Dissertations / Theses on the topic 'Yeast apoptosis'

Heavy metals, such as copper and cadmium have been linked to a number of cellular dysfunctions in single and multicellular organisms that are associated with apoptosis. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cu and Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in YCA1∆ mutant. Other apoptotic markers, including sub-G1 DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. We also show that low levels of copper can induce a similar apoptotic response in yeast within the first hour of exposure. Such cellular responses were verified by analyzing mitochondrial perturbation, generation of superoxide ions, activation of the yeast caspase1, and the eventual fragmentation of nuclear DNA (through TUNEL). In analyzing the response of yeast to the different metals, we also demonstrated that the metal-induced PCD is instigated through the sequential activity of at least two caspase-like proteins (i.e., Yca1 and Atg4), both of which appear to be in involved in the process of inducing mitochondrial stress. The additional caspase-like activity is shown to be derived from an enzyme involved in the latter stages of autophagy (Atg4), and provides an intriguing association of apoptosis with autophagy. Here we also demonstrate that metals such as copper and cadmium causes oxidative damage to mitochondrial proteins. Such oxidative attack is targeted and we show that oxidation of certain crucial proteins is required for apoptosis upon metal exposure. By showing that such targeted protein oxidation is dependent on YCA1 and ATG, we also confirm the finding that in yeast that have been exposed to a heavy metal, YCA1 and ATG are essential for damaging mitochondria and to initiate apoptosis. These novel findings highlight several new perspectives about the mechanism of metal-dependent apoptosis, while opening up future analyses to the power of the yeast model system.

Peroxiredoxins are small ubiquitous cysteine-containing proteins that exhibit high reactivity to hydrogen peroxide. Apart from their role as antioxidants, detoxifying hydrogen peroxide to water, peroxiredoxins have been implicated in other cellular processes, such as protein folding and signalling. Using S. cerevisiae as a model organism, we utilised a variety of techniques to examine previously unexplored links between peroxiredoxins and mitochondrial function. Firstly, we characterised the role of Gpx3 in yeast mitochondria. Proteomic work revealed the presence of Gpx3 in the mitochondrial intermembrane space (IMS) and we characterised when, how and why Gpx3 can be found within the mitochondria. We showed that cells lacking Gpx3 have aberrant mitochondrial morphology and defective protein import capacity and inner membrane potential upon H2O2 stress. Gpx3 translocates to the IMS via a targeting sequence encoded from a non-AUG codon. This provides a novel and unique molecular mechanism that protects mitochondria from the exceptional oxidative stress which their activity imposes. Secondly, we focused on the role of Tsa1 upon protein aggregation-induced stress. Previous studies using the proline analogue AZC to cause protein misfolding revealed that protein aggregates are localised adjacent to mitochondria and mitochondrial ROS are generated in response. We questioned what effect this might have on mitochondrial function and we showed that upon AZC treatment there is a drop in respiratory rate, dependent on Tsa1. We questioned whether Tsa1, like other peroxiredoxins, is involved in regulating signalling cascades and we showed that cells that are lacking Tsa1 have alterations in the activity of the cAMP/PKA pathway. In parallel, we looked for differences both in the proteome and the transcriptome to understand what is the cause of the lethality of a tsa1 strain upon protein aggregation stress. We propose a mechanism where Tsa1 mediates a transcriptional response to protein misfolding stress via the activity of the heat shock transcription factor, Hsf1. Finally, we focused on the role of the mitochondrial peroxiredoxin Prx1. Under conditions where the mitochondrial matrix is oxidised, either genetically or by chemical addition, we showed than an apoptotic pathway is activated, dependent on the redox state of thioredoxin, Trx3. We showed that Trx3 can interact with Prx1 and loss of Prx1 also stops the induction of cell death. Analysis of the interactome of Trx3 unraveled the involvement of Bxl1/Ybh3, the yeast BH3 domain-containing protein and Aim9, a previously uncharacterised protein with kinase-like motifs, in the progression of cell death. The data presented in this thesis widens our understanding of the function of peroxiredoxins and their involvement in the regulation of cellular cascades that ensure correct mitochondrial function and responses to stress.

Saccharomyces cerevisiae exhibits an apoptotic response upon exposure to toxic metals such as cadmium (Cd) and copper (Cu). Preliminary findings indicate that this response is dependent –to some extent- on the presence of a fermentable carbon source, glucose. To investigate this dependency we monitored the apoptotic response to both metals in the presence and absence of glucose and have shown that glucose is absolutely necessary in order to induce apoptosis in yeast at least during the exposure to metal. We have also looked at the biochemical changes that are taking place in yeast when treated with Cd using Fourier Transform Infra-Red (FTIR) Spectroscopy. Our results suggest that there are definitive changes in cellular activities that are discernable at 1660-1640cm-1 (amide I), 1540-1510cm-1 (amide II) and 1140-1080cm-1(DNA absorption bands).

The ability to evade apoptosis is an acquired characteristic associated with many normal and pathophysiological processes. TMEM 85 represents a novel transmembrane domain containing human protein isolated in our previous screen for Bax suppressors, but whose function is currently unknown. Using viability and growth assays, we confirmed that TMEM 85 is anti-apoptotic. Four unique human cDNA sequences containing regions distinct from and of perfect identity to our cDNA were present in the database. Analysis of TMEM 85 suggests that it consists of five exons, alternatively spliced to produce at least four different mRNA's and proteins (TMEM 85v1-v4). RT-PCR analysis using RNA isolated from mice and humane tissues show that all transcripts are expressed. Yeast contain an orthologue of the human TMEM 85v1 protein, YGL213C. Surprisingly, the viability assay indicated that mutants lacking YGL231c do not show a hyper-responsive apoptotic phenotype, however its overexpression shows that it is nevertheless anti-apoptotic. Using a yeast strain expressing chromosomally TAP-tagged YGL231c, we found no up-regulation of the endogenous gene due to stress. The deletion mutant is also known to expresses a synthetically lethal phenotype in the presence of alpha-synuclein. While expression of alpha-synuclein caused significant death in both the wild type and deletion mutants, TMEM 85v2 was unable to exhibit a protective role. These findings demonstrate the complexity of the TMEM 85 gene and its anti-apoptotic function in both yeast and human.

The aim of this thesis is the characterization of a protein involved in apoptosis. Our group has identified an early step common to different forms of intrinsic apoptosis stimuli. This step requires de novo synthesis of a novel Cyclin, Cyclin O, that upon apoptosis induction in lymphoid cells forms active complexes, primarily with Cdk2. Cyclin O expression precedes glucocorticoid and gamma radiation-induced apoptosis in vivo in mouse thymus and its overexpression induces apoptosis in cultured cells. Knocking down the endogenous expression of Cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis while leaving CD95 death receptor mediated apoptosis intact. This data demonstrates that apoptosis induction in lymphoid cells is one of the physiological roles of Cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle. In addition we have identified c-Myb a substrate of Cdk2-Cyclin O complexes and we show that c-Myb is downregulated during apoptosis of lymphoid cells.
L'objectiu d'aquesta tesi és la caracterització d'una proteïna involucrada en l'apoptosi. El nostre grup ha identificat un pas primerenc comú en diversos estímuls apoptòtics de la ruta intrínseca. Aquest pas requereix la síntesi de novo d'una nova Ciclina, Ciclina O, que quan s'indueix apoptosi en cèl·lules limfoides forma complexes actius majoritàriament amb Cdk2. L'expressió de la Ciclina O és prèvia a l'apoptosi induïda per glucocorticoids i radiació gamma i la seva sobreexpressió indueix apoptosi en cultius cel·lulars. La baixada dels nivells d'expressió de la Ciclina O endògena amb shRNA provoca una inhibició de l'apoptosi induïda per glucocorticoids o agents que danyen el DNA, mentre que l'apoptosi mediada pel receptor CD95 es manté intacta. Aquests resultats demostren que la inducció d'apoptosi en cèl·lules limfoides és una de les funcions fisiològiques de la Ciclina O i que no es deu a una pertorbació de processos cel·lulars normals com ara el cicle cel·lular. A més a més, hem identificat c-Myb com a substrat dels complexes Cdk2-Ciclina O i demostrem que els nivells de c-Myb baixen durant l'apoptosis de cèl·lules limfoides.

OmiHtrA2 is a highly conserved mammalian serine protease that belongs to the HtrA family of proteins. Omi shares homology with the bacterially expressed heat shock protease HtrA, which functions as a protease at higher temperatures and a chaperone at lower temperatures. Additionally, Omi shares sequence similarity with the mammalian homologs L56/HtrA1 and PRSP/HtrA3. Omi was first isolated as an interacting protein of Mxi2, an alternatively spliced form of the p38 stress-activated kinase, using a modified yeast two-hybrid system. Omi localizes in the mitochondria and in response to apoptotic stimuli the mature form of this protein translocates to the cytoplasm. In the cytoplasm Omi participates in both the caspase-dependent as well as caspase-independent apoptosis. Additionally, recent studies suggest that Omi may have another unique function, maintaining homeostasis within the mitochondria. In an effort to further elucidate the function of Omi, a yeast two-hybrid screening was performed to isolate novel interacting proteins. This screening identified a novel protein (HOPS), as a specific interactor of Omi. The predicted amino acid sequence of this protein does not provide any information about its potential function in mammalian cells. However, experiments show that HOPS is cleaved in vitro by Omi. Furthermore, in response to apoptotic stimuli, HOPS is also degraded in vivo. This study suggests that HOPS could be a physiological substrate of Omi that is cleaved and removed during apoptosis.
M.S.
Department of Molecular Biology and Microbiology
Health and Public Affairs
Molecular Biology and Microbiology

Programmed cell death in animals, plants and protists is in part regulated by a variety of proteases, including cysteine aspartyl proteases, (caspases, paracaspases and metacaspases), cathepsins, subtilisin-like serine proteases, vacuolar processing enzymes and the proteasome. The role of different proteases in the cell death responses of the fungi is however largely unknown. A greater understanding of the fungal cell death machinery may provide new insights into the mechanisms and evolution of PCD and potentially reveal novel targets for a new generation of antifungal drugs. The role of a metacaspase encoding gene, MCA1, in the cell death response of the human pathogen Candida albicans pathogen has been investigated by functional analysis. MCA1 deletion not only alters the sensitivity of cells to a number of cell death stimuli, it also enhances virulence in an insect model. C. albicans shows altered cell and colony morphology on Lee’s medium. Evidence is presented to suggest that these functions appear to be dependent upon active mitochondria. In this study it has also been shown that key caspase substrates may be conserved between humans and the yeasts Saccharomyces cerevisiae and Candida albicans. Many substrates, particularly those which are essential, have retained their caspase cleavage motifs. 14 protease mutants displayed altered activity against caspase 1, 3, 6 or 8 substrates during acetic acid-induced PCD and caspase 1-like activity appeared to be particularly associated with PCD. Using a novel bioinformatic analysis of experimental LC-MS/MS data, changes in the degradation patterns of the proteome (destructome) following acetic acid-induced cell death have been investigated in wild-type yeast. In addition, potential native substrates of the yeast Mca1 have also been identified. The future challenge is to characterise the destructome of different proteases under a range of cell death conditions. In this way it may be possible to identify key components of the cell death machinery and their substrates and so reveal the most promising targets for future therapeutics.

Le polyomavirus à cellules de Merkel est aujourd’hui reconnu comme l’agent étiologique du carcinome à cellules de Merkel (CCM). Le cycle viral et les mécanismes de l’oncogenèse viro-induite sont peu connus et les connaissances se basent essentiellement sur les études menées notamment sur le polyomavirus SV40. L’objectif des travaux de thèse était d’identifier les interactions entre les protéines virales et les protéines cellulaires lors de l’infection ou dans le contexte du carcinome à cellules de Merkel (CCM). Pour identifier ces interactions, nous avons réalisé des cribles double hybride en levures sur les oncogènes du MCPyV et du BKPyV. Afin valider les interactions obtenues en levures, nous avons utilisé une méthode orthogonale de validation par complémentation en cellules de mammifères reposant sur la restauration de la luciférase de Gaussia princeps. La combinaison de ses deux techniques nous a permis de valider des interactions avec des partenaires cellulaires impliqués dans la régulation du cycle cellulaire ou encore de la voie Akt-mTOR. Les précédents travaux du laboratoire, qui portaient sur l’interactome des protéines mineures de capsides VP2/VP3, avaient également permis d’identifier des interactions avec des protéines de la voie NF-kB. Nous avons alors testé les interactions entre les oncogènes et la protéine mineure de capside VP2 avec des protéines cellulaires impliquées dans cette voie. Ces travaux nous ont conduits à tester l’activation de la voie, l’expression des gènes sous le contrôle de NF-kB et la régulation de l’apoptose. Les résultats obtenus montrent une action de la protéine VP2 sur l’activation de la voie NF-kB et une induction de l’apoptose
The Merkel cell polyomavirus is now recognized as the etiologic agent of Merkel cell carcinoma (MCC). The viral cycle and viro-induced oncogenesis mechanisms are not fully understood and the knowledge is mainly based on the studies carried out particularly on the SV40 polyomavirus. The aim of our work is to identify interactions between viral proteins and cellular proteins during productive infection or in MCC context. To identify these interactions, we performed yeast two hybrid screens on MCPyV and BKPyV oncogenes, as control. To validate the interactions obtained in yeasts, we used an orthogonal method of validation by complementation in mammalian cells based on the restoration of Gaussia princeps luciferase. The combination of these two orthogonal techniques allowed us to validate interactions with cellular partners involved in cell cycle regulation or Akt-mTOR pathway. Previous lab work on VP2/VP3 minor capsid proteins allowed the identification of interactions with NF-kB pathway involved proteins. We examined the interactions between oncogenes, VP2, with the cellular proteins involved in this pathway. This work led us to evaluate pathway activation, genes expression under the control of NF-kB and apoptosis regulation. These results evidenced an action of the VP2 protein on the activation of NF-kB pathway and an induction of apoptosis

Elucidating novel anti-apoptotic regulatory pathways is central to further understanding the molecular basis of several pathologies, including cancer. The Bcl-2 protein family includes central regulators of the apoptotic process in mammalian cells, such as the antiapoptotic Bcl-2 and the pro-apoptotic Bax. Descriptions of apoptotic phenotypes in yeast induced by certain stressful stimuli, including the heterologous expression of Bcl-2 proteins, indicate the presence of an evolutionarily conserved apoptotic programme. We have previously reported the identification of several mammalian cDNAs effective in preventing the lethal effects of heterologous expression of a pro-apoptotic BAX cDNA in yeast (Yang et al., FEMS Yeast Research 2006; 6:751-762). Here we report that one of the Bax suppressors encodes a novel 156 amino acid variant of the human Vps24 protein, Vps24ß, that lacks the N-terminal lipid binding domain of the well characterized 222 residue Vps24 (Vps24a). We demonstrate that the VPS24ß cDNA represents an expressed transcript that is likely produced by alternative splicing of the human VPS24 gene. Vps24a, but not Vps24ß, prevented the temperature and salt sensitive growth defects observed in a yeast mutant lacking a functional VPS24 gene. In contrast, Vps24ß, but not Vps24a, suppressed the inhibitory effects of Bax on yeast growth. Vps24ß protein also suppressed the effects of Bax in mutants lacking other VPS genes suggesting that a functional ESCRT pathway, of which the yeast Vps24p is an essential component, is not required for Vps24ß function. Taken together, we demonstrate that the human VPS24 gene gives rise to two functionally distinct proteins, one of which is involved in the ESCRT pathway and another novel protein that serves an anti-apoptotic role. The apoptotic programme is evolutionarily conserved between yeast and metazoan organisms. The second complete manuscript included herein involves an additional suppressor, named Tsc22(86), that r
La détermination de nouveau cheminements de normalization anti-apoptotique est essentiel à la compréhension de plusieurs pathologies au niveau moléculaire, incluant le cancer et les maladies du coeur. La famille de protéines Bcl-2 inclu des agents de normalization centrales du processus apoptotique présents dans des cellules mammifères, dont l'anti-apoptotique Bcl-2 et le pro-apoptotique Bax. La description des phénotypes apoptotique contenus dans la levure induit par un stimulus aggressif, incluant l'expression hétérologue des protéines Bcl-2, indique la présence d'un programme apoptotique conservé durant l'évolution de ce dernier. Précédemment, nous avons reporté sur l'identification de plusieurs cADNs plutôt efficaces envers la prévention des éffets néfastes concernant l'expression hétérologue d'un BAX cADN pro-apoptotique dans la levure (Yang, Khoury et al., FEMS Yeast Research 2006; 6:751-762). Içi, je reporte le fait qu'un abrogeur Bax est en mesure d'encoder une nouvelle variante d'acide aminé 156 de la protéine humaine Vps24, nommé Vps24ß, qui n'est pas muni du domaine caractéristique d'aggripage de lipides à N-bornes qui se retrouve généralement dans le résidue 222 du Vps24 (Vps24a). Je démontre que le Vps24ß cADN représente une transcription qui est probablement produit par le processus alternatif d'épissement du gène humain Vps24. C'était le Vps24a, et non le Vps24ß, qui a empêché la croissance des défauts sensible au sel et à la température observés dans une affectation de levure qui ne possédait pas un gène Vps24 fonctionnel. Par contre, le Vps24ß, et non le Vps24a, a supprimé les éffets négatives du Bax envers la croissance de la levure. De plus, la protéine Vps24ß a aussi supprimé les éffets du Bax dans des échantillons le levure mutant manquant d'autres gènes VPS, qui suggèrent qu'une voie ESCRT fonctionnelle, ou le Vps24p est essentiel, n'est pas requis pour le Vps24ß. Pris ensembles, no

Regulators of G protein signaling (RGSs) are negative regulators of G protein coupled receptors (GPCRs). Our lab has demonstrated that yeast Saccharomyces cerevisiae is a useful system to study RGS and G protein signaling. Mammalian RGSs can be expressed in yeast and favored to interact with mammalian GPCRs as well.
Based on the observation that human RGS1 causes yeast cell growth arrest, I therefore used RGS1 expressing yeast cells to screen a mouse T cell cDNA library in order to find potential interacting proteins. From the screen, I identified a mouse sphingomyelin synthase 1 (SMS1) cDNA. By using a series of different apoptotic stimuli, such as hydrogen peroxide, osmotic stress, exogenous ceramide and its precursors, high temperature etc., SMS1 expression was found to suppress cell growth arrest and prevent viability decline, indicating that SMS1 represents an anti-apoptotic protein that functions by decreasing the intracellular level of pro-apoptotic ceramide.
Gene analysis further indicated that the SMS1 gene consists of 16 exons spread over a 256kb portion of mouse chromosome 19. It is alternatively spliced to produce 4 different transcripts (SMS1alpha1, SMS1alpha2, SMS1beta and SMS1gamma) and encode 3 different proteins (SMS1alpha, SMS1beta and SMS1gamma). Notably, I found that SMS1beta protein does not interfere with SMS1alpha anti-apoptotic function, although both of these two proteins contain the protein-protein interaction domain, sterile alpha motif (SAM), at their N-terminus.
I also carried out a study to examine GPCR-RGS interactions using the yeast expression system. Our lab had noticed that there was an extra RGS5 related protein that was detected by western blot analysis in the protein extracts prepared from yeast and HEK293 cells expressing RGS5. The size of the band was approximately 2 times the molecular weight of RGS5, indicating the possibility that RGS5 forms a dimer. To further examine this hypothesis, I, therefore, performed a series of experiments, included yeast 2 hybrid assays, to demonstrate that RGS5 does interact with itself. This is the first report that RGS can form a dimer. The implications for this finding are discussed in detail.

Tese de Doutoramento em Ciências.
In the yeast Saccharomyces cerevisiae, acetic acid triggers a mitochondria-mediated death pathway with apoptotic characteristics. In mammalian cells, the mitochondrial outer membrane permeabilization (MOMP), necessary for the release of pro-apoptotic proteins, is a pivotal event for the activation of the apoptotic cascade in numerous cell death pathways. MOMP is thought to be mediated by a complex of proteins that constitute the permeability transition pore (PTP). Since S. cerevisiae possesses orthologues of the components believed to be involved in mammalian PTP composition/regulation, it was used herein to study such proteins both concerning their role in mitochondrial permeabilization and involvement in the course of cell death. The proteins studied were Por1p (yeast voltage-dependent anion channel, VDAC), Cpr3p (mitochondrial cyclophilin) and Aac1/2/3p (ADP/ATP carrier, AAC). We found that during apoptosis triggered by acetic acid deletion of CPR3 has no effect. Absence of Por1p enhances and absence of AAC proteins decreases acetic acidinduced apoptosis indicating an anti- and pro-apoptotic role, respectively, for these proteins. Moreover, the pro-death role of AAC does not require the ADP/ATP translocase activity. Absence of AAC proteins impairs MOMP and release of cytochrome c, which is degraded along with other mitochondrial inner membrane proteins. We observe that, during acetic acid-induced apoptosis, caspase activation is independent of AAC proteins but strongly dependent on the growth phase of the culture. In addition, a strain deleted for the yeast metacaspase YCA1 shows decreased overall caspase activation but still died exhibiting apoptotic features, supporting the existence of an Yca1p-independent apoptotic pathway. Fragmentation and degradation of mitochondria are common events in mammalian apoptosis. Both fragmentation and degradation are able to strongly affect the course of cell death and a relation has been proposed between these events and MOMP/cytochrome c release. Interestingly, por1Δ cells exhibit fragmented mitochondrial reticulum in the absence of any death stimulus. This phenotype however, does not contribute to the apoptosis stimulation observed in por1Δ mutant. We observe that during acetic acidinduced apoptosis the absence of AAC proteins leads to aggregation of fragmented mitochondria and a slower degradation of these organelles. Degradation of mitochondria in response to acetic acid is not due to classical autophagy or mediated by Uth1p-dependent mitophagy. We show that mitochondrial degradation during acetic acid-induced apoptosis is dependent on the protease Pep4p that is released from the vacuole to the cytosol. pep4Δ cells, which are strongly impaired in mitochondria degradation, are sensitized to acetic acid and mainly die by necrosis. This suggests that mitochondria degradation in response to acetic acid helps to sustain the apoptotic process. Taken together, the results show that vacuolar and mitochondrial proteins interfere with mitochondria morphological remodelling and subsequent degradation, suggesting that there is a complex interplay between these organelles in the regulation of yeast cell death. In conclusion, we were able exploring the distinctive ability of yeast to survive without respiration-competent mitochondria to study the involvement of mitochondria and mitochondria-interacting proteins in cell death. Additional studies using this model will undoubtedly further our understanding of the complex cell death processes.
Na levedura Saccharomyces cerevisiae, o ácido acético desencadeia uma via de morte dependente da mitocôndria com características apoptóticas. Nos mamíferos, a permeabilização da membrana mitocondrial externa (PMME), necessária para a libertação de proteínas mitocondriais pró-apoptóticas, constitui um passo crítico na activação do processo de morte apoptótico. Pensa-se que a PMME seja mediada por um complexo proteico que constitui o poro de transição de permeabilidade (PTP). Uma vez que S. cerevisiae possui ortólogos de algumas das proteínas que se pressupõe compor/regular o PTP de mamíferos, o principal objectivo desta tese foi estudar essas proteínas, tanto ao nível do seu papel na permeabilização mitocondrial bem como do seu envolvimento na execução do processo de morte apoptótico. As proteínas estudadas incluem o canal Por1p (canal de aniões dependente da voltagem, VDAC), a ciclofilina mitocondrial Cpr3p e as três isoformas do transportador Aac1/2/3p (antiportador mitocondrial de ATP/ADP, AAC). A interrupção do gene CPR3 não afecta a morte induzida pelo ácido acético. A interrupção do gene POR1 estimula e a ausência dos genes AAC1/2/3 protege as células da morte apoptótica induzida pelo ácido acético indicando um papel anti- e pró-apoptótico, respectivamente, para estas proteínas. A função das proteínas AAC na morte celular apoptótica não depende da actividade de antiporte. Em células tratadas com ácido acético a ausência das proteínas AAC afecta negativamente a PMME e a libertação de citocromo c, o qual juntamente com outra proteína da membrana mitocondrial interna sofre degradação. Observamos que durante o processo apoptótico induzido pelo ácido acético ocorre a activação de caspases e que esta activação é independente das proteínas AAC e fortemente dependente da fase de crescimento da cultura. Adicionalmente, uma estirpe interrompida na metacaspase de levedura, YCA1, que exibe um decréscimo na activação total de caspases, desencadeia uma morte celular apoptótica. Esta observação suporta a existência de uma via de morte apoptótica independente de Yca1p. A fragmentação e a degradação mitocondrial são eventos comuns no processo apoptótico em mamíferos. Estes eventos podem ter um forte impacto no decurso da morte celular tendo sido proposta uma relação entre estes e a PMME/libertação de citocromo c. Curiosamente, a ausência da Por1p origina uma elevada percentagem de células com o retículo mitocondrial fragmentado mesmo na ausência de qualquer estímulo externo. Este fenótipo, contudo, não parece contribuir para a estimulação da apoptose exibida pela estirpe por1Δ. Durante o processo de morte apoptótico induzido pelo ácido acético, a ausência das proteínas AAC leva à formação de agregados mitocondriais associada a uma menor degradação destes organelos. A degradação mitocondrial durante a apoptose induzida pelo ácido acético não é devida à activação da autofagia clássica, nem mediada pela mitofagia dependente de Uth1p. A degradação mitocondrial induzida pelo ácido acético é dependente da protease Pep4p, que é libertada do vacúolo para o citosol. A estirpe pep4Δ que exibe uma degradação mitocondrial manifestamente diminuída é sensível ao ácido acético, e direcciona o processo de morte para uma forma necrótica. Estes dados sugerem que a degradação mitocondrial favorece a manutenção do processo apoptótico. Globalmente, os resultados evidenciam que proteínas vacuolares e mitocondriais interferem com a morfologia mitocondrial e subsequente degradação e sugerem uma interacção e regulação complexa entre estes organelos durante o processo apoptótico em leveduras. Concluindo, a capacidade de sobrevivência da levedura em condições em que a respiração mitocondrial está afectada ou ausente permitiu a sua utilização como um excelente modelo para a pesquisa do papel de proteínas mitocondriais, ou de proteínas que interactuam com a mitocôndria no processo de morte celular. Estudos adicionais utilizando este modelo irão seguramente contribuir para uma melhor compreensão dos processos de morte celular.
Fundação para Ciência e a Tecnologia (FCT) - grant SFRH/BD/13497/2003

Dissertação de mestrado em Genética Molecular
The vacuole is the largest organelle of yeast cells and is functionally equivalent to animal lysosome and plant vacuole. Vacuoles are the most acidic organelles of the cell, and play major roles in protein degradation, ion and metabolite storage, as well as in ion homeostasis, response to nutrient deprivation, osmotic and ionic stress, autophagy and even in apoptosis. Lysosome membrane permeabilization and the consequent release of lysosomal proteases, namely cathepsins, are now widely accepted to trigger apoptosis. Of all lysosomal cathepsins, the aspartic cathepsin D was the first identified protein with apoptogenic properties. Acetic acid was shown to induce apoptosis in yeast, associated with changes in mitochondria and release of pro-apoptotic proteins. Pep4p, a vacuolar yeast protease orthologue of the lysosomal human cathepsin D was also shown to be involved in acetic acid-induced apoptosis. The ultimate objective of the present study was to contribute to the understanding of the crosstalk between the vacuole and mitochondria in yeast programmed cell death induced by acetic acid. For these purposes the effect of acetic acid in isolated vacuoles and whole cells with different genetic backgrounds, namely on vacuole membrane permeabilization as well on the release of Pep4p and their relation with alterations in vacuole function, was investigated. The functional characterization of isolated vacuoles as well the effect of acetic acid on vacuolar function was monitored with fluorescent probes combined with flow cytometry and fluorescence microscopy. Functional characterization of isolated vacuoles was also monitored through the activity of V-H+-ATPase by spectrofluorimetry. Epifluorescence microscopy imaging showed that the vacuolar membrane stains strongly with the styryl dye FM1-43 and that most of the vacuoles accumulate Ca2+, as assessed with Fluo-4 AM. Flow cytometry analysis of vacuole samples incubated with these fluorescent probes confirmed a well-defined population of intact and functional vacuoles. Consistently spectrofluorimetric assays with the pH-sensitive probe ACMA suggested that the isolated vacuoles were intact and functional, the vacuolar membrane being able to generate and maintain a pH gradient through a concanamycin A-sensitive V-H+-ATPase. The addition of acetic acid induced the release from the vacuole lumen of an EGFP-Pep4p fusion protein. Changes in fluorescence of vacuoles stained with acridine orange and Fluo-4 AM suggest that the acid induces a transient perturbation of vacuolar pH and Ca2+ release. It has been described that release of Ca2+ is an event involved in cell death, as well as the release of H+ and consequent cytosol acidification. The main novelty of the present study with isolated vacuoles is the finding that acetic acid is able to directly induce a partial permeabilization of the vacuolar membrane, similar to LMP in mammalian cells, without the involvement of other organelles or triggering upstream pathways.
O vacúolo é o maior organelo da levedura e é funcionalmente equivalente ao lisossoma das células animais e ao vacúolo das células vegetais. Este organelo, cujo lúmen tem o pH mais ácido, participa em processos celulares importantes tais como degradação proteica e armazenamento de iões e metabolitos, bem como na homeostase iónica, resposta à privação de nutrientes, stress osmótico e iónico, autofagia e mesmo na apoptose. Sabe-se que a permeabilização da membrana lisossomal (LMP) e a consequente libertação de proteases lisossomais, nomeadamente de catepsinas, induz apoptose. De todas as proteases lisossomais, a catepsina D foi a primeira proteína a ser identificada com propriedades apoptóticas. Foi demonstrado que o ácido acético induz apoptose em leveduras, associada a alterações mitocondriais e à libertação de proteínas pró-apoptóticas. Foi igualmente demonstrado que a protease vacuolar Pep4p, ortóloga da catepsina D humana, está envolvida na apoptose induzida por ácido acético. O presente estudo teve como principal objectivo contribuir para a compreensão da interligação entre o vacúolo e a mitocôndria na morte celular programada induzida por ácido acético na levedura Saccharomyces cerevisiae. Para tal foi investigado o efeito do ácido acético em vacúolos isolados e em células inteiras de leveduras com diferentes backgrounds genéticos, nomeadamente no que respeita à permeabilização da membrana vacuolar bem como à libertação de Pep4p e sua relação com alterações nas funções do vacúolos. A caracterização funcional de vacúolos isolados bem como o efeito da adição de ácido acético na função vacuolar foram realizadas por citometria de fluxo e microscopia de fluorescência associada à utilização de diferentes sondas fluorescentes. A caracterização funcional dos vacúolos isolados foi complementada pela determinação da actividade da V-H+-ATPase por espectrofluorimetria. Os estudos de microscopia de fluorescência mostraram uma marcação forte da membrana vacuolar com a sonda lipofílica FM1-43 e que a maioria dos vacúolos acumulavam Ca2+ com base na marcação pela sonda Fluo-4 AM. A análise por citometria de fluxo de amostras de vacúolos incubadas com estas sondas fluorescentes evidenciou uma população bem definida de vacúolos intactos e funcionais. Consistentemente os ensaios de espectrofluorimetria com a sonda ACMA, sensível ao pH, sugeriram que os vacúolos isolados se encontravam intactos e funcionais, dado que a membrana vacuolar era capaz de gerar e manter um gradiente de pH através da actividade da bomba V-H+-ATPase, sensível à concanamicina A. A adição de ácido acético promoveu a libertação da proteína de fusão EGFP-Pep4p em vacúolos isolados e induziu variações de fluorescência de vacúolos marcados com laranja de acridina e com Fluo-4 AM indicativas de uma perturbação transitória do pH vacuolar e de uma libertação de Ca2+. Está descrito que a libertação de Ca2+ e de H+, com a consequente acidificação do citosol, está envolvida na morte celular. Estes novos resultados em vacúolos isolados permitem concluir que o ácido acético é capaz de induzir directamente uma permeabilização parcial da membrana vacuolar, semelhante à LMP em mamíferos, sem o envolvimento de outros organelos ou sem a activação de vias a montante.
This work was supported by the following projects: (PTDC/BIA-BCM/69448/2006) and by PTDC/AGR-ALI/100636/2008

Doctoral Thesis (PhD Programm on Molecular and Environmental Biology)
Lysosomal cathepsins play a crucial role in cell homeostasis by participating in the degradation of heterophagic and autophagic material. Additionally, following their release into the cytosol, these proteases are involved in pro-apoptotic and anti-apoptotic processes, particularly the aspartic cathepsin D (CatD). Indeed, CatD released into the cytosol triggers a mitochondrial apoptotic cascade. However, CatD can have anti-apoptotic effects in some cellular types and specific contexts. Therefore, targeting this apoptosis regulator in therapies for apoptosis deficiency-associated diseases, such as cancer, requires detailed elucidation of its mechanisms of action. Understanding the molecular mechanisms connecting lysosomal to mitochondrial membrane permeabilization is thus particularly relevant. More recently, vacuolar membrane permeabilization and consequent release of vacuolar proteins into the cytosol was also observed in yeast. It was demonstrated, that Pep4p (yeast CatD), a pepsin-like aspartic protease found in the yeast vacuole and ortholog to human CatD, is released from the vacuole during hydrogen peroxide- or actin stabilization-induced apoptosis. It also translocates into the cytosol during acetic acidinduced apoptosis, and is required for autophagy-independent degradation of mitochondria and for increased cell survival in response to this acid. Furthermore, acetate in colorectal carcinoma (CRC) cells seems to behave as acetic acid in yeast, triggering lysosomal membrane permeabilization (LMP), CatD release and mitochondria-dependent apoptosis. Recently, we found that CatD is involved in autophagy-independent degradation of damaged mitochondria, which renders CRC cells more resistant to apoptosis induced by acetate. These observations, combined with the hints provided by the yeast cell model, support the idea that LMP associated with the release of CatD protects CRC cells from mitochondrial dysfunction during acetate-induced apoptosis through its involvement in degradation of damaged mitochondria. Thus, it has become apparent that the approaches with yeast have already provided and can further offer new perspectives for an enhanced understanding of the role of CatD in mammalian apoptosis, as well of the molecular basis of the crosstalk between the lysosome and mitochondria. Thereafter, we set out to exploit acetic acid-induced apoptosis in Saccharomyces cerevisiae to study the yeast vacuolar protease Pep4p, both concerning its role in mitochondrial degradation and its involvement in the course of apoptosis. In this thesis, it is shown that the protective role of Pep4p in acetic acid-induced apoptosis is independent of the yeast voltage dependent channel Por1p (which has no role on mitochondrial degradation) but dependent on AAC proteins, the yeast adenine nucleotide translocator. Moreover, it has shown that both the Pep4p anti-apoptotic function and its role in mitochondrial degradation depend on Pep4p proteolytic activity. In this study, we also demonstrated that the pro-survival role of Pep4p in acetic acid-induced apoptosis is dependent on mitochondrial respiratory function, and that deficiency in mitochondrial respiration suppresses its role in mitochondrial degradation. Altogether, these results contributed to unveil a novel pro-survival function of CatD in autophagy-independent mitochondrial degradation, which can lead to enhanced cell survival in CRC cells undergoing acetate-induced apoptosis. Moreover, these studies reinforce the use of yeast as a valuable model to elucidate the role of CatD in mammalian apoptosis, as well as the molecular mechanisms involved in the crosstalk between the lysosome and mitochondria.
As catepsinas lisossomais têm um papel crucial na homeostasia celular, participando na degradação de material hetero- e autofágico. Adicionalmente, estas proteases estão envolvidas em processos pró- e anti-apoptóticos após a sua libertação para o citosol, particularmente a catepsina aspártica D (CatD). Com efeito, uma vez no citosol, a CatD desencadeia a cascata apoptótica mitocondrial. Contudo, esta protease pode ter um papel anti-apóptótico. A utilização deste regulador apoptótico como alvo molecular na terapia de doenças associadas a deficiências no processo apoptótico requer portanto uma elucidação detalhada dos seus mecanismos de acção. Por este motivo, a compreensão dos mecanismos moleculares que conectam a permeabilização da membrana lisossomal (PML) à permeabilização da membrana mitocondrial é particularmente relevante. Mais recentemente, observou-se que a permeabilização da membrana vacuolar e consequente libertação de proteases vacuolares para o citosol também ocorre na levedura. Foi demonstrado que a Pep4p, a protease aspártica encontrada no vacúolo da levedura e ortóloga da CatD humana, é libertada do vacúolo para o citosol durante a apoptose induzida pelo peróxido de hidrogénio ou estabilização da actina. Esta protease também é translocada para o citosol durante a apoptose induzida pelo ácido acético desempenhando um papel crucial na degradação mitocondrial independente da autofagia e na sobrevivência celular em resposta a este ácido. Adicionalmente, o acetato em linhas celulares derivadas do carcinoma colorectal (CRC) comporta-se de modo análogo ao ácido acético na levedura, induzindo PML, libertação da CatD e apoptose dependente da mitocôndria. Nós demonstrámos recentemente que a CatD está envolvida na degradação mitocondrial independente da autofagia, o que torna as células do CRC mais resistentes à apoptose induzida pelo acetato. Estas observações, mais as indicações obtidas através do modelo de levedura, reforçam a ideia de que a PML associada à libertação da CatD protege as células do CRC de uma disfunção mitocondrial durante a apoptose induzida pelo acetato, através do seu envolvimento na degradação de mitocôndrias danificadas. Tornou-se então aparente que as abordagens na levedura forneceram informação importante e podem vir a oferecer perspectivas adicionais, contribuindo assim para uma melhor compreensão do papel da CatD na apoptose em mamíferos, bem como das bases moleculares do “crosstalk” entre o lisossoma e a mitocôndria. Por conseguinte, decidimos explorar o modelo da apoptose induzida pelo ácido acético na Saccharomyces cerevisiae para estudar a protease vacuolar da levedura Pep4p, relativamente ao seu papel na degradação mitocondrial e consequentemente seu envolvimento na apoptose. Nesta tese, mostra-se que o papel protector da Pep4p na apoptose induzida pelo ácido acetico é independente do canal de aniões dependente da voltagem de levedura Por1p (que por sua vez não desempenha um papel na degradação mitocondrial), mas é dependente das proteínas AAC, o antiportador mitocondrial de ATP/ADP da levedura. Também foi demonstrado que a função anti-apoptótica da Pep4p, bem como o seu papel na degradação mitocondrial dependem da sua actividade proteolítica. Neste estudo, foi também demonstrado que o papel protector da Pep4p na apoptose induzida pelo ácido acético é dependente da função respiratória mitocondrial, e também que a deficiência na respiração mitocondrial suprime o papel da Pep4p na degradação mitocondrial. Em conjunto, os resultados aqui descritos contribuíram para revelar uma nova função da CatD na degradação mitocondrial independente da autofagia, que pode conduzir a um aumento da sobrevivência nas células do CRC durante a apoptose induzida pelo acetato. Além disso, estes estudos reforçam o uso da levedura como modelo para elucidar o papel da CatD na apoptose de mamíferos, bem como os mecanismos moleculares envolvidos no “crosstalk” entre o lisossoma e a mitocôndria.
A autora deste trabalho usufruiu de uma bolsa da Fundacao para a Ciencia e a Tecnologia (FCT), com a referencia SFRH/BD/73139/2010 co-financiada pelo Programa Operacional Potencial Humano (POPH) do Quadro de Referencia Estrategico Nacional (QREN), comparticipado pelo fundo Social Europeu e por fundos nacionais

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

Philosophiae Doctor - PhD
Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa protein that is implicated in mRNA processing and ubiquitination functions and has been shown to be highly up-regulated in a number of cancers. In humans and mice,RBBP6 interacts with both tumour suppressors p53 and pRb, suggesting that it is involved in regulation of transcription, induction of apoptosis and cell cycle control. Knock-out of an RBBP6 homologue PACT resulted in p53 dependent cell cycle arrest and apoptosis. Although the biological functions of RBBP6 remain largely unclear, it is possible that its functions are mediated through interaction with other cellular proteins. Since it is possible to unveil novel functions of a target protein through identifying its interacting protein partners,this study aims to further characterize the functions of RBBP6 through identifying novel protein interacting partners using a yeast 2-hybrid screen.In order to identify interaction partners of RBBP6, two well characterized domains of RBBP6, the N-terminal ubiquitin-like DWNN domain and RING finger domain, were used as baits in a yeast 2-hybrid screen of a human testis cDNA library. Putative interactors were verified using in vitro and in vivo immunoprecipitation assays. The RING finger domain was shown to interact with transcriptional factors Y-Box binding protein 1 (YB-1) and zinc finger and BTB containing protein 38 (zBTB38), resulting in their ubiquitination. In the case of YB-1 ubiquitination was correlated with a decrease in the intra-cellular levels of YB-1, suggesting that ubiquitination leads to degradation in the proteosome. The DWNN domain was shown to interact with a splicing associated small nuclear ribonucleoprotein polypeptide G (snRPG) and heat shock protein 70 (Hsp70).The results of this work suggest that, at least in the case of YB-1 and zBTB38,RBBP6 plays a role in the regulation of gene expression by ubiquitination of transcription factors, causing them to be degraded in the proteosome. The study provides further evidence of RBBP6’s involvement in mRNA splicing through its interaction with snRPG. The interaction with Hsp70 suggests a possible role in protein quality control similar to that played by other E3 ligases such as Parkin and CHIP.

La mort cellulaire programmée (PCD pour Programmed Cell Death) est un processus essentiel aux cellules. Le PCD a d’abord été caractérisé dans le développement cellulaire et peut être divisé en plusieurs groupes selon les caractéristiques observées. L’apoptose, un sous-groupe du PCD, est caractérisé par plusieurs distinctions morphologiques et signalétiques attribué tout d’abord aux organismes complexes pour son rôle dans le développement et dans le maintien de l’intégrité tissulaire. Depuis la dernière décennie, de nombreuses études font état de l’existence d’un programme apoptotique dans des organismes unicellulaires comme les levures. Ce programme apoptotique a surtout été étudié chez les levures Saccharomyces cerevisiae et Schizosaccharomyces pombe et partage certaines caractéristiques avec l’apoptose des mammifères. Par contre, l’apoptose associé aux levures est distinct à certains égards entre autre par l’absence de certains homologues présents chez les mammifères. L’intérêt au niveau de l’étude du phénomène apoptotique chez les levures est sans cesse grandissant par la facilité avec laquelle les levures peuvent être utilisées comme système modèle. L’apoptose peut être induit dans les cellules de différentes façons en réponse à des stimuli internes ou externes. L’accumulation de protéines mal repliées au niveau du réticulum endoplasmique (RE) causant un stress est un inducteur bien caractérisé de la voie apoptotique. La signalisation de l’apoptose dans un cas de stress au RE fait appel aux transducteurs des signaux de la voie du UPR ( Unfolded Protein Response). Récemment, il a été montré que la calnexine, une chaperone transmembranaire du RE connue et caractérisée surtout pour ses fonctions d’aide au repliement des protéines et au contrôle de qualité, joue un rôle dans la transduction du signal apoptotique en réponse au stress du RE chez mammifères. Le rôle de la calnexine dans ce cas consiste principalement en l’échafaudage pour le clivage par la caspase 8 de la protéine apoptotique Bap31. Nous avons tout d’abord démontré que le stress du RE et que la déficience en inositol, un précurseur essentiel de nombreuses molécules signalétiques, sont deux inducteurs de l’apoptose chez la levure S. pombe. Ces deux voies semblent induire l’apoptose par deux voies distinctes puisque seule la voie de la déficience en inositol induit l’apoptose de façon dépendante à la métacaspase Pca1p. La calnexine, essentielle à la viabilité chez la levure S. pombe, est impliquée dans ces deux phénomènes apoptotiques. L’apoptose induit par le stress du RE nécessite une version de la calnexine ancrée à la membrane du RE pour être optimal. De façon opposée, l’apoptose induit par une déficience en inositol nécessite la présence de la queue cytosolique ancrée à la membrane de la calnexine pour être retardé. Ces deux actions différentes imputables à une même protéine laisse croire à une double fonction pro et anti-apoptotique de celle-ci. Suite à la découverte de l’existence d’un clivage endogène de la calnexine en situation normale de croissance, un modèle a été élaboré expliquant les rôles distincts de la calnexine dans ces deux voies apoptotiques. Ce modèle fait état d’un rôle associé au clivage de la calnexine dans l’apoptose.
Programmed Cell Death (PCD) is an essential process to the cells. PCD was first characterized in cell development and can be separated in sub-groups depending of cell death characteristics observed. Apoptosis is one of the PCD sub-groups that was first associated to complex organisms for its roles in cell development and in maintenance of tissues integrity. The apoptotic pathway is characterized by specific morphological and signalization characteristics. In the last ten years, numerous studies demonstrated the existence of apoptosis in unicellular organisms such as yeast. This apoptotic program was extensively studied in the two yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe and share characteristics with the mammalian one. However, yeast apoptosis is distinctive at many points as yeast do not encodes all the mammalian homologues of the apoptotic pathway. Although yeast and mammalian apoptosis seems to differs, the interest about yeast apoptosis is growing given that yeast is an excellent and easily tractable model system. External and internal stimuli can induce apoptosis by different ways. Accumulation of unfolded or incompletely folded proteins in the endoplasmic reticulum (ER) causing ER stress is a well-known inducer of the apoptotic pathway. Signalization of ER-stress induced apoptosis involves the same transducers than the UPR (Unfolded Protein Response) pathway. It was recently shown that calnexin, a transmembrane chaperone of the ER, is implicated in ER-stress apoptosis in mammalian cells. In this particular case, it was demonstrated that calnexin acts as a scaffold in the cleavage of the apoptotic protein Bap31 by caspase 8. We demonstrated that ER stress and inositol deficiency, a precursor of many important signalization molecules, are two situations leading to apoptosis in the yeast S. pombe. These two pathways leading to apoptosis seems to differ as only inositol deficiency is dependant of the yeast metacaspase Pca1p. We also demonstrated that S. pombe calnexin, essential for cell viability of this yeast, takes part in these two apoptotic process. ER stress induced apoptosis needs a calnexin anchors to the ER membrane to be efficient. However, apoptosis induced by inositol starvation needs the calnexin C-terminal tail with the transmembrane domain to be delayed. These two opposite actions from the same protein lead to the hypothesis that calnexin encodes both pro and anti-apoptotic functions. By the discovery that calnexin is cleaved under normal culture conditions, a model was elaborated explaining the distinctive roles of calnexin in these two apoptotic pathways. This model proposed a role of calnexin cleavage to apoptosis.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina)
Cell death has long been considered crucial for proper tissue shaping during embryonic development and for normal turnover of the cells in several tissues. Moreover, mis-regulation of apoptosis has been implicated in a diversity of abnormal functions and in the progression and development of diseases. Indeed, a decrease in apoptosis has been associated with ageing and tumour progression, whereas an increase in apoptosis is mainly associated with ageing-related neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s diseases. The life and death switch is among others regulated by interactions between pro- and anti-apoptotic Bcl-2 family members, including Bax and Bcl-xL, respectively. Bax plays a central role in apoptosis, as it is involved in the formation of pores within the mitochondria outer membrane through which apoptogenic factors, including cytochrome c, are released and originate a cascade of events that culminate in cell death. Given the importance of this pro-apoptotic protein in cell death, its potential as a therapeutic target was quickly recognized, and its mode of action and regulation have been studied extensively. However, many questions still remain, and thus further understanding of Bax regulation was the general purpose of this thesis. To accomplish this, we took advantage of the genetically tractable yeast Saccharomyces cerevisiae, whose genome is devoid of genes coding apparent homologues of the human Bcl-2 family. Therefore, the heterologous expression of human Bax allows its study without interference of the apoptotic network. To achieve our goal, we first optimized the conditions for expression of human Bax (Bax α) in yeast and characterized the phenotype of expression of Bax α and of an active form, Bax c-myc. Then, conditions that led to Bax α activation were found, and its regulation through different proteins was explored. Herein, we describe for the first time that sub-lethal concentrations of acetic acid trigger Bax α-mediated cell death without disturbing plasma membrane integrity and mitochondrial mass but increasing superoxide anion accumulation and release of cytochrome c, which is partially reverted by the anti-apoptotic protein Bcl-xL. This finding allows us to mimic what happens in human cells, without the need to use non-natural mutants of Bax, such as phosphomimetic or non-phosphorylatable, or mitochondrial tagged versions. Thus, yeast continuously proves to be a valuable tool to express human Bax and perform studies regarding its function, regulation and interaction with other members of the Bcl-2 protein family and other partners.
A morte celular é um evento fundamental para a correta formação dos tecidos durante o desenvolvimento embrionário e para o normal funcionamento das células. Devido ao seu papel fundamental a nível fisiológico, desregulações neste processo estão associadas a uma grande diversidade de funções anormais e, consequentemente, ao desenvolvimento e progressão de várias doenças. De facto, uma diminuição da morte celular relaciona-se com envelhecimento e a progressão de tumores, enquanto que um aumento está associado a doenças neurodegenerativas incluindo a doença de Alzheimer, Parkinson e Huntington. O destino de uma dada célula é regulado, entre outras formas, por interações entre membros pro-apoptóticos e anti-apoptóticos da família de proteínas Bcl-2, nomeadamente a Bax e a Bcl-xL, respetivamente. A proteína Bax está envolvida na formação de poros na membrana mitocondrial externa, através do qual são libertados fatores apoptóticos, incluindo o citocromo c, originando uma cascata de eventos que culmina na morte da célula. Considerando o papel importante desta proteína na morte celular, as suas capacidades como alvo terapêutico emergiram rapidamente. Por este motivo, o seu modo de ação e regulação têm sido extensivamente estudados. Contudo, várias questões permanecem eminentes e, por isso a melhor compreensão da sua regulação é o foco principal desta tese. Assim, escolhemos a levedura Saccharomyces cerevisiae, cujo genoma não possui genes codificantes de homólogos da família de proteínas humanas Bcl-2. Deste modo, a expressão heteróloga da proteína Bax humana na levedura permite o seu estudo sem interferência dos restantes membros da família. Para alcançarmos o nosso objetivo, primeiramente otimizamos as condições para expressão da proteína na levedura, e caracterizamos o seu fenótipo de expressão, bem como o de uma forma ativa, Bax c-myc. De seguida, determinamos condições que nos permitiram a ativação da proteína Bax humana e a sua regulação por outras proteínas. Nesta tese, descrevemos pela primeira vez que concentrações sub-letais de ácido acético desencadeiam um processo de morte celular mediado por Bax, sem provocar alterações na integridade da membrana plasmática e na massa mitocondrial, mas aumentando a acumulação de anião superóxido e causando a libertação de citocromo c, parcialmente revertida pela proteína anti-apoptótica Bcl-xL. Estes resultados permitiram-nos mimetizar o que acontece em células humanas, sem a necessidade de usar mutantes de Bax artificiais, tais como os mutantes fosfomiméticos e não fosforiláveis ou versões endereçadas para a mitocôndria. Deste modo, a levedura continua a revelar o seu valor como modelo celular para expressar a proteína Bax humana e realizar estudos no que toca à sua função, regulação e interação com outros membros da família Bcl-2 e outros parceiros.
Fundação para a Ciência e Tecnologia (FCT) através do projeto UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) e dos financiamentos PD/BD/128032/2016 no âmbito do Programa Doutoral em Biologia Aplicada e Ambiental (DP_AEM) e SFRH/BPD/89980/2012.