Biomedical subjects

Frank Madeo

Publications and source records attributed to Frank Madeo.

At least 19 recordsLinked to original sources

PubMed2006

Why yeast cells can undergo apoptosis: death in times of peace, love, and war.

The purpose of apoptosis in multicellular organisms is obvious: single cells die for the benefit of the whole organism (for example, during tissue development or embryogenesis). Although apoptosis has also been shown in various microorganisms, the reason for this cell death program has remained unexplained. Recently published studies have now described yeast apoptosis during aging, mating, or exposure to killer toxins (Fabrizio, P., L. Battistella, R. Vardavas, C. Gattazzo, L.L. Liou, A. Diaspro, J.W. Dossen, E.B. Gralla, and V.D. Longo. 2004. J. Cell Biol. 166:1055-1067; Herker, E., H. Jungwirth, K.A. Lehmann, C. Maldener, K.U. Frohlich, S. Wissing, S. Buttner, M. Fehr, S. Sigrist, and F. Madeo. 2004. J. Cell Biol. 164:501-507, underscoring the evolutionary benefit of a cell suicide program in yeast and, thus, giving a unicellular organism causes to die for.

Adaptation, Physiological↗

PubMed2006

Yeast unravels epigenetic apoptosis control: deadly chat within a histone tail.

An important paper by David Allis and colleagues (Ahn et al., 2006 [this issue of Molecular Cell]) describes a specific deacetylation/phosphorylation crosstalk at the histone H2B tail required for apoptosis induction in yeast, thus giving first insights into the poorly understood epigenetic regulation of cell death.

Acetylation↗

PubMed2006

Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis.

One major problem concerning the electrophoresis of mitochondria is the heterogeneity of mitochondrial appearance especially under pathological conditions. We show here the use of zone electrophoresis in a free flow electrophoresis device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the structural properties of the organelles by hyperosmotic stress resulted in broad separation profiles. Conversely untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity accompanied by a different outer membrane proteome composition could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. To demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48(S565G) strain carries a mutation in the CDC48 gene that is an essential participant in the endoplasmic reticulum-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of subpopulations of a mitochondrial preparation allowing a further detailed functional analysis.

Adenosine Triphosphatases↗

PubMed2006

Crucial mitochondrial impairment upon CDC48 mutation in apoptotic yeast.

Mutation in CDC48 (cdc48(S565G)), a gene essential in the endo-plasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48(S565G) yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48(S565G) cells. Mitochondrial dysfunction in the cdc48(S565G) strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCP-dependent apoptosis.

Adenosine Triphosphatases↗

PubMed2006

Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures.

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G(0) state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.

Apoptosis↗

PubMed2006

MMI1 (YKL056c, TMA19), the yeast orthologue of the translationally controlled tumor protein (TCTP) has apoptotic functions and interacts with both microtubules and mitochondria.

The yeast orthologue of mammalian TCTP is here proposed to be named Mmi1p (microtubule and mitochondria interacting protein). This protein displays about 50% amino acid sequence identity with its most distantly related orthologs in higher organisms and therefore probably belongs to a small class of yeast proteins which have housekeeping but so far incompletely known functions needed for every eukaryotic cell. Previous investigations of the protein in both higher cells and yeast revealed that it is highly expressed during active growth, but transcriptionally down-regulated in several kinds of stress situations including starvation stress. In human cells, TCTP presumably has anti-apoptotic functions as it binds to Bcl-XL in vivo. TCTP of higher cells was also shown to interact with the translational machinery. It has acquired an additional function in the mammalian immune system, as it is identical with the histamine releasing factor. Here, we show that in S. cerevisiae induction of apoptosis by mild oxidative stress, replicative ageing or mutation of cdc48 leads to translocation of Mmi1p from the cytoplasm to the mitochondria. Mmi1p is stably but reversibly attached to the outer surface of the mitochondria and can be removed by digestion with proteinase K. Glutathionylation of Mmi1p, which is also induced by oxidants, is not a prerequisite or signal for translocation as shown by replacing the only cysteine of Mmi1p by serine. Mmi1p probably interacts with yeast microtubules as deletion of the gene confers sensitivity to benomyl. Conversely, the deletion mutant displays resistance to hydrogen peroxide stress and shows a small but significant elongation of the mother cell-specific lifespan. Our results so far indicate that Mmi1p is one of the few proteins establishing a functional link between microtubules and mitochondria which may be needed for correct localization of mitochondria during cell division.

Adenosine Triphosphatases↗

PubMed2006

Apoptosis-inducing factor: vital and lethal.

Apoptosis-inducing factor (AIF) is a NADH oxidase with a local redox function that is essential for optimal oxidative phosphorylation and for an efficient anti-oxidant defense. The absence of AIF can cause neurodegeneration, skeleton muscle atrophy and dilated cardiomyopathy. In many models of apoptosis, AIF translocates to the nucleus, where it induces chromatin condensation and DNA degradation. The nuclear localization of AIF can be inhibited by blocking upstream signals of apoptosis. The contribution of AIF to cell death depends on the cell type and apoptotic insult and is only seen when caspases are inhibited or not activated. It is unknown to what extent and through which mechanisms AIF contributes to the induction of cell death. Here, we discuss recent progress in the quest to understand the contribution of AIF to life and death.

Animals↗

PubMed2006

The inhibitor-of-apoptosis protein Bir1p protects against apoptosis in S. cerevisiae and is a substrate for the yeast homologue of Omi/HtrA2.

Inhibitor-of-apoptosis proteins (IAPs) play a crucial role in the regulation of metazoan apoptosis. IAPs are typically characterized by the presence of one to three baculovirus IAP repeat (BIR) domains that are essential for their anti-apoptotic activity. Bir1p is the sole BIR-protein in yeast and has been shown to participate in chromosome segregation events. Here, we show that Bir1p is a substrate for Nma111p, which is the homologue of the human pro-apoptotic serine protease Omi/HtrA2 and which is known to mediate apoptosis in yeast. Bir1p is a cytoplasmic and nuclear protein, and yeast cells lacking bir1 are more sensitive to apoptosis induced by oxidative stress. Consistently, overexpression of Bir1p reduces apoptosis-like cell death, whereas this protective effect can be antagonized in vivo by simultaneous overexpression of Nma111p. Moreover, chronologically aged cells that constitutively overexpress Bir1p show a delayed onset of cell death. Therefore, Bir1p, like its closest metazoan homologues deterin and survivin, has dual functions: it participates in chromosome segregation events and cytokinesis and exhibits anti-apoptotic activity.

Aging↗

PubMed2006

Defects in N-glycosylation induce apoptosis in yeast.

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Defects of N-glycosylation in humans lead to congenital disorders. The pivotal step of this pathway is the transfer of the evolutionarily conserved lipid-linked core-oligosaccharide to the nascent polypeptide chain, catalysed by the oligosaccharyltransferase. One of its nine subunits, Ost2, has homology to DAD1, originally characterized in hamster cells as a defender against apoptotic death. Here we show that ost mutants, such as ost2 and wbp1-1, display morphological and biochemical features of apoptosis upon induction of the glycosylation defect. We observe nuclear condensation, DNA fragmentation as well as externalization of phosphatidylserine. We also demonstrate induction of caspase-like activity, both determined by flow cytometric analysis and in cell-free extracts. Similarly, the N-glycosylation inhibitor tunicamycin in combination with elevated temperature is able to challenge the apoptotic cascade. Heterologous expression of anti-apoptotic human Bcl-2 diminishes caspase activation, improves survival of cells and suppresses the temperature-sensitive growth defect of wbp1-1. Furthermore, accumulation of reactive oxygen species occurs in response to defective glycosylation. As deletion of the metacaspase YCA1 does not seem to abrogate glycosylation-induced apoptosis, we postulate a different proteolytic process to be involved in this death pathway.

Anti-Bacterial Agents↗

PubMed2006

Evidence for ORC-dependent repression of budding yeast genes induced by starvation and other stresses.

The highly conserved origin recognition complex (ORC) is required for repressing genes in the silent mating type loci of budding yeast. Here we report that at a non-permissive temperature, the temperature-sensitive orc2-1 mutation induces the expression of more than 500 genes, the majority of which are also induced during starvation of wild-type cells. Many genes induced by starvation or by the orc2-1 mutation are also induced by inactivation of proteins required for chromatin-mediated repression of transcription. Genes induced by the orc2-1 mutation, starvation, or inactivation of repressor proteins, map near ORC-binding loci significantly more frequently compared to all genes. Genes repressed by starvation map near ORC-binding sites less frequently compared to all genes, which suggests they have been evolutionarily excluded from regions of repressive chromatin near ORC-binding sites. Deletion of sequences containing ORC-binding sites near the DAL2 and DAL4 genes in the DAL gene cluster, which are induced by either the orc2-1 mutation or by starvation, constitutively activates these genes and abolishes their activation by the orc2-1 mutation. Our findings suggest a role for ORC in the repression of a large number of budding yeast genes induced by starvation or other aspects of a deleterious environment.

Apoptosis↗

PubMed2005

Yeast caspase 1 links messenger RNA stability to apoptosis in yeast.

During the past years, yeasts have been successfully established as models to study the mechanisms of apoptotic regulation. We recently showed that mutations in the LSM4 gene, which is involved in messenger RNA decapping, lead to increased mRNA stability and apoptosis in yeast. Here, we show that mitochondrial function and YCA1, which encodes a budding yeast metacaspase, are necessary for apoptosis triggered by stabilization of mRNAs. Deletion of YCA1 in yeast cells mutated in the LSM4 gene prevents mitochondrial fragmentation and rapid cell death during chronological ageing of the culture, diminishes reactive oxygen species accumulation and DNA breakage, and increases resistance to H2O2 and acetic acid. mRNA levels in lsm4 mutants deleted for YCA1 are still increased, positioning the Yca1 budding yeast caspase as a downstream executor of cell death induced by mRNA perturbations. In addition, we show that mitochondrial function is necessary for fast death during chronological ageing, as well as in LSM4 mutated and wild-type cells.

Apoptosis↗

PubMed2005

A comparison of the aging and apoptotic transcriptome of Saccharomyces cerevisiae.

In this paper, we present the results of global transcript analysis by the microarray technique of senescent and apoptotic yeast cells. We compared young daughter and old mother cells isolated by elutriation centrifugation, and non-apoptotic and apoptotic cells induced either by a temperature shift of the cdc48(S565G) temperature-sensitive mutant or of the orc2-1 temperature-sensitive mutant. The majority of all genes found to be differentially regulated in these three physiological situations was upregulated, indicating that a cellular death process was initiated rather than an unspecific shut-down of gene expression due to immediate killing. The functional classes of genes upregulated in all three conditions were largely the same, although individual genes were in many cases not identical. The largest group of genes involved were nuclear genes coding for mitochondrial components or functions, which is understandable given the fact that apoptosis can be triggered by mitochondrially generated oxygen radicals and that mitochondria play an important role in the execution of apoptosis. Other functional classes consisted of genes involved in DNA damage response, in cell cycle regulation and checkpoints, in DNA repair, and in membrane lipid and cell wall synthesis. These functional classes represent the response of the cell to the known cellular insults, which occur during aging and apoptosis. As we have shown previously, final-stage senescent yeast mother cells (of the wild-type) are apoptotic.

Apoptosis↗

PubMed2005

Viral killer toxins induce caspase-mediated apoptosis in yeast.

In yeast, apoptotic cell death can be triggered by various factors such as H2O2, cell aging, or acetic acid. Yeast caspase (Yca1p) and cellular reactive oxygen species (ROS) are key regulators of this process. Here, we show that moderate doses of three virally encoded killer toxins (K1, K28, and zygocin) induce an apoptotic yeast cell response, although all three toxins differ significantly in their primary killing mechanisms. In contrast, high toxin concentrations prevent the occurrence of an apoptotic cell response and rather cause necrotic, toxin-specific cell killing. Studies with Deltayca1 and Deltagsh1 deletion mutants indicate that ROS accumulation as well as the presence of yeast caspase 1 is needed for apoptosis in toxin-treated yeast cells. We conclude that in the natural environment of toxin-secreting killer yeasts, where toxin concentration is usually low, induction of apoptosis might play an important role in efficient toxin-mediated cell killing.

Apoptosis↗

PubMed2005

High throughput functional genomics: identification of novel genes with tumor suppressor phenotypes.

We have used a combination of high throughput functional genomics, computerized database mining and expression analyses to discover novel human tumor suppressor genes (TSGs). A genome-wide high throughput cDNA phenotype screen was established to identify genes that induce apoptosis or reduce cell viability. TSGs are expressed in normal tissue and frequently act by reduction of growth of transformed cells or induce apoptosis. In agreement with that and thus serving as platform validation, our pro-apoptotic hits included genes for which tumor suppressing activities were known, such as kangai1 and CD81 antigen. Additional genes that so far have been claimed as putative TSGs or associated with tumor inhibitory activities (prostate differentiation factor, hRAS-like suppressor 3, DPH2L1-like and the metastasis inhibitor Kiss1) were confirmed in their proposed TSG-like phenotype by functionally defining their growth inhibitory or pro-apoptotic function towards cancer cells. Finally, novel genes were identified for which neither association with cell growth nor with apoptosis were previously described. A subset of these genes show characteristics of TSGs because they (i) reduce the growth or induce apoptosis in tumor cells; (ii) show reduced expression in tumor vs. normal tissue; and (iii) are located on chromosomal (LOH-) loci for which cancer-associated deletions are described. The pro-apoptotic phenotype and differential expression of these genes in normal and malignant tissue make them promising target candidates for the diagnosis and therapy of various tumors.

Apoptosis↗

PubMed2004

AIF deficiency compromises oxidative phosphorylation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.

Adenosine Triphosphate↗

PubMed2004

An AIF orthologue regulates apoptosis in yeast.

Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).

Apoptosis↗

PubMed2004

Chronological aging leads to apoptosis in yeast.

During the past years, yeast has been successfully established as a model to study mechanisms of apoptotic regulation. However, the beneficial effects of such a cell suicide program for a unicellular organism remained obscure. Here, we demonstrate that chronologically aged yeast cultures die exhibiting typical markers of apoptosis, accumulate oxygen radicals, and show caspase activation. Age-induced cell death is strongly delayed by overexpressing YAP1, a key transcriptional regulator in oxygen stress response. Disruption of apoptosis through deletion of yeast caspase YCA1 initially results in better survival of aged cultures. However, surviving cells lose the ability of regrowth, indicating that predamaged cells accumulate in the absence of apoptotic cell removal. Moreover, wild-type cells outlast yca1 disruptants in direct competition assays during long-term aging. We suggest that apoptosis in yeast confers a selective advantage for this unicellular organism, and demonstrate that old yeast cells release substances into the medium that stimulate survival of the clone.

Aging↗

PubMed2004

A glutamate receptor-interacting protein homolog organizes muscle guidance in Drosophila.

During Drosophila embryogenesis, developing muscles extend growth-cone-like structures to navigate toward specific epidermal attachment sites. Here, we show that the homolog of Glutamate Receptor-Interacting Proteins (DGrip) acts as a key component of proper muscle guidance. Mutations in dgrip impair patterning of ventral longitudinal muscles (VLMs), whereas lateral transverse muscles (LTMs) that attach to intrasegmental attachment sites develop normally. Myoblast fusion, stabilization of muscle contacts, and general muscle function are not impaired in the absence of DGrip. Instead, the proper formation of cellular extensions during guidance fails in dgrip mutant VLMs. DGrip protein concentrates at the ends of VLMs while these muscles guide toward segment border attachment sites. Conversely, LTMs overexpressing DGrip form ectopic cellular extensions that can cause attachment of these muscles to other muscles at segment borders. Our data suggest that DGrip participates in the reception of an attractive signal that emanates from the epidermal attachment sites to direct the motility of developing muscles. This dgrip phenotype should be valuable to study mechanistic principles of Grip function.

Animals↗