Peroxiredoxins are ubiquitous, thiol-specific proteins that have multiple functions in stress protection, including oxidative stress. Tsa1 is the major yeast peroxiredoxin and we show that it functions as a specific antioxidant to protect against oxidative stress caused by nascent protein misfolding and aggregation. Yeast mutants lacking TSA1 are sensitive to misfolding caused by exposure to the proline analogue azetidine-2-carboxylic acid (AZC). AZC promotes protein aggregation and its toxicity to a tsa1 mutant is caused by reactive oxygen species (ROS). Generation of [rho0] cells lacking mitochondrial DNA rescues the tsa1 mutant AZC sensitivity indicating that mitochondria are the source of ROS. Inhibition of nascent protein synthesis with cycloheximide prevents AZC-induced protein aggregation and abrogates ROS generation confirming that aggregate formation causes ROS production. Protein aggregation is accompanied by mitochondrial fragmentation and we show that Tsa1 localizes to the sites of protein aggregation, which are formed adjacent to mitochondria. Further investigation reveals that AZC-induced protein aggregation leads to an inhibition of mitochondrial respiration and the depolarisation of the mitochondrial membrane. Remarkably, this was entirely dependent on the presence of Tsa1. We show that the effects of protein aggregation on mitochondrial function are mediated by the Ras/PKA pathway and that Tsa1 appears to influence the activity of this pathway through its effects on the yeast phosphodiesterase, Pde2. Together, these data indicate a new role for peroxiredoxins in the response to ROS, generated as a result of protein misfolding and aggregate formation. Finally, we analysed the characteristics of proteins found within protein aggregates, isolated from different conditions during the course of the study. Our results highlight the differences between proteins that aggregate under normal, mid-exponential growth conditions (physiological aggregates) and those which aggregate during cellular stress. We were able to establish the characteristics of an archetypical physiological aggregate, through an assessment of a range of properties, identifying factors that significantly differed from genomic expectations. Furthermore, our observations indicate that, in general, cellular stress reduces the threshold of metrics associated with protein aggregation propensity. We also found that different stresses result in the aggregation of proteins that are, largely, physicochemically indistinct from one another, regardless of the mode of toxicity. Finally we show that a significant number of proteins, identified in our protein aggregates, were also present in protein aggregates isolated from aged C. elegans. This suggests that the factors and components of protein aggregates are conserved.

A redução incompleta de oxigênio a água durante a respiração celular resulta na formação de Espécies Reativas de Oxigênio (EROs), compostos oxidantes que podem, em determinadas situações, gerar quadros sérios de estresse oxidativo celular. células aeróbicas são equipadas com um complexo sistema de defesas anti-oxidantes para lidar com esta situação. Peroxirredoxinas constituem uma família de enzimas anti-oxidantes com a habilidade de remover hidroperóxidos à custa de um agente redutor que contenha tióis, protegendo biomoléculas de danos oxidativos. Ahp1 e Tsa1 são as duas peroxirredoxinas mais abundantes da levedura Saccharomyces cerevisiae, podendo cada uma constituir até 1% do total de proteínas solúveis celulares. Neste trabalho, foi realizada a caracterização de algumas propriedades bioquímicas de Ahp1, principalmente no que diz respeito às suas preferências por substratos e sensibilidade à inativação por hidroperóxidos, as quais foram comparadas com as de Tsa1. Diversos cristais de Ahp1 foram obtidos e difrataram com resolução máxima de 1.8A. Diversas estratégias para a resolução de sua estrutura tridimensional foram adotadas, sem sucesso. Por outro lado, a estrutura tridimensional preliminar de Tsa1 foi resolvida, buscando relacionar as características funcionais da proteína com a sua organização espacial. Em uma outra linha, relações entre a regulação do gene AHP1 em condições fisiológicas específicas com a de outros genes que codificam enzimas anti-oxidantes foram reveladas. Ahp1, em conjunto com as outras Prxs Tsa2 e Prx1 parece desempenhar um papel importante na defesa contra o estresse oxidativo em situações nas quais os peroxissomos estão ativos e não possuem catalase. / The incomplete reduction of oxygen to water during respiration results in the formation of Reactive Oxygen Species (ROS), oxidizing compounds, which can generate severe cellular oxidative stress. Aerobic cells are equipped with a complex defense system to cope with this condition. Peroxiredoxins make up a family of antioxidant enzymes with the ability to remove hydroperoxides at the expense of a thiol-containing reducing agent, thereby protecting biomolecules from oxidative damage. Ahp1 and Tsa1 are the two most abundant peroxiredoxins in the yeast Saccharomyces cerevisiae, each one making up to 1% of the cellular soluble proteins. In this work, we carried out a biochemical characterization of some of Ahp1\'s properties, particularly of its substrate preferences and sensitivity to inactivation by hydroperoxides, which have been compared to those of Tsa1. In one approach, Ahp1 crystals were obtained and diffracted at 1.8A resolution. Several methods were unsuccessfully employed in the attempt to solve Ahp1 structure. On the other hand, the three-dimensional structure of Tsa1 was solved, aiming to correlate its functional properties with its spatial organization. In another part of the work, relations between the regulation of AHP1 and other Prx genes were revealed. Ahp1, together with Tsa2 and Prx1, appears to carry out an important role in the cellular defense against oxidative stress, in conditions where peroxisomes are active and devoid of catalase.

A redução incompleta de oxigênio a água durante a respiração celular resulta na formação de Espécies Reativas de Oxigênio (EROs), compostos oxidantes que podem, em determinadas situações, gerar quadros sérios de estresse oxidativo celular. células aeróbicas são equipadas com um complexo sistema de defesas anti-oxidantes para lidar com esta situação. Peroxirredoxinas constituem uma família de enzimas anti-oxidantes com a habilidade de remover hidroperóxidos à custa de um agente redutor que contenha tióis, protegendo biomoléculas de danos oxidativos. Ahp1 e Tsa1 são as duas peroxirredoxinas mais abundantes da levedura Saccharomyces cerevisiae, podendo cada uma constituir até 1% do total de proteínas solúveis celulares. Neste trabalho, foi realizada a caracterização de algumas propriedades bioquímicas de Ahp1, principalmente no que diz respeito às suas preferências por substratos e sensibilidade à inativação por hidroperóxidos, as quais foram comparadas com as de Tsa1. Diversos cristais de Ahp1 foram obtidos e difrataram com resolução máxima de 1.8A. Diversas estratégias para a resolução de sua estrutura tridimensional foram adotadas, sem sucesso. Por outro lado, a estrutura tridimensional preliminar de Tsa1 foi resolvida, buscando relacionar as características funcionais da proteína com a sua organização espacial. Em uma outra linha, relações entre a regulação do gene AHP1 em condições fisiológicas específicas com a de outros genes que codificam enzimas anti-oxidantes foram reveladas. Ahp1, em conjunto com as outras Prxs Tsa2 e Prx1 parece desempenhar um papel importante na defesa contra o estresse oxidativo em situações nas quais os peroxissomos estão ativos e não possuem catalase. / The incomplete reduction of oxygen to water during respiration results in the formation of Reactive Oxygen Species (ROS), oxidizing compounds, which can generate severe cellular oxidative stress. Aerobic cells are equipped with a complex defense system to cope with this condition. Peroxiredoxins make up a family of antioxidant enzymes with the ability to remove hydroperoxides at the expense of a thiol-containing reducing agent, thereby protecting biomolecules from oxidative damage. Ahp1 and Tsa1 are the two most abundant peroxiredoxins in the yeast Saccharomyces cerevisiae, each one making up to 1% of the cellular soluble proteins. In this work, we carried out a biochemical characterization of some of Ahp1\'s properties, particularly of its substrate preferences and sensitivity to inactivation by hydroperoxides, which have been compared to those of Tsa1. In one approach, Ahp1 crystals were obtained and diffracted at 1.8A resolution. Several methods were unsuccessfully employed in the attempt to solve Ahp1 structure. On the other hand, the three-dimensional structure of Tsa1 was solved, aiming to correlate its functional properties with its spatial organization. In another part of the work, relations between the regulation of AHP1 and other Prx genes were revealed. Ahp1, together with Tsa2 and Prx1, appears to carry out an important role in the cellular defense against oxidative stress, in conditions where peroxisomes are active and devoid of catalase.

Chez Arabidopsis, l’enveloppe nucléaire constitue un site de nucléation des microtubules à partir des complexes à gamma-tubuline. Conservées des plantes à l'Homme, les protéines GIPs/MZT1 ont été initialement découvertes comme partenaires d’AtGCP3. J’ai consacré ma thèse à la caractérisation moléculaire et fonctionnelle des AtGIPs et de leurs partenaires à l’interface nucléocytoplasmique. Mes résultats confirment l’appartenance des GIPs aux complexes à gamma-tubuline, et démontrent leur association entre elles et avec TSA1 (TonSoKu [TSK]-Associating protein 1) et l'histone centromérique CenH3. Les interactions génétiques entre les gènes GIPs, TSA1 et TSK révèlent des anomalies sévères à l'échelle de l'organisme, des cellules et des noyaux. Les mutants gip1gip2 démontrent une diminution de la cohésion des régions centromériques. L’ensemble de nos résultats suggère un rôle des AtGIPs dans un continuum nucléocytoplasmique inédit, la régulation de l'architecture nucléaire et du centromère. / In Arabidopsis, the nuclear envelope is a nucleation center where gamma-tubulin complexes initiate the polymerization of microtubules. Conserved from plants to humans, GIPs/MZT1 proteins were initially discovered as AtGCP3 interacting partners. Our investigations were devoted to the molecular and functional characterization of AtGIPs and their associated proteins at the nucleocytoplasmic interface. We confirmed that AtGIPs are integral components of gamma-tubulin complexes, and showed that they interact with each other, TSA1 (TonSoKu [TSK]-Associating protein 1) and centromeric histone H3 (CenH3). Genetic interactions between GIPs, TSA1 and TSK reveal severe defects at the organism, cellular and nuclear scales. gip1gip2 mutants exhibit a decrease of centromeric and pericentromeric cohesion. Altogether, this is the first evidence for the role of a gamma–tubulin complex component in the structural maintenance of centromeric regions, and in defining nuclear morphology and architecture.