial mass affecting cell death. In addition, disruption of single genes or generation of respiratory deficiency (via abrogation of mitochondrial DNA) assists in revealing connections between metabolism and apoptosis. In this minireview, we discuss recent studies using the potential of the yeast model to provide new insights into the processes of stress defense, cell death and longevity.

Pathological neuronal inclusions of the 43-kDa TAR DNA-binding protein (TDP-43) are implicated in dementia and motor neuron disorders; however, the molecular mechanisms of the underlying cell loss remain poorly understood. Here we used a yeast model to elucidate cell death mechanisms upon expression of human TDP-43. TDP-43-expressing cells displayed markedly increased markers of oxidative stress, apoptosis, and necrosis. Cytotoxicity was dose- and age-dependent and was potentiated upon expression of disease-associated variants. TDP-43 was localized in perimitochondrial aggregate-like foci, which correlated with cytotoxicity. Although the deleterious effects of TDP-43 were significantly decreased in cells lacking functional mitochondria, cell death depended neither on the mitochondrial cell death proteins apoptosis-inducing factor, endonuclease G, and cytochrome c nor on the activity of cell death proteases like the yeast caspase 1. In contrast, impairment of the respiratory chain attenuated the lethality upon TDP-43 expression with a stringent correlation between cytotoxicity and the degree of respiratory capacity or mitochondrial DNA stability. Consistently, an increase in the respiratory capacity of yeast resulted in enhanced TDP-43-triggered cytotoxicity, oxidative stress, and cell death markers. These data demonstrate that mitochondria and oxidative stress are important to TDP-43-triggered cell death in yeast and may suggest a similar role in human TDP-43 pathologies.

To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.

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.

‘7th International Meeting on Yeast Apoptosis’ in Graz, Austria, 9–13 September 2009From 9 to 13 September 2009, more than 100 scientistsfrom 22 different countries came together in the lovely city ofGraz in Styria/Austria to share information and experiences,as well as to discuss their data on ‘killing’ yeast at the7th International Meeting on Yeast Apoptosis. This year’smeeting was organised by Frank Madeo and colleagues, whodid not miss the chance to create a chilling atmosphereaccompanied by wine tasting, folk music and traditional foodin the typical Austrian countryside, as well as on a viewpointin the old city centre of Graz, to make their stay as glamorousas possible and share their two passions, science anddissoluteness.This year, the participating groups reported great deliver-ables from their recently published and ongoing work on celldeath,stressdefenceandageinginyeast.Severalresearcherspresented their work with humanised yeast models toinvestigate cell-death pathways and possible drug therapiesfor treating neurodegenerative diseases and cancer.The opening lecture was given by the most cited cell-deathresearcher Guido Kroemer (Paris, France) who presented anoverviewintotheprogressofcell-deathresearchinthelast20years, including the turnabout from a rather ‘nucleocentricsystem’ to the ‘mitochondriocentric system’ of programmedcell death (PCD) in multiple organisms. In his lecture, hefocused on the evolutionary development of PCD and on theemerging concept that any kind of protein that has previouslybeen specifically implicated in cell-death pathways has aphylogenetically conserved apoptosis-unrelated function.Consequently, he hinted at the increasing importance of theyeast system to clarify pathways and discover factors, whichwould support current views and generate new views onageing, cell death and diseases.Cell-Death Pathways and Key PlayersAsapoptoticmarkersweredescribedinyeastforthefirsttime,a dozen years ago

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

We analyzed the role of the peroxisomal peroxiredoxin Pmp20 of the yeast Hansenula polymorpha. Cells of a PMP20 disruption strain (pmp20) grew normally on substrates that are not metabolized by peroxisomal enzymes, but showed a severe growth defect on methanol, the metabolism of which involves a hydrogen peroxide producing peroxisomal oxidase. This growth defect was paralleled by leakage of peroxisomal matrix proteins into the cytosol. Methanol-induced pmp20 cells accumulated enhanced levels of reactive oxygen species and lipid peroxidation products. Moreover, the fatty acid composition of methanol-induced pmp20 cells differed relative to WT controls, suggesting an effect on fatty acid homeostasis. Plating assays and FACS-based analysis of cell death markers revealed that pmp20 cells show loss of clonogenic efficiency and membrane integrity, when cultured on methanol. We conclude that the absence of the peroxisomal peroxiredoxin leads to loss of peroxisome membrane integrity and necrotic cell death. (C) 2008 Elsevier Inc. All Fights reserved

alpha-Synuclein is one of the principal toxic triggers of Parkinson disease, an age-associated neurodegeneration. Using old yeast as a model of alpha-synuclein expression in post-mitotic cells, we show that alpha-synuclein toxicity depends on chronological aging and results in apoptosis as well as necrosis. Neither disruption of key components of the unfolded protein response nor deletion of proapoptotic key players (including the yeast caspase YCA1, the apoptosis-inducing factor AIF1, or the serine protease OMI) did prevent alpha-synuclein-induced cell killing. However, abrogation of mitochondrial DNA (rho(0)) inhibited alpha-synuclein-induced reactive oxygen species formation and subsequent apoptotic cell death. Thus, introducing an aging yeast model of alpha-synuclein toxicity, we demonstrate a strict requirement of functional mitochondria.

Since hyperphosphorylation of protein tau is a crucial event in Alzheimer's disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation.

Programmed cell death of lipid-laden macrophages is a prominent feature of atherosclerotic lesions and mostly ascribed to accumulation of excess intracellular cholesterol. The present in vitro study investigated whether intracellular triacylglycerol (TG) accumulation could activate a similar apoptotic response in macrophages. To address this question, we utilized peritoneal macrophages isolated from mice lacking adipose triglyceride lipase (ATGL), the major enzyme responsible for TG hydrolysis in multiple tissues. In Atgl(-/-) macrophages, we observed elevated levels of cytosolic Ca(2+) and reactive oxygen species, stimulated cytochrome c release, and nuclear localization of apoptosis-inducing factor. Fragmented mitochondria prior to cell death were indicative of the mitochondrial apoptosis pathway being triggered as a consequence of defective lipolysis. Other typical markers of apoptosis, such as externalization of phosphatidylserine in the plasma membrane, caspase 3 and poly(ADP-ribose) polymerase cleavage, were increased in Atgl(-/-) macrophages. An artificial increase of cellular TG levels by incubating wild-type macrophages with very low density lipoprotein closely mimicked the apoptotic phenotype observed in Atgl(-/-) macrophages. Results obtained during the present study define a novel pathway linking intracellular TG accumulation to mitochondrial dysfunction and programmed cell death in macrophages.

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.

Cell cycle control plays a central role in the development and homeostasis of multicellular organisms. Mis-regulation of cell cycles often leads to abnormal tissue sizes and functions in development and can cause various diseases.1 Even though many pathways controlling the cell cycle machinery have been characterized, the signals regulating the cell cycle during development remain poorly understood. The stereotyped nuclear division pattern in the early Drosophila embryonic development has been a good model system to study how development and cell cycle are co-regulated.2 Drosophila embryos develop as a syncytium and undergo thirteen rounds of synchronous mitosis without cytokinesis generating about six thousand nuclei in a common cytoplasm. Subsequently, embryos pause in the interphase of cycle 14 for approximately 1 hour when cellularization, a specialized form of cytokinesis, encloses nuclei into cell membranes. After cellularization, embryos undergo rapid morphogenetic changes (gastrulation) and individual cells resume mitosis asynchronously forming different mitotic domains. The transition from the early synchronous division to cell cycle pause, onset of morphogenetic change and asynchronous cell cycle is similar to the transition observed in other metazoa and is known as “mid-blastula transition” (MBT). The mechanism controlling the onset of the MBT seems to monitor the nucleocytoplasmic (N/C) ratio,3,4 i.e., the ratio of DNA content to unknown cytoplasmic factor(s). During the early syncytial cycles, this ratio is low enough to allow the next nuclear division, such that the DNA content of the embryo continues to double with each cycle. It has been proposed that when this ratio passes a pre-set threshold, all the phenomena of MBT, including cell cycle arrest, take place. However, how nuclei sense or measure the N/C ratio and how they achieve the synchronous divisions preceding the MBT remain unknown. To address these questions, we have used a series of compound fly stocks to generate embryos with different amount of DNA, thus different values of N/C ratio during early divisions.5 We found that the threshold for the N/C ratio to trigger MBT is about 70% of wild-type DNA (Fig. 1A): above this threshold, embryos behave like wild-type embryo, pausing at interphase of cycle 14; below the threshold, embryos undergo one extra mitosis and pause in the interphase of cycle 15. We were intrigued to observe that embryos with N/C values close to the threshold frequently form mitotic patches, with one part pausing in one cycle and the rest undergoing an extra mitosis and pausing in the next cycle. Embryos from cross C(3R);F(3L) × C(3)EN, which gave highest percentage of patchy embryos, were collected, stained with Hoechst dye to visualize DNA and examined for geometric characteristics of the mitotic patches. Cycle 15 patches occupy on average 74.5% of the whole c14/c15 embryo area with standard deviation of ±12.7% (n = 12) while the position of the patches is variable (Fig. 1B). To explain the formation of the mitotic patches, we consider three possible models. First, in what we refer to as the “independence model,” individual nuclei make independent decisions for whether entering next division cycle. If the N/C ratio is measured and the decision to pause is made immediately at the MBT, the nuclear distributions in patchy embryos with threshold DNA contents should show a salt and pepper pattern. However the large mitotic patches actually observed are not compatible with such a model. Using a statistical mechanics model to analyze the distribution of random cluster sizes on a lattice,6 we show that the observed pattern of nuclear density is incompatible with the independent decisions made at cycle 14 (p value < 10^–240). An alternative explanation for the large patch size is that the N/C ratio is measured at an earlier stage and “remembered” in all the progeny of a given nucleus, resulting in a large patch of coherent lineage-related nuclei. To test this possibility, we introduced the Histone-GFP transgene into the compound stocks used to produce embryos with threshold DNA content and used live-imaging to determine if two sister nuclei descending from the same mother nucleus immediately prior to the MBT cell cycle decision always divide in the same way as would be predicted in the lineage model. We monitored the dynamics of patch formation in five embryos and focused our analysis on the nuclei at the boundary between the patches, since these nuclei can provide the required information to link lineage and different division behaviors. As shown in Figure 1C and Supplementary Movie 1, the mother nucleus labeled with a red dot divides into two daughter nuclei. Subsequently, one of the daughter nuclei divides again while the other arrests in interphase. This is the case for 4 out of 11 boundary nuclei we tracked. This observation argues that the decision to divide is made immediately before the MBT and that the coherent behavior of adjacent nuclei cannot be attributed to their common lineage. Instead, the coherent behavior suggests that the decision to arrest the cell cycle may be made collectively by neighboring nuclei through the exchange of some unknown molecule(s). Alternatively, it may reflect subtle local inhomogeneities in the cytoplasmic component of the N/C ratio that only impact the cell cycle decision when DNA content is close to threshold values. Figure 1 Patchy cell cycle pause during Drosophila mid-blastula transition. (A) Cell cycle behaviors of embryos with different amount of DNA compared with wild-type. Below the threshold of 70%, embryos undergo one extra division. Above the threshold, embryos pause ... We favor the communication model because it provides the embryo with a mechanism ensuring coherent behavior across all nuclei during MBT even if individual nuclei make errors in the measurement of the N/C ratio or if the level of the cytoplasmic component is subject to local fluctuations. Communication between nuclei would dampen this randomness and may also synchronize the behavior of individual nuclei during the earlier stage of embryonic development, when nuclei undergo synchronous wave-like cleavage divisions. Similar community effects have also been suggested to generate homogeneity during Xenopus embryonic development and organismal level organization for cell cycle regulation may be a general feature of many organisms.7 It would be interesting to know the molecular nature of the exchanged information and if such inter-nuclear or inter-cellular signaling pathways are conserved. We suggest that molecular and genetic approaches might reveal the nature of the exchange molecules.

Neurodegeneration is characterized by the disease-specific loss of neuronal activity, culminating in the irreversible destruction of neurons. Neuronal cell death can proceed via distinct subroutines such as apoptosis and necrosis, but the underlying molecular mechanisms remain poorly understood. Saccharomyces cerevisiae is an established model for programmed cell death, characterized by distinct cell death pathways conserved from yeast to mammals. Recently, yeast models for several major classes of neurodegeneration, namely alpha-synucleinopathies, polyglutamine disorders, beta-amyloid diseases, tauopathies, and TDP-43 proteinopathies, have been established. Heterologous expression of the human proteins implicated in these disorders has unraveled important insights in their detrimental function, pointing to ways in which yeast might advance the mechanistic dissection of cell death pathways relevant for human neurodegeneration.

Disturbance of peroxisome function can lead to various degenerative diseases during ageing. Here, we show that in yeast deletion of PEX6, encoding a protein involved in a key step of peroxisomal protein import, results in an increased accumulation of reactive oxygen species and an enhanced loss of viability upon acetic acid treatment and during early stationary phase. Cell death of ageing-like yeast cells lacking PEX6 does not depend on the apoptotic key players Yca1p and Aif1p, but instead shows markers of necrosis. Thus, we conclude that loss of peroxisomal function leads to a form of necrotic cell death.

This spring, more than a hundred scientists from around the world gathered in Canterbury, the historic city in the county of Kent in South East England, to attend the eighth International Meeting on Yeast Apoptosis (IMYA). As with every IMYA conference since its inception in 2002, the feeling of being part of a community that is almost a family was evident. In addition, this year's meeting has shown that the field of yeast programmed cell death (PCD) is growing, not only in numbers, but also in its thematic scope.

The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast.