CBMA

Centro de Biologia Molecular e Ambiental

Centre of Molecular and Environmental Biology

Programmed cell death


Research highlights

The main goal of our research group is to understand the complex Programmed Cell Death (PCD) regulatory network of living organisms. Particularly, we aim to identify/characterise apoptosis regulators that may be attractive molecular targets for the development of novel therapeutics of Human pathologies associated with cell death dysfunctions.

We have formerly identified common features between a mitochondria-dependent death pathway in yeast and the mammalian apoptosis programme, namely the release of cytochrome c (cyt c) and of the yeast orthologue of the human AIF. These findings, together with studies from other labs, led to the recognition that PCD processes are highly conserved from yeast to mammals and to the exploitation of yeast as a model to comprehend basic mechanisms of cell death in higher eukaryotes. In this line we intend pursuing our well established acetic acid induced apoptosis model as an approach to achieve our main research purposes which focus on the two following areas: i) study of the intrinsic cell death machinery and apoptosis signalling pathways and ii) characterization of mammalian apoptosis regulatory proteins.

We found that the absence of orthologs of mammalian proteins putatively involved in mitochondrial outer membrane permeabilization (MOMP) and cyt c release, affects these processes in yeast committed to apoptosis. Using yeast genetics we showed that while deletion of POR1 (voltage-dependent anion channel, VDAC) promotes apoptosis triggered by acetic acid, CPR3 (mitochondrial cyclophilin) deletion had no effect. Moreover, absence of AAC proteins impaired MOMP and cyt c release caused by acetic acid.

Absence of AAC suppresses MOMP and cytochrome c release in acetic acid-induced cell death. A. Cytochrome c release and enzymatic activities were measured in mitochondria isolated from untreated cells and acetic acid-treated (180 mM, 200 min) cells of wild-type and aac1/2/3D A. Cytochrome c content in mitochondria assessed by redox spectra analysis of isolated mitochondria from untreated cells (full lines) and acetic acid-treated cells (dotted lines). B. Enzymatic assays of matricial CS and intermembrane space AK.

We also showed that autophagy is not active in cells undergoing acetic acid-induced apoptosis and is therefore not responsible for mitochondrial degradation. Furthermore, we implicate the vacuolar protease Pep4p and AAC proteins in mitochondrial degradation and found that Pep4p is released from the vacuole into the cytosol in response to acetic acid treatment.

 

Mitochondrial degradation is impaired in the absence of AAC proteins. A. Quantification of mitochondrial degradation for W303 (full line) and aac1/2/3D (dotted line) mutant along treatment with 180 mM acetic acid, assessed by loss of GFP fluorescence by flow cytometry. Values are means+SD of five independent experiments. *P0.05; unpaired t-test. B. Western blots for Tom22p and Por1p from W303 and aac1/2/3D mutant cell lysates prepared at the indicated times after treatment with 180 mM acetic acid as a measure of mitochondrial degradation.Quantification is shown below. Cytosolic phosphoglycerate kinase (Pgk1p) level was used to normalize protein amount loaded on the gel. Values are means+SD of three independent experiments; P 0.001 for Porin1 and P0.05 for Tom22 (aac1/2/3D versus WT cells); two-way ANOVA. C. Release of the Pep4p protease from the vacuole due to acetic acid treatment (AA; 180 mM, 60 min) was assessed in aac1/2/3D cells expressing a PEP4–EGFP fusion protein. Release of PEP4–EGFP to the cytosol (indicated by the arrow heads) was not affected by the absence of AAC proteins. Bar, 10 mm.


S. cerevisiae aac1/2/3 and pep4 mutant strains show alteration from a tubular mitochondrial morphology to a clustered pattern upon acetic acid treatment. Mitochondrial membrane potential in S. cerevisiae W303 and aac1/2/3D cells, non-treated and treated with acetic acid (AA; 180 mM, 30 min), was examined by Mito-Tracker Red staining (middle panels) and compared with mitochondrial matrix-targeted GFP (left panels) in the merged images (right panels). Bar, 4 mm.

AAC-deleted cells also show a decrease in mitochondrial degradation in response to acetic acid but are not defective in Pep4p release. Therefore, AAC proteins seem to affect mitochondrial degradation at a step subsequent to Pep4p release, possibly triggering degradation through their involvement in mitochondrial permeabilization. Moreover, absence of AAC and Pep4 proteins is associated with the formation of mitochondrial clusters during acetic acid-induced apoptosis. The finding that both mitochondrial AAC proteins and the vacuolar Pep4p interfere with mitochondrial degradation suggests a complex regulation and interplay between mitochondria and the vacuole in yeast apoptosis, giving a new insight to the contribution of such proteins to the death pathway.

In another work, we demonstrated that Isc1p, the yeast ortholog of the neutral mammalian sphingomyelinase, plays a key role in H2O2 resistance and chronological lifespan through modulation of iron levels and apoptosis. A yeast mutant lacking Isc1p revealed more resistant to acetic acid indicating that ceramide may contribute to acid-induced apoptosis.

In the scope of a collaborative project with Porto University and using a mammalian PKC expression system in yeast we found that coleon U is a potent and selective activator of the pro-apoptotic nPKCd and e. Moreover we showed that translocation of these nPKCs to the nucleus and the enhancement of a metacaspase- and mitochondrial-dependent pathway was responsible for the observed coleon U-induced growth inhibition in yeast expressing the nPKCd or e.

Using yeasts co-expressing the human wild-type p53 and a mammalian PKC isoform we showed a differential regulation of the p53 inhibitory effects on yeast growth and cell cycle progression as well of the p53 phosphorylation state by PKC isoforms. While PKC-a and PKC-z reduced and did not interfere with the p53 effects, respectively, PKC-d and -e markedly increased p53 activity through p53 phosphorylation.

Our optimised and improved protocol for single cell gel electrophoresis (comet assay) allowed us to reproducibly measure H2O2-induced DNA damage in dying yeast. DNA repair was quantified as diminishing comet tail length with time after oxidative stress removal and followed first-order kinetics. This is the first reported use of the comet assay for quantitative measurement of DNA repair.

Samples obtained by the application of the yeast comet in untreated cells (control) and treated with 10 mM H2O2.

Ongoing and future work

Following our finding on the involvement of Pep4p in mitochondrial degradation we aim to dissect the molecular mechanisms underlying Pep4p function at the mitochondrial level and during the course of apoptosis. In particular we intend to: i) ascertain the role of Pep4p in mitochondrial alterations and release of apoptotic factors occurring during acetic acid-induced cell death and ii) identify Pep4p-mediated pathways, namely those involved in mitochondrial degradation. We are currently developing protocols to elucidate Pep4p-mediated pathways through the identification of genetic and physical interactions of Pep4p with other proteins.

Aiming to elucidate the signalling pathways involved in the apoptotic process we are developing a project on the role of ceramide in acetic acid-induced apoptosis. The results obtained in the yeast model will be complemented by studies with colorectal carcinoma cell lines. This study is supported by an ongoing FCT project.

Regarding our research in an international collaborative project on the role of mammalian apoptotic regulators (proteins of the Bcl-2 family, PKC isoforms) we showed that PKCa increases the translocation and insertion of Bax c-myc, an active mitochondrial form of Bax, into the outer membrane of yeast mitochondria. This event leads to enhanced mitochondrial dysfunction and cell death. Currently we are studying the modulation of wild type Bax by four major mammalian PKC forms (a, d, e and z) and the involvement of specific phosphorylation/dephosphorylation pathways.

Evidence is missing that the DNA repair phenotype that is observed in the yeast comet assay depends on the DNA repair machinery. For this purpose, we are investigating whether the DNA repair can be abolished by the absence of the cdc9 ligase (catalyses phosphodiester bond formation in the last step of DNA repair). Initial results with a conditional mutant of the cdc9 DNA ligase confirm that DNA repair observed in the comet assay is indeed CDC9 dependent.

 

Key References

Pereira, C., Chaves, S., Alves, S., Salin, B., Camougrand, N., Manon, S., Sousa, M. J., Côrte-Real, M. 2010. Mitochondrial degradation in acetic acid-induced yeast apoptosis: the role of Pep4 and the ADP/ATP carrier. Mol Microbiol 76:1398-410.

Azevedo, F., Marques, F., Fokt, H., Oliveira, R., Johansson, B. 2010. Measuring oxidative DNA damage and DNA repair using the yeast comet assay. Yeast (in press).

Coutinho, I., Pereira, G., Leão, M., Gonçalves, J., Côrte-Real, M., Saraiva, L. 2009. Differential regulation of p53 function by protein kinase C isoforms revealed by a yeast cell system. FEBS Letters 583 (2009) 3582–3588.

Coutinho, I., Pereira, G., Simões, M.F., Côrte-Real, M., Gonçalves, J., Saraiva, L. 2009. Selective activation of protein kinase C-delta and -epsilon by 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U). Biochem Pharmacol 78(5):449-59.

Pereira, C., Silva, R.D., Saraiva, L., Johansson, B., Sousa, M.J., Côrte-Real, M. 2008. Mitochondria dependent apoptosis in yeast. BBA - Molecular Cell Research, Special Issue: Apoptosis in Yeast 1783:1286-1302.

Pereira, C., Camougrand, N., Manon, S., Sousa, M.J., Côrte-Real, M. 2007. ADP/ATP carrier is required for mitochondrial outer membrane permeabilization and cytochrome c release in yeast apoptosis. Molecular Microbiolology 66(3), 571-82. Commented at Molecular Microbiology, MicroCommentary by Lawen A. Another piece of the puzzle of apoptotic cytochrome c release, 66(3):553-6.

Almeida, T., Marques, M., Mojzita, D., Amorim, M.A., Silva, R.D., Almeida, B., Rodrigues, P., Ludovico,P., Hohmann, S., Moradas-Ferreira, P., Côrte-Real, M., Costa, V. 2007. Isc1p plays a key role in hydrogen peroxide resistance and chronological lifespan through modulation of iron levels and apoptosis. Mol Biol Cell 19:865-876.

Silva, R. D., Manon, S., Gonçalves, J., Saraiva, L., Côrte-Real, M. Modulation of Bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase C?.. Exp Cell Res (under revision).

 

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