Vanessa Palermo

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.

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.

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.

Apoptosis and aging in mitochondrial morphology mutants ofS. cerevisiae

Cell viability during chronological aging and after apoptotic stimuli in some yeast mutants with altered mitochondrial morphology was followed; a function for the corresponding genes in the apoptotic process was assessed.MDM30 andDNM1, the genes encoding an F-box protein and the dynamin-related GTPase, respectively, are involved in triggering aging and apoptosis. In contrast,YME1, encoding a subunit of the mitochondrial inner membrane i-AAA proteinase complex, has a protective role in these processes.FIS1, the mitochondrial fission gene, might play a protective role after an apoptotic insult while it seems to promote cell death in aging cells.

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.

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. Copyright

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 rho0 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.

A Kluyveromyces lactis mutant in the essential gene KlLSM4 shows phenotypic markers of apoptosis

We report the study of Kluyveromyces lactis cells expressing a truncated form of KlLSM4, a gene ortholog to LSM4 of Saccharomyces cerevisiae which encodes an essential protein involved in both pre-mRNA splicing and mRNA decapping. We had previously demonstrated that the first 72 amino acids of the K. lactis Lsm4p (KlLsm4Deltap) can restore cell growth in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, cells showed a remarkable loss of viability in stationary phase. Here we report that cells expressing KlLsm4Deltap presented clear apoptotic markers such as chromatin condensation, DNA fragmentation, accumulation of reactive oxygen species, and showed increased sensitivity to different drugs. RNA analysis revealed that pre-mRNA splicing was almost normal while mRNA degradation was significantly delayed, pointing to this as the possible step responsible for the observed phenotypes.

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

Background: A new mutant (K351N) of p53 in the tetramerization domain impairs cisplatin-mediated apoptosis in a cisplatin-resistant ovarian carcinoma cell line. Results: Defects in monoubiquitination of the p53 K351N mutant cause its nuclear accumulation. Conclusion: K351N mutation impairs p53 targeting to mitochondria and transcription-independent apoptosis. Significance: K351N mutation of p53 is critical in contributing to and maintaining the resistance to cisplatin. 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.