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.

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.

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.

The expanding role of yeast in cancer research and diagnosis: insights into the function of the oncosuppressors p53 and BRCA1/2.

When the glucose supply is high, despite the presence of oxygen, Saccharomyces cerevisiae uses fermentation as its main metabolic pathway and switches to oxidative metabolism only when this carbon source is limited. There are similarities between glucose-induced repression of oxidative metabolism of yeast and metabolic reprogramming of tumor cells. The glucose-induced repression of oxidative metabolism is regulated by oncogene homologues in yeast, such as RAS and Sch9p, the yeast homologue of Akt. Yeast also undergoes an apoptosis-like programmed cell death process sharing several features with mammalian apoptosis, including oxidative stress and a major role played by mitochondria. Evasion of apoptosis and sustained proliferative signaling are hallmarks of cancer. This, together with the possibility of heterologous expression of human genes in yeast, has allowed new insights to be obtained into the function of mammalian oncogenes/oncosuppressors. Here, we elaborate on the similarities between tumor and yeast cells underpinning the use of this model organism in cancer research. We also review the achievements obtained through heterologous expression in yeast of p53, BRCA1, and BRCA2, which are among the best-known cancer-susceptibility genes, with the aim of understanding their role in tumorigenesis. Yeast-cell-based functional assays for cancer genetic testing will also be dealt with.

p53 death signal is mainly mediated by Nuc1(EndoG) in the yeast Saccharomyces cerevisiae.

The tumor suppressor p53 plays a central role in the regulation of cellular growth and apoptosis. In the yeast Saccharomyces cerevisiae, the overexpression of the human p53 leads to growth inhibition and apoptotic cell death on minimal medium. In the present work, we show that p53-expressing cells are more susceptible to cell death after an apoptotic stimulus such as H2O2. The analysis of mutants involved in yeast apoptosis-like death suggests that the observed cell death is Yca1 independent and mainly mediated through Nuc1p.

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.

The cancer-associated K351N mutation affects the ubiquitination and the translocation to mitochondria of p53 protein.

Stress-induced monoubiquitination of p53 is a crucial event for the nuclear-cytoplasm-mitochondria trafficking and transcription-independent pro-apoptotic functions of p53. Although an intact ubiquitination pathway and a functional nuclear export sequence are required for p53 nuclear export, the role of specific residues within this region in regulating both processes remains largely unknown. Here we characterize the mechanisms accounting for the nuclear accumulation of a new point mutation (Lys-351 to Asn) in the nuclear export sequence of p53 identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution abrogates the monoubiquitination of p53 induced by both Mdm2 and MSL2 E3-ligases. As a consequence, cells expressing p53 K351N mutant showed defects in cisplatin-induced translocation of p53 to mitochondria, Bax oligomerization, and mitochondrial membrane depolarization. These data identify K351N as a critical mutation of p53 that contributes to the development and maintenance of resistance to cisplatin.

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.

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell.

Voltage-dependent anion-selective channels (VDACs) are pore-forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin-1. We swapped the VDAC3 N-terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N-terminus with the VDAC1 N-terminus caused the chimaera to become more active than VDAC1. The VDAC2 N-terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N-terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.

Characterization of human VDAC isoforms: a peculiar function for VDAC3?

VDACs are a family of pore-forming proteins mainly located in the mitochondrial outer membrane. In mammals three isoforms exist. In this work we review the information available about them with the addition of new results. We have compared the human VDACs transformed in a yeast strain lacking the endogenous porin. VDAC1 and 2 are able to complement the lack of porin in mitochondrial respiration and modulation of ROS. VDAC3 has a limited ability to support the mitochondrial respiration and has no influence in the control of ROS production. The over-expression of VDAC isoforms in wild type yeast strain led to a dramatic sensitivity to oxidative stress, especially for VDAC3, and a shorter lifespan in respiratory conditions. Real-time PCR comparison of the isoforms indicated that in HeLa cells VDAC1 is 10 times more abundant than VDAC2 and 100 times than VDAC3. The over-expression of any single isoform caused a 10 times increase of the transcripts of VDAC2 and VDAC3, while VDAC1 is not changed by the over-expression of the other isoforms. Models of VDAC2 and VDAC3 isoform structure showed that they could be made of a 19-strand beta-barrel and an N-terminal sequence with variable features. In this work we show for the first time a functional characterization of VDAC3 in a cellular context.

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.

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1beta in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy.

BACKGROUND: Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1beta (IL1beta), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1beta] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Deltayca1 [PIR4-IL1beta], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Deltayca1 mutant strain. RESULTS: Our results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1beta] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1beta] did not achieve a high cell density. The Deltayca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kd value (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Deltayca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Deltayca1 mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high. CONCLUSIONS: The paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with S. cerevisiae BY4741 [PIR4-IL1beta] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Deltayca1 disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process.

HIR1, the co-repressor of histone gene transcription of Saccharomyces cerevisiae, acts as a multicopy suppressor of the apoptotic phenotypes of the LSM4 mRNA degradation mutant.

We previously have reported that Saccharomyces cerevisiae mutants expressing Kllsm4Delta1, a truncated form of the KlLSM4 gene, as well as mutants in genes of the mRNA-decapping pathway, show phenotypic markers of apoptosis, increased temperature sensitivity and reduced growth in the presence of different drugs and oxidative stressing agents, such as acetic acid and H(2)O(2). To isolate multicopy extra-genic suppressors of these defects, we transformed the Kllsm4Delta1 mutant with a yeast DNA library and we selected a series of clones showing resistance to acetic acid. One of these clones carried a DNA fragment containing the HIR1 gene that encodes a transcriptional co-repressor of histone genes. The over-expression of HIR1 in the Kllsm4Delta1 mutant prevented rapid cell death during chronological aging, reduced nuclei fragmentation and increased resistance to H(2)O(2). Transcription analysis revealed that the expression of histone genes was lowered in the mutant over-expressing HIR1, indicating a relationship between the latter gene and apoptosis.

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.