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Distribuição dos recursos metabólicos entre proliferação e manutenção celular: um estudo em tempo real e in vivo por calorimetria em Saccharomyces cerevesiae
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A Saccharomyces cerevisiae (S. cerevisiae), usada tradicionalmente na produção de bebidas alcoólicas e pão, é um organismo unicelular eucariota que partilha a maioria dos processos e
funções de eucariotas superiores, apresentando ainda um crescimento rápido em meio líquido. Isto torna a S. cerevisiae um dos modelos mais simples e práticos para o estudo de células eucariotas. Além disso, o metabolismo respiro-fermentativo da S. cerevisiae durante a fase de crescimento exponencial apresenta semelhanças com o “efeito de Warburg”, observado em algumas células com proliferação rápida, como as células cancerosas. Esta semelhança, torna a S. cerevisiae um modelo interessante para o estudo do metabolismo das células cancerosas, destacando a importância vital do estudo do metabolismo da S. cerevisiae.
A S. cerevisiae é um organismo bastante versátil, tendo a capacidade de adaptar o seu metabolismo e taxa de crescimento em resposta a alterações no meio de cultura, tal como a disponibilidade de nutrientes, e situações de stress, tais como stress oxidativo ou fome. Elevados níveis de oxidantes, como o peróxido de hidrogénio (H2O2), criam uma situação de elevado stress oxidativo caraterizada por extensas lesões celulares, apesar de doses baixas deste oxidante estarem associadas a efeitos de sinalização intracelular e poderem induzir uma resposta adaptativa nas células, protegendo-as de um possível stress posterior. Assim, o H2O2 consegue modular o metabolismo de forma dependente da sua concentração. O objetivo geral desta Tese foi estudar a dinâmica do metabolismo da S. cerevisiae na resposta a diferentes meios de cultura e ao H2O2. A microcalorimetria de fluxo foi a ferramenta central desse estudo, permitindo seguir em tempo real a energia dissipada por todas as reações que têm lugar na célula. Porque todas as reações bioquímica que acontecem na célula são acompanhadas por transferências de calor, as medidas calorimétricas são um bom indicador da actividade metabólica. Além de ser uma técnica extremamente sensível, a microcalorimetria apresenta diversas vantagens sob outros métodos, uma vez que permite medidas em tempo real e não é invasiva nem destrutiva. Neste trabalho, a questão da natureza uniforme ou dinâmica do metabolismo de S. cerevisiae ao longo da fase exponencial foi investigada através de microcalorimetria fluxo e de medidas auxiliares como densidade celular, consumo de oxigénio e consumo de glucose. Isto envolveu acompanhar o crescimento da S. cerevisiae em diferentes meios de cultura e situações de stress, nomeadamente stress oxidativo. Ao contrário de estudos anteriores, em que foram utilizadas estirpes prototróficas, este trabalho baseou-se numa estirpe auxotrófica (BY4741) amplamente usada atualmente em estudos laboratoriais por ser a estirpe wild-type da coleção de mutantes de S. cerevisiae. Observou-se que a potência dissipada, P, associada à atividade metabólica celular depende significativamente do meio de cultura utilizado. Estudos de crescimento realizados com glucose a 2% em dois meios de cultura diferentes, nomeadamente, um meio composto por extrato de levedura (yeast extract), peptona e dextrose (YPD) e um meio sintético completo (SC) mostraram que a potência máxima dissipada pela BY4741 durante a fase exponencial é consideravelmente mais elevada no meio rico YPD (662 ± 11 μW (n=4)) do que no meio menos rico SC (365 ± 17 μW (n=5)). Os perfis das curvas calorimétricas referentes aos dois meios são também diferentes. Em YPD a potência dissipada pela BY4741 aumenta exponencialmente até se atingir um máximo, diminuindo, em seguida, abruptamente para o valor estacionário típico das células em fase estacionária. Em meio SC observam-se dois máximos: o primeiro, é causado pela depleção do aminoácido auxotrófico metionina no meio de cultura; o segundo é semelhante ao observado no meio YPD, sendo também seguido de uma diminuição rápida de potência para valores típicos da fase estacionária. Estes resultados evidenciam a incapacidade do meio SC, amplamente utilizado na seleção de mutantes auxotróficos, para proporcionar um crescimento exponencial sem constrangimentos. A análise das curvas de crescimento revelou que, independentemente do meio de cultura, a potência dissipada por célula é máxima durante a transição entre as fases lag e exponencial, sendo o valor atingido em meio SC superior ao observado no meio YPD. Isto indica que a actividade metabólica é máxima na transição entre estas duas fases e mais elevada no meio mais pobre (SC). O perfil de consumo de O2 sugere que a diminuição contínua da potência por célula durante a fase exponencial, pode ser devida a uma diminuição contínua do metabolismo respiratório, sendo que este tipo de metabolismo tem uma entalpia associada muito superior à do metabolismo fermentativo.
De forma a otimizar as condições de exposição da S. cerevisiae ao H2O2, investigaram-se os efeitos de diferentes doses deste oxidante na respetiva curva calorimétrica de crescimento. Observou-se um efeito hormético do H2O2 à concentração de 150 μM, evidenciado por um aumento de P imediatamente após a adição de H2O2, contrastando com a diminuição observada para concentrações superiores (0.3 – 1.0 mM), possivelmente devido a morte celular. Para as concentrações mais elevadas observou-se ainda uma paragem temporária da curva calorimétrica após a exposição ao H2O2. A fim de definir os efeitos de condições de elevado stress oxidativo na capacidade proliferativa de culturas de S. cerevisiae em crescimento exponencial, analisou-se a viabilidade celular de culturas expostas a uma dose letal (1 mM ) de H2O2 ao longo do tempo. Observou-se que a cinética de morte da S. cerevisiae segue um decaimento exponencial bifásico: a primeira fase é observada imediatamente após a adição de H2O2 e tem uma duração de aproximadamente 3h; a segunda fase inicia-se após a população se ter adaptado a elevadas concentrações de H2O2, tornando-se mais resistente, e é evidenciada por uma diminuição do número de células mortas por unidade de tempo, para a mesma concentração de 1 mM. O mesmo protocolo usando células previamente adaptadas a uma dose subletal de H2O2 produziu resultados semelhantes, o que é indicativo de que mesmo células adaptadas a doses subletais de H2O2 necessitam de mais adaptação para conseguir sobreviver em condições de maior stress oxidativo. A adaptação com doses subletais de H2O2 diminuiu a morte celular na população reduzindo a duração da primeira fase, i.e. menos tempo necessário para que as células se conseguissem adaptar à dose letal de H2O2. Interessantemente, as células conseguiram retomar o crescimento exponencial mesmo na presença de tais concentrações elevadas de H2O2, implicando uma capacidade aprimorada de lidar com o stress oxidativo por parte das células. Curiosamente, o mutante ZWF1Δ, deletado para o enzima 6P-glucose desidrogenase (G6PDH), revelou-se extremamente resistente ao stress oxidativo, evidenciado por uma primeira fase pós adição de H2O2 bastante curta e uma segunda fase com uma taxa de morte celular muito baixa. Além do mais, o típico protocolo de adaptação ao H2O2, nesta estirpe não surte qualquer efeito ao nível da sobrevivência. Contudo, esta estirpe apenas consegue retomar o crescimento exponencial depois de todo o H2O2 no meio ter sido consumido. No mutante ZWF1Δ, a produção de NADPH encontra-se drasticamente afetada devido à ausência do G6PDH, que é o primeiro enzima da via dos fosfatos de pentose (PPP), principal via de formação de NADPH. Em conjunto, os resultados obtidos para as duas estirpes sugerem um papel central da via PPP na resposta antioxidante a doses letais de H2O2. Foi estudado o efeito de doses não letais de H2O2 no metabolismo de culturas de S. cerevisiae em crescimento exponencial, através de 3 adições bolus de 150 μM H2O2. A potência dissipada pela população aumentou ligeiramente imediatamente depois de cada uma das adições, possivelmente como resultado de um aumento do catabolismo associado à atividade dos enzimas antioxidantes. As 3 adições bolus de H2O2 produziram um aumento de cerca de 14% na entalpia associada ao metabolismo da cultura de S. cerevisiae até ao primeiro máximo da curva calorimétrica em meio SC. Este aumento não se deveu apenas à exposição ao H2O2, mas também à fome de metionina, uma vez que o H2O2 não produziu qualquer alteração na entalpia do metabolismo em culturas a crescer em meio extra suplementado em metionina e outros aminoácidos, onde a limitação de metionina é eliminada. Durante este trabalho, um novo método de doseamento de glucose foi desenvolvido. Este método revelou ser altamente específico para a glucose e consiste na reação do glucose oxidase (GO) com a glucose presente na amostra, consumindo O2 no processo. Este consumo foi quantificado num elétrodo de O2 e relaciona-se proporcionalmente com a concentração de glucose da amostra. Este método apresenta uma gama de linearidade larga, indo dos 0 μM aos 260 μM de glucose. No geral, o trabalho microcalorimétrico levado a cabo nesta Tese veio confirmar e alargar observações anteriores de que as alterações metabólicas que ocorrem ao longo da fase exponencial favorecem a glicólise aeróbia em função da respiração mitocondrial. Observou-se também que o metabolismo da S. cerevisiae é bastante dependente do meio de cultura e dos nutrientes que
integram o meio para além da fonte de carbono. Foi ainda detetado um efeito de hormese a uma baixa concentração de H2O2 (150 μM) e demonstrou-se que o H2O2 em doses letais mata as células de uma forma bifásica.
Saccharomyces cerevisiae (S. cerevisiae), used traditionally in the making of brewers and baking, is a eukaryotic unicellular organism that shares most of the biologic processes and functions of higher eukaryotes and is able to grow rapidly in liquid media. This makes it one of the simplest and practical models for the study of the eukaryotic cell. Moreover, because the respiro-fermentative metabolism during the exponential growth phase of this yeast shows some resemblance with the “Warburg effect” observed in some fast-proliferating cells such as cancer cells. This resemblance, makes S. cerevisiae an interesting model for the study of cancer metabolism, highlighting the vital importance of studying S. cerevisiae’s metabolism. S. cerevisiae is a very versatile organism, which is able to adapt its metabolism and growth rate in response to changes in the growth medium, such as nutrient availability, and stress situations, such as oxidative stress or starvation. High levels of oxidants, such as hydrogen peroxide (H2O2), create a situation of high oxidative stress characterized by extensive cellular damages, although low doses of this oxidant have been associated with intracellular signaling and can induce an adaptive response in cells protecting them from subsequent high oxidative stress conditions. Thus H2O2 can modulate metabolism in a concentration dependent fashion. The overall aim of this thesis is to study the dynamics of S. cerevisiae metabolism in response to different growth media and to H2O2. A suitable tool for the study of yeast metabolism is flow microcalorimetry as it allows following in real-time the dissipated energy resulting from all reactions of the metabolism taking place in the cell. Because all biochemical reactions and processes taking place in the cell are accompanied by heat transfer, heat measurements provide an all-inclusive surrogate of metabolic activity. Besides being extremely sensitive, microcalorimetry has several advantages over other methods used in the study of metabolism since it is a probe-free, non-invasive and non-destructive technique that allows measurements in real-time. In this work, the question of uniform or dynamic nature of S. cerevisiae metabolism over the exponential phase was investigated by flow microcalorimetry supplemented by cell density evolution and oxygen and glucose consumption measurements. This involved following the yeast growth in different culture media and stress situations, namely oxidative stress. As opposed to previous studies in which prototrophic strains were used, an auxotrophic strain (BY4741), which is widely used in current laboratory studies, was selected. It was found that the dissipated power, P, associated to the cellular metabolic activity significantly depends on the culture medium used. Growth studies carried out with 2% glucose in two different common media, namely Yeast Extract, Peptone and Dextrose (YPD) and Synthetic Complete (SC) showed that the maximum power dissipated by BY4741 during the exponential phase was considerably higher in the rich YPD medium (662 ± 11 μW (n=4)) than in the less rich SC medium (365 ± 17 μW (n=5)). The calorimetric power-time profiles were also different in the two media. When BY474 was grown in YPD, the power increased exponentially until a maximum was reached and then abruptly changed to the approximately steady value typical of the stationary phase. When growth was carried out on SC medium two maximums were noted: the first was caused by the depletion of the amino acid methionine in the synthetic medium; the second one was similar to that observed in the other media and was also followed by a fast drop to the stationary phase value. These results outline the failure of SC medium, widely used in the selection of auxotrophic mutants in genetic approaches, to provide an unconstrained exponential growth. Analysis of complete growth curves revealed that, regardless of the medium used, the power dissipated per cell is maximal during the transition from the lag to the exponential phase, but this maximum is higher in the poorer SC than in the richer YPD medium. This indicates that metabolic activity is maximal during transition between these two phases and higher in the poorer culture medium. Results from O2 consumption assays suggest that the power per cell decreases continually in both media along the exponential phase possibly as a consequence of the decreasing respiratory metabolism, which is associated with a much higher enthalpy than glycolysis. To optimize exposure conditions to H2O2, the effects of different doses of this oxidant on the calorimetric curve of S. cerevisiae’s growth were investigated. A hormetic behaviour was noted at 150 μM H2O2, for which an increase in P was observed immediately after the addition of H2O2, while for most concentrations H2O2 addition led to a decrease in P associated with cell death. For the higher concentrations a temporary arrest of the calorimetric curve was also observed. In order to define the effects of high oxidative stress conditions on the proliferative capacity of growing cultures of S. cerevisiae, cellular viability over time following exposure to a lethal dose of H2O2 was assayed. It was found that the killing kinetics of H2O2 follows a biphasic exponential decay: a first phase is observed immediately after the addition of 1 mM (lethal dose) H2O2 until 3 hours; a second phase follows after the population has become adapted to high H2O2 concentration, becoming more resistant, as evidenced by a decrease in the number of killed cells per unit of time, with the same concentration of 1 mM. The same protocol using cells previously adapted to a sublethal dose of H2O2 produced similar results, indicative that even cells adapted to low doses of H2O2 need further adaptation in order to survive in higher stress situations. Adaptation reduced cellular death by decreasing the first phase duration, i.e. less time was needed for cells adapted to sublethal doses of H2O2 to become adapted to high lethal H2O2 doses. Interestingly, cells were able to resume their exponential growth even in the presence of such high concentrations of H2O2, implying an enhanced capacity to deal with the oxidative stress. Curiously, the ZWF1Δ mutant, in which the enzyme glucose-6P dehydrogenase is deleted, exhibit the same two phases of exponential decay and revealed an extreme resistance to oxidative stress, as evidenced by a shorter first phase and a much lower death rate during the second phase of the killing kinetics, following exposure to a lethal dose of H2O2. However, this strain can only resume exponential growth when all the H2O2 in the medium was consumed and the adaptation protocol has no effect in the survival rate of this strain. In ZWF1Δ mutants, NADPH production is severely affected because of G6PDH deletion. This is the first enzyme of the pentose phosphate pathway (PPP), which corresponds to the main pathway for NADPH production. Overall, the results obtained for both strains suggest a central role for PPP in the antioxidant response to lethal doses of H2O2. The effect of non-lethal doses of H2O2 on the metabolism of growing S. cerevisiae was studied via three bolus additions of 150 μM H2O2. The power dissipated by the overall population increased slightly immediately after every addition of H2O2 possibly as a result of increased catabolism associated with antioxidant enzymatic activity. The three bolus additions produced an increase in the total heat dissipated by the growing culture of S. cerevisiae until the first maximum in SC media. This increase was not solely caused by H2O2 exposure but also due to methionine starvation, since H2O2 did not change the heat dissipated when the cells were grown in methionine extra-supplemented SC medium, where methionine limitation was eliminated. A new method to determine glucose concentration was developed during this work. This method was found to be highly specific to glucose and consisted of the glucose oxidase reaction with the glucose in the sample consuming O2 in the process. This consumption was quantified in an O2 electrode and was proportionally related to the glucose concentration in the sample. This method has a wide range extending from 0 μM to 260 μM glucose. Overall, the flow microcalorimetry work carried out in this Thesis confirmed and extended previous observations that metabolic changes occurring during the exponential growth phase of S. cerevisiae favors aerobic glycolysis over mitochondrial respiration. It was also noted that metabolism is quite dependent on the culture medium and the nutrients contained in the medium besides the carbon source. A hormetic effect at a low dose of H2O2 (150 μM H2O2) was also detected, and it was further demonstrated that high oxidative H2O2 stress kills yeast in a biphasic exponential decay fashion.
Saccharomyces cerevisiae (S. cerevisiae), used traditionally in the making of brewers and baking, is a eukaryotic unicellular organism that shares most of the biologic processes and functions of higher eukaryotes and is able to grow rapidly in liquid media. This makes it one of the simplest and practical models for the study of the eukaryotic cell. Moreover, because the respiro-fermentative metabolism during the exponential growth phase of this yeast shows some resemblance with the “Warburg effect” observed in some fast-proliferating cells such as cancer cells. This resemblance, makes S. cerevisiae an interesting model for the study of cancer metabolism, highlighting the vital importance of studying S. cerevisiae’s metabolism. S. cerevisiae is a very versatile organism, which is able to adapt its metabolism and growth rate in response to changes in the growth medium, such as nutrient availability, and stress situations, such as oxidative stress or starvation. High levels of oxidants, such as hydrogen peroxide (H2O2), create a situation of high oxidative stress characterized by extensive cellular damages, although low doses of this oxidant have been associated with intracellular signaling and can induce an adaptive response in cells protecting them from subsequent high oxidative stress conditions. Thus H2O2 can modulate metabolism in a concentration dependent fashion. The overall aim of this thesis is to study the dynamics of S. cerevisiae metabolism in response to different growth media and to H2O2. A suitable tool for the study of yeast metabolism is flow microcalorimetry as it allows following in real-time the dissipated energy resulting from all reactions of the metabolism taking place in the cell. Because all biochemical reactions and processes taking place in the cell are accompanied by heat transfer, heat measurements provide an all-inclusive surrogate of metabolic activity. Besides being extremely sensitive, microcalorimetry has several advantages over other methods used in the study of metabolism since it is a probe-free, non-invasive and non-destructive technique that allows measurements in real-time. In this work, the question of uniform or dynamic nature of S. cerevisiae metabolism over the exponential phase was investigated by flow microcalorimetry supplemented by cell density evolution and oxygen and glucose consumption measurements. This involved following the yeast growth in different culture media and stress situations, namely oxidative stress. As opposed to previous studies in which prototrophic strains were used, an auxotrophic strain (BY4741), which is widely used in current laboratory studies, was selected. It was found that the dissipated power, P, associated to the cellular metabolic activity significantly depends on the culture medium used. Growth studies carried out with 2% glucose in two different common media, namely Yeast Extract, Peptone and Dextrose (YPD) and Synthetic Complete (SC) showed that the maximum power dissipated by BY4741 during the exponential phase was considerably higher in the rich YPD medium (662 ± 11 μW (n=4)) than in the less rich SC medium (365 ± 17 μW (n=5)). The calorimetric power-time profiles were also different in the two media. When BY474 was grown in YPD, the power increased exponentially until a maximum was reached and then abruptly changed to the approximately steady value typical of the stationary phase. When growth was carried out on SC medium two maximums were noted: the first was caused by the depletion of the amino acid methionine in the synthetic medium; the second one was similar to that observed in the other media and was also followed by a fast drop to the stationary phase value. These results outline the failure of SC medium, widely used in the selection of auxotrophic mutants in genetic approaches, to provide an unconstrained exponential growth. Analysis of complete growth curves revealed that, regardless of the medium used, the power dissipated per cell is maximal during the transition from the lag to the exponential phase, but this maximum is higher in the poorer SC than in the richer YPD medium. This indicates that metabolic activity is maximal during transition between these two phases and higher in the poorer culture medium. Results from O2 consumption assays suggest that the power per cell decreases continually in both media along the exponential phase possibly as a consequence of the decreasing respiratory metabolism, which is associated with a much higher enthalpy than glycolysis. To optimize exposure conditions to H2O2, the effects of different doses of this oxidant on the calorimetric curve of S. cerevisiae’s growth were investigated. A hormetic behaviour was noted at 150 μM H2O2, for which an increase in P was observed immediately after the addition of H2O2, while for most concentrations H2O2 addition led to a decrease in P associated with cell death. For the higher concentrations a temporary arrest of the calorimetric curve was also observed. In order to define the effects of high oxidative stress conditions on the proliferative capacity of growing cultures of S. cerevisiae, cellular viability over time following exposure to a lethal dose of H2O2 was assayed. It was found that the killing kinetics of H2O2 follows a biphasic exponential decay: a first phase is observed immediately after the addition of 1 mM (lethal dose) H2O2 until 3 hours; a second phase follows after the population has become adapted to high H2O2 concentration, becoming more resistant, as evidenced by a decrease in the number of killed cells per unit of time, with the same concentration of 1 mM. The same protocol using cells previously adapted to a sublethal dose of H2O2 produced similar results, indicative that even cells adapted to low doses of H2O2 need further adaptation in order to survive in higher stress situations. Adaptation reduced cellular death by decreasing the first phase duration, i.e. less time was needed for cells adapted to sublethal doses of H2O2 to become adapted to high lethal H2O2 doses. Interestingly, cells were able to resume their exponential growth even in the presence of such high concentrations of H2O2, implying an enhanced capacity to deal with the oxidative stress. Curiously, the ZWF1Δ mutant, in which the enzyme glucose-6P dehydrogenase is deleted, exhibit the same two phases of exponential decay and revealed an extreme resistance to oxidative stress, as evidenced by a shorter first phase and a much lower death rate during the second phase of the killing kinetics, following exposure to a lethal dose of H2O2. However, this strain can only resume exponential growth when all the H2O2 in the medium was consumed and the adaptation protocol has no effect in the survival rate of this strain. In ZWF1Δ mutants, NADPH production is severely affected because of G6PDH deletion. This is the first enzyme of the pentose phosphate pathway (PPP), which corresponds to the main pathway for NADPH production. Overall, the results obtained for both strains suggest a central role for PPP in the antioxidant response to lethal doses of H2O2. The effect of non-lethal doses of H2O2 on the metabolism of growing S. cerevisiae was studied via three bolus additions of 150 μM H2O2. The power dissipated by the overall population increased slightly immediately after every addition of H2O2 possibly as a result of increased catabolism associated with antioxidant enzymatic activity. The three bolus additions produced an increase in the total heat dissipated by the growing culture of S. cerevisiae until the first maximum in SC media. This increase was not solely caused by H2O2 exposure but also due to methionine starvation, since H2O2 did not change the heat dissipated when the cells were grown in methionine extra-supplemented SC medium, where methionine limitation was eliminated. A new method to determine glucose concentration was developed during this work. This method was found to be highly specific to glucose and consisted of the glucose oxidase reaction with the glucose in the sample consuming O2 in the process. This consumption was quantified in an O2 electrode and was proportionally related to the glucose concentration in the sample. This method has a wide range extending from 0 μM to 260 μM glucose. Overall, the flow microcalorimetry work carried out in this Thesis confirmed and extended previous observations that metabolic changes occurring during the exponential growth phase of S. cerevisiae favors aerobic glycolysis over mitochondrial respiration. It was also noted that metabolism is quite dependent on the culture medium and the nutrients contained in the medium besides the carbon source. A hormetic effect at a low dose of H2O2 (150 μM H2O2) was also detected, and it was further demonstrated that high oxidative H2O2 stress kills yeast in a biphasic exponential decay fashion.
Descrição
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016
Palavras-chave
H2O2 Metabolismo Microcalorimetria Fome S. cerevisiae Teses de mestrado - 2016