Yeast Lsm Pro-Apoptotic Mutants Show Defects in Autophagy.

LSM4 is an essential yeast gene encoding a component of different LSM complexes involved in the regulation of mRNA splicing, stability, and translation. In previous papers, we reported that the expression in S. cerevisiae of the K. lactis LSM4 gene lacking the C-terminal Q/N-rich domain in an Lsm4 null strain S. cerevisiae (Sclsm4Δ1) restored cell viability. Nevertheless, in this transformed strain, we observed some phenotypes that are typical markers of regulated cell death, reactive oxygen species (ROS), and oxidated RNA accumulation. In this paper, we report that a similar truncation operated in the S. cerevisiae LSM4 gene confers on cells the same phenotypes observed with the K. lactis lsm4Δ1 gene. Up until now, there was no evidence of the direct involvement of LSM4 in autophagy. Here we found that the Sclsm4Δ1 mutant showed a block in the autophagic process and was very sensitive to nitrogen starvation or treatment with low doses of rapamycin, an inducer of autophagy. Moreover, both during nitrogen starvation and aging, the Sclsm4Δ1 mutant accumulated cytoplasmic autophagy-related structures, suggesting a role of Lsm4 in a later step of the autophagy process.

The C-terminal region of yeast ubiquitin-protein ligase Not4 mediates its cellular localization and stress response.

Transient modification of the environment involves the expression of specific genes and degradation of mRNAs and proteins. How these events are linked is poorly understood. CCR4-NOT is an evolutionary conserved complex involved in transcription initiation and mRNA degradation. In this paper, we report that the yeast Not4 localizes in cytoplasmic foci after cellular stress. We focused our attention on the functional characterization of the C-terminus of the Not4 protein. Molecular dissection of this region indicates that the removal of the last 120 amino acids, does not affect protein localization and function, in that the protein is still able to suppress the thermosensitivity observed in the not4Δ mutant. In addition, such shortened form of Not4, as well its absence, increases the transcription of stress-responsive genes conferring to the cell high resistance to the oxidative stress. On the contrary, the last C-terminal 211 amino acids are required for proper Not4 localization at cytoplasmic foci after stress. This truncated version of Not4 fails to increase the transcription of the stress genes, is more stable and seems to be toxic to cells undergoing oxidative stress.

RNA stability and metabolism in regulated cell death, aging and diseases.

The stability of RNAs represents a crucial point for cell life in that these molecules code for proteins and also play structural and regulatory functions. In this review, we will mainly focus on RNA stability and its connection with cell death and aging. In addition, we will consider the interaction of RNAs with ribonucleoprotein complexes, such as P-bodies and stress granules, as well as the role of non-coding RNAs. Finally, we will mention some correlations between RNA and diseases, considering yeast as a simple model system for the study of human cancer and neurodegenerative disorders.

Annurca apple (M. pumila Miller cv Annurca) extracts act against stress and ageing in S. cerevisiae yeast cells.

BACKGROUND: During the past years, a number of studies have demonstrated the positive effect of apple on ageing and different diseases such as cancer, degenerative and cardiovascular diseases. The unicellular yeast Saccharomyces cerevisiae represents a simple eukaryotic model to study the effects of different compounds on lifespan. We previously demonstrated that apple extracts have anti-ageing effects in this organism because of their antioxidant properties. In particular, the effect is related to the presence in this fruit of polyphenols, which give a large contribution to the antioxidant activity of apples. METHODS: We we used a clonogenic assay to assess the viability and the resistance to oxidative stress of S. cerevisiae cells in the presence of Annurca apple extracts. The production of ROS and the aberrant morphology of nuclei were detected by cell staining with the fluorescent dies Dihydrorhodamine 123 and DAPI, respectively. Mitochondrial morphology was analyzed by following the localization of the mito-GFP protein into the mitochondrial matrix. RESULTS: In this study, we show that apple extracts can increase yeast lifespan, reduce the levels of reactive oxygen species and cell sensitivity to oxidative stress, and prevent nuclei and mitochondria fragmentation protecting cells from regulated cell death. CONCLUSIONS: In this paper, by using the yeast S. cerevisiae as a model, we have demonstrated that Annurca extracts possess antioxidant properties thanks to which the extracts can reduce the intracellular ROS levels and have anti-apoptotic functions thus prolonging cell lifespan. These results contribute to knowledge on the effects of natural compounds on ageing and support the use of yeast as a model organism for the development of simple tests to assess the effectiveness of bioactive substances from natural sources.

Increased levels of RNA oxidation enhance the reversion frequency in aging pro-apoptotic yeast mutants.

Despite recent advances in understanding the complexity of RNA processes, regulation of the metabolism of oxidized cellular RNAs and the mechanisms through which oxidized ribonucleotides affect mRNA translation, and consequently cell viability, are not well characterized. We show here that the level of oxidized RNAs is markedly increased in a yeast decapping Kllsm4Δ1 mutant, which accumulates mRNAs, ages much faster that the wild type strain and undergoes regulated-cell-death. We also found that in Kllsm4Δ1 cells the mutation rate increases during chronological life span indicating that the capacity to handle oxidized RNAs in yeast declines with aging. Lowering intracellular ROS levels by antioxidants recovers the wild-type phenotype of mutant cells, including reduced amount of oxidized RNAs and lower mutation rate. Since mRNA oxidation was reported to occur in different neurodegenerative diseases, decapping-deficient cells may represent a useful tool for deciphering molecular mechanisms of cell response to such conditions, providing new insights into RNA modification-based pathogenesis.

External and internal triggers of cell death in yeast.

In recent years, yeast was confirmed as a useful eukaryotic model system to decipher the complex mechanisms and networks occurring in higher eukaryotes, particularly in mammalian cells, in physiological as well in pathological conditions. This article focuses attention on the contribution of yeast in the study of a very complex scenario, because of the number and interconnection of pathways, represented by cell death. Yeast, although it is a unicellular organism, possesses the basal machinery of different kinds of cell death occurring in higher eukaryotes, i.e., apoptosis, regulated necrosis and autophagy. Here we report the current knowledge concerning the yeast orthologs of main mammalian cell death regulators and executors, the role of organelles and compartments, and the cellular phenotypes observed in the different forms of cell death in response to external and internal triggers. Thanks to the ease of genetic manipulation of this microorganism, yeast strains expressing human genes that promote or counteract cell death, onset of tumors and neurodegenerative diseases have been constructed. The effects on yeast cells of some of these genes are also presented.

NEM1 acts as a suppressor of apoptotic phenotypes in LSM4 yeast mutants.

Saccharomyces cerevisiae mutants in the essential gene LSM4, involved in messenger RNA decapping, and expressing a truncated form of the LSM4 gene of the yeast Kluyveromyces lactis (Kllsm4Δ1), show premature aging accompanied by the presence of typical markers of apoptosis and high sensitivity to oxidative stressing agents. We isolated multicopy extragenic suppressors of these defects, transforming the Kllsm4Δ1 mutant with a yeast DNA library and selecting clones showing resistance to acetic acid. Here we present one of these clones, carrying a DNA fragment containing the NEM1 gene (Nuclear Envelope Morphology protein 1), which encodes the catalytic subunit of the Nem1p-Spo7p phosphatase holoenzyme. Nem1p regulates nuclear growth by controlling phospholipid biosynthesis and it is required for normal nuclear envelope morphology and sporulation. The data presented here correlate the mRNA metabolism with the biosynthesis of phospholipids and with the functionality of the endoplasmic reticulum.

Intracellular NADPH levels affect the oligomeric state of the glucose 6-phosphate dehydrogenase.

In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD(+)(H)/NADP(+)(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP(+) bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP(+), also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.

Apple can act as anti-aging on yeast cells.

In recent years, epidemiological and biochemical studies have shown that eating apples is associated with reduction of occurrence of cancer, degenerative, and cardiovascular diseases. This association is often attributed to the presence of antioxidants such as ascorbic acid (vitamin C) and polyphenols. The substances that hinder the presence of free radicals are also able to protect cells from aging. In our laboratory we used yeast, a unicellular eukaryotic organism, to determine in vivo efficacy of entire apples and their components, such as flesh, skin and polyphenolic fraction, to influence aging and oxidative stress. Our results indicate that all the apple components increase lifespan, with the best result given by the whole fruit, indicating a cooperative role of all apple components.

Hypothesis: is yeast a clock model to study the onset of humans aging phenotypes?

In this paper we report the growth and aging of yeast colonies derived from single cells isolated by micromanipulation and seeded one by one on separated plates to avoid growth interference by surrounding colonies. We named this procedure clonal life span, and it could represent a third way of studying aging together with the replicative life span and chronological life span. In this study we observed over time the formation of cell mass similar to the human "senile warts" (seborrheic keratoses), the skin lesions that often appear after 30 years of life and increase in number and size over the years. We observed that similar signs of aging appear in yeast colonies after about 27 days of growth and increase during aging. In this respect we hypothesize to use yeast as a clock to study the onset of human aging phenotypes.

mRNA stability and control of cell proliferation.

Most of the studies on cell proliferation examine the control of gene expression by specific transcription factors that act on transcriptional initiation. In the last few years, it became evident that mRNA stability/turnover provides an important mechanism for post-transcriptional control of gene expression. In eukaryotes, mRNAs are mainly degraded after deadenylation by decapping and exosome pathways. Mechanisms of mRNA surveillance comprise deadenylation-independent pathways such as NMD (nonsense-mediated decay), when mRNAs harbour a PTC (premature termination codon), NSD (non-stop decay, when mRNAs lack a termination codon, and NGD (no-go decay), when mRNA translation elongation stalls. Many proteins involved in these processes are conserved from bacteria to yeast and humans. Recent papers showed the involvement of proteins deputed to decapping in controlling cell proliferation, virus replication and cell death. In this paper, we will review the newest findings in this field.

Yeast lsm pro-apoptotic mutants show defects in S-phase entry and progression.

Expression of the histone genes is tightly coupled to rates of DNA synthesis in yeast and histone mRNAs are modulated both transcriptionally and post-transcriptionally. Trf4 and Trf5, poly(A) polymerases, that mediates polyadenylation and consequent degradation) and Rrp6, an exosome component, play a role in the regulation of histone mRNA levels. In this paper we show that in the mRNA degradation mutant Kllsm4Δ1, histone mRNAs are induced early in the S-phase and maintained at high level all along the entire cell cycle due to a delay in the exit from S-phase and/or entry into M-phase. The overexpression of the HIR1 gene (Histone transcriptional repressor), previously isolated as a multicopy suppressor of the apoptotic phenotypes observed in Kllsm4Δ1, can also restore the normal cycling of histone genes expression. We also found that low doses of hydroxyurea neutralize the onset of the apoptotic phenotypes in Kllsm4Δ1, as well in another mRNA decapping mutants (lsm1) and, in addition, increase the chronological lifespan in both strains suggesting that an entry delay into the S phase can recover some cellular defects in decapping mutants.

Acetyl-L-carnitine protects yeast cells from apoptosis and aging and inhibits mitochondrial fission.

In this work we report that carnitines, in particular acetyl-l-carnitine (ALC), are able to prolong the chronological aging of yeast cells during the stationary phase. Lifespan extension is significantly reduced in yca1 mutants as well in rho(0) strains, suggesting that the protective effects pass through the Yca1 caspase and mitochondrial functions. ALC can also prevent apoptosis in pro-apoptotic mutants, pointing to the importance of mitochondrial functions in regulating yeast apoptosis and aging. We also demonstrate that ALC attenuates mitochondrial fission in aged yeast cells, indicating a correlation between its protective effect and this process. Our findings suggest that ALC, used as therapeutic for stroke, myocardial infarction and neurodegenerative diseases, besides the well-known anti-oxidant effects, might exert protective effects also acting on mitochondrial morphology.

The transdehydrogenase genes KlNDE1 and KlNDI1 regulate the expression of KlGUT2 in the yeast Kluyveromyces lactis.

KlNDE1 and KlNDI1 code for two inner mitochondrial membrane transdehydrogenases involved in the maintenance of the intracellular NAD(P)H redox balance. The function of these genes during the utilization of fermentative and respiratory carbon sources was studied. During growth in glucose, deletion of KlNDE1 and KlNDI1 led to an altered kinetic of ethanol and glycerol accumulation compared with the wild type; in addition, KlndiDelta was unable to grow in respiratory substrates. Northern analysis and GFP-fusion experiments showed that KlNDE1 and KlNDI1 regulate the expression of KlGUT2, a component of the glycerol-3-phosphate shuttle. Moreover, both genes seem to be involved in the biogenesis of the mitochondrial tubular network.

New arylthioindoles and related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell growth inhibition, and molecular modeling studies.

New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27-29, 36, 39, and 41 showed approximately 50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 microM, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC(50)'s in the 78-220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition.

PGK1, the gene encoding the glycolitic enzyme phosphoglycerate kinase, acts as a multicopy suppressor of apoptotic phenotypes in S. cerevisiae.

In a previous paper we reported the construction of a S. cerevisiae strain lacking the essential gene LSM4, which could survive by the introduction of a truncated form of the orthologous gene from Kluyveromyces lactis. This strain showed apoptotic hallmarks and other phenotypes, including an increased sensitivity to caffeine and acetic acid. The suppression of the latter phenotype by overexpressing yeast genes allowed the isolation of PGK1, the gene encoding the glycolytic enzyme phosphoglycerate kinase. This gene restored normal ageing, oxygen peroxide resistance and nuclear integrity in the mutant. Other phenotypes, such as caffeine sensitivity and glycerol utilization, were also suppressed.

Oxidation of Cys278 of ADH I isozyme from Kluyveromyces lactis by naturally occurring disulfides causes its reversible inactivation.

The inactivation of the homotetrameric cytosolic alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) by naturally occurring disulfides, oxidized glutathione, cystine and cystamine, was studied. The inactivation was fully reversed by dithiothreitol. The nicotinamide coenzyme, but not the substrate ethanol, protected KlADH I from inactivation. Gel filtration experiments and SDS-PAGE analysis, also, revealed that enzyme inactivation coincides with inter-subunits disulfide bond formation which are noticeably enhanced after prolonged oxidation with GSSG. Moreover, oxidized KlADH I, as its reduced state, retained the tetrameric stucture and appears mainly as a dimer under non-reducing SDS-PAGE. Conversely, KlADH I Cys278Ile mutant is unaffected by disulfides treatment. Therefore, in vitro, KlADH I wild-type could exist in two reversible forms: reduced (active) and oxidized (inactive), in which the Cys278 residues of each tetramer are linked by disulfide bonds. The redox state of KlADH I could represent the path for modulating its activity and then a regulatory step of glycolysis under hypoxic conditions. It might be hypothesized that KlADH I could represent an important target in redox signaling of Kluyveromyces lactis cell by inhibiting, under oxidative stress, the glycolytic pathway in favor of the pentose-phosphate shunt to restore its reducing potential.

Characterization of KlGUT2, a gene of the glycerol-3-phosphate shuttle, in Kluyveromyces lactis.

KlGUT2 encodes the mitochondrial component of the glycerol-3-phosphate shuttle in Kluyveromyces lactis, a dehydrogenase involved in the maintenance of the NADH redox balance and in glycerol utilization. Deletion of KlGUT2 led to glycerol accumulation during growth in glucose and growth retardation in ethanol. In addition, KlGUT2 deletion altered the expression of other mitochondrial dehydrogenases that contribute to the maintenance of the intracellular redox balance, suggesting a rerouting of ethanol oxidation from the cytoplasm to the mitochondria. Finally, Northern analysis showed that KlGUT2 has two transcripts: one constitutively expressed and dependent on HGT1, the high-affinity hexose transporter gene, and the other induced under respiratory conditions.