A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2017. / Glutathione transferase P1-1 (GSTP1-1) is an enzyme belonging to the glutathione transferases superfamily of enzymes responsible for xenobiotic detoxification metabolism in the cells. It has been shown recently that GSTP1-1 performs a distinct function from its family members in that it acts as a carrier of the glutathione in the reactivation and glutathionylation of oxidised peroxiredoxin 6 (Prdx6). Prdx6 is a peroxidase belonging to the peroxiredoxin superfamily. The family functions to reduce organic peroxides which are sources of oxidative stress. Prdx6, however, differs from its family members as it is a bi-functional enzyme and it only contains one cysteine in its catalytic centre. The interaction of GSTP1-1 with Prdx6 has proven to be vital for the functioning of the Prdx6. The recombinant Prdx6 and GSTP1-1 proteins have been over-expressed and purified to homogeneity. The secondary structure of the proteins was studied using circular dichroism which has shown that GSTP1-1 is predominantly alpha helical and Prdx6 is mainly alpha helical with aspects of a beta sheet. The tertiary structural analysis has been carried out using tryptophan fluorescence which revealed that in both proteins the tryptophans are partially exposed to solvent. Furthermore, the quaternary structure was analysed using size exclusion-HPLC which indicated that the proteins are homodimeric in solution (both ~50 kDa). This study will present the findings on the overall characterisation and the implications of the findings on the interaction of these proteins. / LG2018

Pro-inflammatory cytokines released from immune cells infiltrating the endocrine pancreas in Type 1 Diabetes (T1D) induce the generation of reactive oxygen and nitrogen species (ROS/RNS). Cytokines are in part cytotoxic to ƓÒ-cells via the production of peroxynitrite (ONOO-). ƓÒ-cell are weakly protected against the toxicity of ROS/RNS because of limited expression of antioxidant enzymes. The purpose of this study was to evaluate the expression and regulation of Peroxiredoxins (Prdxs/PRDXs), a new family of antioxidant enzymes in islet ƓÒ-cell. Peroxiredoxin 5 (Prdx5) is ubiquitously expressed in mammals and it exhibits a range of cellular roles including cytoprotective antioxidant defence. Human PRDX5 possesses a peroxynitrite reductase activity but its role in ƓÒ-cell defence was not investigated yet. In a first set of experiments, the localization of the Prdx family was analyzed in rodent pancreas. Prdx1 was preferentially found in the non-b-cells of the islet and in exocrine tissue. Prdx2, Prdx3 and Prdx5 were present in b and non-b-cells, while Prdx4 and Prdx6 were poorly expressed. Then, we investigated the modulation of Prdx mRNA and protein expression levels by cytokines in adult rat isolated islets. Prdx1, Prdx2 and Prdx3 expression was not modified while Prdx5 mRNA was upregulated. However, Prdx5 protein was downregulated, which could involve ubiquitination and proteasomal degradation. Little is known about the PRDX antioxidant enzyme expression in human islets. In a second set of experiments, we investigated the expression and regulation of the 6 PRDXs in human islet preparations facing the context of T1D pathogenesis. PRDX 2, 3, 5, 6 were observed in the exocrine part of the pancreas. PRDX2 and PRDX6 were preferentially expressed in islet ƓÑ cells rather than in ƓÒ cells. PRDX3 and PRDX5 were localized in ƓÑ cells as well as in ƓÒ cells. PRDX4 was detected neither in exocrine nor in endocrine tissue. Islets exposed to a mixture of cytokines showed a downregulation of PRDX2, 3, 5, 6 mRNA expression, as was also the case for PRDX5 protein. This study demonstrated that a clear difference between human and rodent species does exist in terms of tissue localization, expression and regulation of Prdxs by cytokines. Finally, we performed Prdx5 overexpression or silencing in insulin secreting cell line INS-1E. Overexpression of Prdx5 was effective against a stress induced by SIN-1 but not against the cytokines mixture. On the opposite, silencing Prdx5 expression decreased the cell viability. Then, the hypothesis that the vulnerability of islets to cytokines mixture was due to the Prdx5 downregulation was not demonstrated. However, the modification of Prdx5 expression would in part be responsible for the high sensitivity of ƓÒ-cell to peroxynitrite. In conclusion, this study featured the presence of some Prdxs/PRDXs in islet cells, and the regulation of their expression by cytokines. They intervene in protection against ONOO- toxicity but their implication against cytokine agression remain to be more precisely evaluated.

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 família Tiol Peroxidases (TPxs - Peroxirredoxinas e Glutationa peroxidases) purificadas definitivamente reduzem peróxidos rapidamente (peroxinitrito, ONOOH/ONOO; peróxido de hidrogênio, H2O2), mas nenhuma evidência direta desta atividade foi demonstrada em células vivas. Isto é particularmente importante pois o ciclo catalítico da atividade peróxido redutase de TPxs depende de sucessivas reações de trocas de tióis que podem limitar a velocidade de redução do peróxido. Neste trabalho, esta questão foi investigada em Saccharomyces cerevisiae (Sc) por meio de cinética de competição com um indicador fluorescente que é específico para ONOO (ácido borônico de cumarina; CBA), com a expectativa de que quanto maior a atividade peroxinitrito redutase, menor a oxidação do indicador. Também foi investigado o papel de duas peroxirredoxinas (Prxs) específicas na remoção deste peróxido. O estudo mostrou que a oxidação do indicador CBA dependente de ONOO foi sempre significativamente maior em células de Saccharomyces cerevisiae deficientes em TPxs (cepa 8) relativo a cepa nativa (WT). Além disso, a transfecção do gene que codifica a Prx mais abundante em Saccharomyces cerevisiae (Tsa1) na cepa 8 diminui parcialmente a oxidação de CBA. Além disso, a oxidação de CBA foi maior na cepa deficiente apenas da peroxirredoxina Tsa1 (a mais abundante da família) relativo à cepa WT, mostrando a relevância desta isoforma especificamente. De forma adversa, a oxidação de CBA na cepa deficiente da peroxirredoxina Tsa2 foi semelhante à cepa WT. Também, foi constatado que o processo de remoção de ONOO é catalítico (e não estequiométrico) para crescentes fluxos de peroxinitrito em todas as cepas e condições utilizadas no estudo. Finalmente, o estudo sugere que células possuem sistemas catalíticos peroxinitrito redutase redundantes, já que a própria cepa 8 apresenta e pode modular esta atividade. Estes resultados confirmam a expectativa da relevância de TPxs na remoção de ONOO e por extensão de outros peróxidos biologicamente relevantes e são a primeira evidência direta e em tempo real da atividade peroxinitrito redutase de TPxs em células. / The purified Thiol Peroxidases family (TPxs - Peroxiredoxins and Glutathione peroxidases) rapidly reduces peroxides (peroxynitrite, ONOOH/ONOO-, hydrogen peroxide, H2O2), but no direct evidence of this activity has been demonstrated in living cells. This is particularly important since the catalytic cycle of the TPxs peroxide reductase activity depends on successive thiol exchange reactions, which may limit the rate of peroxide reduction. In this work, this question was investigated in Saccharomyces cerevisiae (Sc) by competition kinetics using a fluorescent indicator that is specific for ONOO- (coumarin boronic acid; CBA). It is expected that the higher the peroxynitrite reductase activity, the lower the oxidation of the indicator. The role of two specific peroxiredoxins (Prxs) in the removal of this peroxide has also been investigated. The study showed that the oxidation of ONOO- dependent CBA indicator was always significantly higher in TPxs-deficient Saccharomyces cerevisiae cells (strain 8) compared to the native strain (WT). In addition, the transfection of the gene encoding the most abundant Prx into Saccharomyces cerevisiae (Tsa1) in the 8 strain partially diminishes CBA oxidation. Besides that, CBA oxidation was greater in the deficient strain only of the peroxiredoxin Tsa1 (the most abundant in the family) compared to the WT strain, showing the relevance of this isoform specifically. On the other hand, CBA oxidation in the deficient strain of the Tsa2 peroxiredoxin was similar to the WT strain. Also, it was found that the ONOO- removal process is catalytic (and not stoichiometric) for increasing peroxynitrite fluxes in all strains and conditions used in the study. Finally, the study suggests that cells have redundant peroxynitrite reductase catalytic systems, since the 8 strain itself presents and can modulate this activity. These results confirm the expectation of the relevance of TPxs in the removal of ONOO- and by extension of other biologically relevant peroxides and are the first direct and real-time evidence of peroxynitrite reductase activity of TPxs in cells.

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.

Pseudomonas aeruginosa é uma gamma-proteobacteria ubíqua, sendo a principal causa de infecção hospitalar dentre todos os patógenos relacionados com pneumonia em UTI. A defesa do hospedeiro se dá por vários mecanismos como a liberação, por fagócitos, de espécies reativas de oxigênio, como o ânion superóxido (O2?-), peróxido de hidrogênio (H2O2) e o radical hidroxila (OH?) para combater o patógeno. LsfA pertence à família das peroxirredoxinas (Prx) e ao sub-grupo de Prxs que contém somente uma cisteína catalítica (denominadas 1-Cys Prx). Prxs são enzimas capazes de remover peróxidos (incluindo peroxinitrito) em velocidades muito elevadas. Além disso, LsfA está relacionada a patogenicidade de P. aeruginosa. Dentro desse contexto, o objetivo do presente trabalho é a caracterização bioquímica e estrutural de LsfA; que pode possibilitar a identificação de inibidores dessa enzima antioxidante. Por outro lado, caracterizando cineticamente reações de oxido-redução de LsfA e caracterizando seus mecanismos de ação, podemos identificar seus substratos biológicos. Dessa maneira, utilizando diferentes técnicas, determinamos as constantes de segunda ordem de LsfA com H2O2 (na ordem de 107 M -1.s -1); para terc-butilhidroperóxido (na ordem de 106 M-1.s-1) e peroxinitrito (na ordem de 107 M-1.s-1). A redução de LsfA por ascorbato foi descrita previamente por nosso grupo (na ordem de 103 M-1.s-1); e aqui apresentamos dados preliminares sobre a redução dessa 1-Cys Prx por GSH. Além disso, fomos capazes de determinar a estrutura cristalográfica de LsfA em sua forma oxidada e superoxidada, com resolução de 2.6 e 2.0 ? respectivamente; que, como esperado, se apresentou no estado dimérico, em ambos os casos. Descrevemos aqui características sobre a estrutura do sítio ativo de LsfA, que apresenta mais eletronegativo, com a cisteína peroxidásica desprotonada, e mais hidrofóbico. Na estrutura de LsfA superoxidada, observamos a co-cristalização dessa enzima com uma molécula de polietileno glicol que pode estar mimetizando um substrato. Portanto, esses estudos levantaram importantes informações estruturais e bioquímicas de uma enzima antioxidante envolvida com a virulência de P. aeruginosa / Pseudomonas aeruginosa is a ubiquous gamma-proteobacteria that is the main cause of hospitalar infections among all pathogens related with pneumonia. Host defenses against pathogens are mainly by phagocytes, which releases reactive oxygen species, such as superoxide (O2?-), hydrogen peroxide (H2O2) and hydroxyl radical (OH?) to fight against pathogen. LsfA belongs to peroxiredoxins (Prx) family; and to the 1-Cys Prx sub-group (Prx6 sub-family) that possess only one catalytic cysteine. Prx are enzymes can remove peroxides with extremely high efficiency. LsfA was already related with P. aeruginosa virulence. So, the aim of the present work is the structural and biochemical characterization of LsfA, which may enable the discovery of inhibitors. Furthermore, the investigation of the kinetics and the mechanism of catalysis of LsfA may give insights on the chemical nature of its biological substrates. Therefore, using different techniques; the second order rate constants of LsfA with H2O2 (107 M -1.s -1), tert-butylhydroperoxide (106 M -1.s -1) and peroxynitrite (107 M -1.s -1) were determined. Our group has already determined the rate constant between ascorbate and LsfA (103 M -1.s -1) and preliminary data on the reduction of this 1-Cys Prx by glutathione is described. Furthermore, two crystallographic structures of LsfA were elucidated in distinct oxidative states (sulfenic and sulfonic acid in the CP), both in the dimeric state; at 2.6 and 2.0 ? resolution respectively. Features in the LsfA active site are also described here, such as poor exposure to the solvent. In the LsfA crystal structure where Cp is hyperoxidized to sulfinic acid, we observed the presence of an electronic density compatible with a PEG molecule that might be mimicking one of the possible substrates. Therefore, relevant structural and biochemical information were gained with our studies about an antioxidant enzyme involved with P aeruginosa virulence

A família Tiol Peroxidases (TPxs - Peroxirredoxinas e Glutationa peroxidases) purificadas definitivamente reduzem peróxidos rapidamente (peroxinitrito, ONOOH/ONOO; peróxido de hidrogênio, H2O2), mas nenhuma evidência direta desta atividade foi demonstrada em células vivas. Isto é particularmente importante pois o ciclo catalítico da atividade peróxido redutase de TPxs depende de sucessivas reações de trocas de tióis que podem limitar a velocidade de redução do peróxido. Neste trabalho, esta questão foi investigada em Saccharomyces cerevisiae (Sc) por meio de cinética de competição com um indicador fluorescente que é específico para ONOO (ácido borônico de cumarina; CBA), com a expectativa de que quanto maior a atividade peroxinitrito redutase, menor a oxidação do indicador. Também foi investigado o papel de duas peroxirredoxinas (Prxs) específicas na remoção deste peróxido. O estudo mostrou que a oxidação do indicador CBA dependente de ONOO foi sempre significativamente maior em células de Saccharomyces cerevisiae deficientes em TPxs (cepa 8) relativo a cepa nativa (WT). Além disso, a transfecção do gene que codifica a Prx mais abundante em Saccharomyces cerevisiae (Tsa1) na cepa 8 diminui parcialmente a oxidação de CBA. Além disso, a oxidação de CBA foi maior na cepa deficiente apenas da peroxirredoxina Tsa1 (a mais abundante da família) relativo à cepa WT, mostrando a relevância desta isoforma especificamente. De forma adversa, a oxidação de CBA na cepa deficiente da peroxirredoxina Tsa2 foi semelhante à cepa WT. Também, foi constatado que o processo de remoção de ONOO é catalítico (e não estequiométrico) para crescentes fluxos de peroxinitrito em todas as cepas e condições utilizadas no estudo. Finalmente, o estudo sugere que células possuem sistemas catalíticos peroxinitrito redutase redundantes, já que a própria cepa 8 apresenta e pode modular esta atividade. Estes resultados confirmam a expectativa da relevância de TPxs na remoção de ONOO e por extensão de outros peróxidos biologicamente relevantes e são a primeira evidência direta e em tempo real da atividade peroxinitrito redutase de TPxs em células. / The purified Thiol Peroxidases family (TPxs - Peroxiredoxins and Glutathione peroxidases) rapidly reduces peroxides (peroxynitrite, ONOOH/ONOO-, hydrogen peroxide, H2O2), but no direct evidence of this activity has been demonstrated in living cells. This is particularly important since the catalytic cycle of the TPxs peroxide reductase activity depends on successive thiol exchange reactions, which may limit the rate of peroxide reduction. In this work, this question was investigated in Saccharomyces cerevisiae (Sc) by competition kinetics using a fluorescent indicator that is specific for ONOO- (coumarin boronic acid; CBA). It is expected that the higher the peroxynitrite reductase activity, the lower the oxidation of the indicator. The role of two specific peroxiredoxins (Prxs) in the removal of this peroxide has also been investigated. The study showed that the oxidation of ONOO- dependent CBA indicator was always significantly higher in TPxs-deficient Saccharomyces cerevisiae cells (strain 8) compared to the native strain (WT). In addition, the transfection of the gene encoding the most abundant Prx into Saccharomyces cerevisiae (Tsa1) in the 8 strain partially diminishes CBA oxidation. Besides that, CBA oxidation was greater in the deficient strain only of the peroxiredoxin Tsa1 (the most abundant in the family) compared to the WT strain, showing the relevance of this isoform specifically. On the other hand, CBA oxidation in the deficient strain of the Tsa2 peroxiredoxin was similar to the WT strain. Also, it was found that the ONOO- removal process is catalytic (and not stoichiometric) for increasing peroxynitrite fluxes in all strains and conditions used in the study. Finally, the study suggests that cells have redundant peroxynitrite reductase catalytic systems, since the 8 strain itself presents and can modulate this activity. These results confirm the expectation of the relevance of TPxs in the removal of ONOO- and by extension of other biologically relevant peroxides and are the first direct and real-time evidence of peroxynitrite reductase activity of TPxs in cells.

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.

As peroxirredoxinas (Prx) são enzimas antioxidantes que se destacam pela capacidade de decompor uma grande variedade de hidroperóxidos com elevada eficiência (106-108M-1s-1), mantendo essas moléculas em níveis adequados à homeostase celular. Entretanto, já foi demonstrado que em diversos tipos tumorais os níveis de Prx são extremamente aumentados e experimentos envolvendo sua inativação resultam na diferenciação ou apoptose de células tumorais. Recentemente, foi descoberto um diterpenóide denominado adenantina que seria o primeiro inibidor para as Prx1 e Prx2 de humanos e foi demonstrada que sua aplicação em células de leucemia mieloide aguda promoveu diferenciação ou apoptose dessas células. Nesse contexto, o presente trabalho apresenta duas vertentes: 1) A caracterização das alterações estruturais e funcionais promovidas pela ligação da adenantina ao sítio ativo das Prx utilizando Tsa1 de Saccharomyces cerevisiae como modelo biológico, em função da sua alta similaridade com Prx2 de humanos; 2) Avaliação da atividade antitumoral dose dependente de adenantina sobre as linhagens celulares REH e MOLT-4 de leucemia linfoide aguda. No que concerne à primeira linha de investigação, nossos resultados revelam que Tsa1 é suscetível à inibição por adenantina, uma vez que o tratamento reduziu em ~66 % a velocidade de decomposição de peróxido de hidrogênio. Adicionalmente, a mutação da Thr44 de Tsa1, pertencente à chamada tríade catalítica, por uma Ser resultou em uma proteína mais suscetível a alterações na estrutura secundária e à inibição da atividade peroxidásica em função da ligação com adenantina, apresentando uma diminuição de ~85% na velocidade de reação. Características semelhantes foram observadas para a proteoforma Tsa2 de S. cerevisiae, que carreia naturalmente a substituição da Thr44 pela Ser. Análises de sequências de Prx em bancos de dados revelaram que majoritariamente proteínas contendo Ser são encontradas em organismos procariotos, muitos deles patogênicos. Finalmente, demonstramos por meio de ensaios citotoxicidade que as bactérias Staphylococcus aureus e Staphylococcus epidermidis, que possuem uma Ser na tríade catalítica, têm seu crescimento inibido pelo tratamento com adenantina (IC50 de 460µM e 77µM, respectivamente), enquanto que para Escherichia coli, que possui Thr nessa posição, a toxicidade da adenantina foi bastante baixa (não foi possível determinar o IC50 nas condições utilizadas). Dessa forma, os dados apresentados neste trabalho demonstram o potencial da utilização da adenantina tanto como antibiótico quanto como antileucêmico. / Peroxiredoxins (Prx) are antioxidant enzymes which stand out due the ability to decompose a wide variety of hydroperoxides with high efficiency (106-108M-1s-1) maintaining these molecules at suitable levels to cellular homeostasis and participating in several signaling events. However, it has been shown that, in many tumor types, Prx levels are extremely increased and experiments involving its inactivation have resulted in differentiation or apoptosis of tumor cells. It was recently found a diterpenoid, called adenanthin, that would be the first human Prx1 and Prx2 inhibitor and it was demonstrated that its application in acute myeloid leukemia cells was able to promote differentiation or apoptosis. In this context, this work presents two lines of research: 1) Characterization of structural and functional changes promoted by adenanthin binding to Prx active site using Tsa1 from Saccharomyces cerevisiae as biological model, due to its high similarity to human Prx2. 2) Evaluation of adenanthin dose-dependent antitumor activity over the acute lymphoid leukemia cell lines REH and MOLT-4. As regards the first line of research, our result reveal that Tsa1 is susceptible to inhibition by adenanthin, since the treatment with this binder reduced the hydrogen peroxide decomposition velocity in ~ 66%. In addition, the replacement of Thr44 from Tsa1, aminoacid belonging to the so-called catalytic triad, by a Ser resulted in a protein more susceptible to alterations in secondary structure and to peroxidase activity inhibition in function of adenanthin binding, presenting ~85% of decrease in reaction velocity. Similar characteristics were observed for Tsa2 proteoform from S. cerevisiae, which naturally carries the substitution of Thr44 by Ser. Prx sequences analyzes in databases revealed that mostly Ser-containing proteins are found in prokaryotic organisms, many of them pathogenic ones. Finally, we demonstrate through cytotoxicity assays that the bacteria Staphylococcus aureus and Staphylococcus epidermidis, which have a Ser in catalytic triad, have their growth inhibited by adenanthin treatment (IC50 of 460µM and 77µM, respectively), whereas for Escherichia Coli, which has Thr at that position, the tocyxicity of adenanthin was quite low (it was not possible to determine the IC50 under the used conditions). Regarding the second line of investigation, we found that adenanthin is able to induce the death of leukemic cell lines REH and MOLT-4, and for the last one, there was an unexpected proliferation of cells treated by the longest incubation period (72 hours), characterizing a possible indication of differentiation process. In this sense, the data presented here demonstrate the potential of adenanthin use in both antibiotic and antileukemic treatments.

Peroxirredoxinas (Prxs) são enzimas que desempenham papéis centrais no metabolismo redox celular, são proteínas abundantes reduzindo peróxidos com uma velocidade extraordinária. As Prxs estão presentes em todos os grupos taxonômicos, possuem várias isoformas com diferentes funções, tais como antioxidantes, chaperonas moleculares e reguladores da transdução de sinal. São peroxidases tióis dependentes baseadas em um resíduo de cisteína catalítico podendo ser divididas em 1-Cys ou 2-Cys, dependendo do número de resíduos de cisteínas envolvidos na catálise. Inicialmente a redução das Prxs foi descrita por ser estritamente dependente de tióis, no entanto nosso grupo demonstrou que o ascorbato também poderia reduzir o sulfênico intermediário das 1-Cys Prx (1-Cys Prx-SOH) em diferentes organismos. Esses dados representam um quebra no paradigma antioxidante tiól específico, mostrando que o ascorbato pode reduzir os ácidos sulfênicos nas 1-Cys Prx. Para obter evidências que o ascorbato possa ser considerado um redutor biológico das 1-Cys Prx, foi necessário determinar a constante de segunda ordem, uma vez que em células, outros compostos podem competir com este antioxidante. Devido a problemas técnicos, este foi um desafio por muitos anos. Neste trabalho, descrevemos a caracterização cinética da redução de ácidos sulfênicos por ascorbato em diferentes proteínas. Primeiramente, a redução das 1-Cys Prx-SOH foi analisado usando a enzima de A. fumigatus (AfPrxA) que apresenta similaridade de 37% com a Prdx6 (1-Cys Prx humana) e foi considerada uma enzima bastante estável. Inicialmente, realizamos a análise bi-substrato utilizando eletrodos específicos para H2O2 (Free Radical Analyzer 4100 da World Precision Instruments Inc.), através de uma abordagem de estado estacionário. AfPrxA decompõem H2O2 em uma reação ascorbato dependente com boa eficiência (kcat/KM asc = 7.4 x 103 M-1s-1), através de um mecanismo Bi-Bi Ping-Pong. Para apoiar ainda mais esses resultados, desenvolvemos uma abordagem cinética independente, baseada na competição entre diclorofenolindofenol (DCPIP) e ácidos sulfênicos por ascorbato. DCPIP é um sensor redox, cuja a coloração azul é perdida quando reduzido. Primeiramente, confirmamos que o DCPIP foi reduzido por ascorbato com uma constante de segunda ordem de 718 M-1s-1, similar a valores descritos na literatura anteriormente. Este método validou nossas condições de ensaio, permitindo determinar a constante de segunda ordem de reação entre AfPrxA-SOH e ascorbato (1,5 x 103 M-1s-1) a pH 7,44, 25ºC através da abordagem de pseudo-primeira ordem. Portanto, por dois métodos diferentes, demonstramos que o ascorbato reduz a AfPrxA-SOH com constantes de velocidade na ordem de 103 M-1s-1. Este ensaio também nos permitiu determinar as constantes de segunda ordem para as 1-Cys Prx de diferentes organismos como bactéria, levedura, planta e mamíferos. Em todos os casos, as constantes de velocidade foram na ordem de 103 M-1s-1. Posteriormente, analisamos se a 2-Cys Prx de levedura (Tsa1), que possui uma mutação na cisteína de resolução por um resíduo de serina (Tsa1-C170S) poderia adquiri atividade ascorbato dependente. Novamente, a forma ácido sulfênico da Tsa1-C170S foi reduzida por ascorbato na mesma ordem de grandeza de 103 M-1s-1. Em adição, decidimos investigar a redução dos Cys-SOH por ascorbato em outras proteínas como Gliceraldeído 3-fosfato desidrogenase (GAPDH) e Papaína que também possuem um ácido sulfênico como produto da oxidação. A constante de segunda ordem obtida da reação com ascorbato foi novamente à mesma ordem de grandeza (103 M-1s-1). Em conclusão, a redução de ácidos sulfênicos presentes nas Prxs por ascorbato pode ser considerada relevante em compartimentos subcelulares em que este redutor esteja presente em grandes quantidades. Além disso, o ascorbato pode reduzir ácidos sulfênicos de outras proteínas, e esta interação pode representar uma nova via na biologia redox que ainda precisa ser explorada in vivo / Peroxiredoxins (Prxs) are enzymes that play central roles in cellular redox metabolism, since they reduce peroxides with extraordinary rates and are abundant proteins. Prxs are found in all kingdoms with multiple isoforms, performing multiple functions such as antioxidants, molecular chaperones, and regulators of signal transduction. Prxs are Cys-based, thiol-dependent peroxidases with remarkable catalytic efficiency that can be divided into 1-Cys or 2-Cys, depending on the number of Cys residues involved in catalysis. Initially, reduction of Prxs was described to be strictly dependent on thiols, but later we showed that ascorbate can also reduce the sulfenic intermediate of 1-Cys Prx (1-Cys Prx-SOH) from various organisms. These data represented a breakthrough in the thiol-specific antioxidant paradigm, as ascorbate can also reduce sulfenic acids in 1-Cys Prx. To gain evidences that ascorbate could be a biological reductant for 1-Cys Prx it was necessary to determinate the respective second order rate constant, since in cells other compounds might compete with this antioxidant. Due to technical issues, this was a challenge for many years. In this work, we describe the kinetic characterization of sulfenic acid reduction by ascorbate in several proteins. Firstly, the reduction of 1-Cys Prx-SOH by ascorbate was analyzed using an enzyme from A. fumigatus (AfPrxA) that is 37% similar to PRDX6 (human 1-Cys Prx) and was quite stable. Initially, a steady-state, bi-substrate approach was followed by means of a specific H2O2 electrode (Free Radical Analyzer 4100, World Precision Instruments Inc.). AfPrxA decomposed H2O2 in an ascorbate dependent manner with good efficiency (kcat/KM asc = 7.4 x103 M-1s-1), through a Bi-Bi Ping-Pong mechanism. To further support these findings, we developed an independent, competitive kinetic approach based on the competition between dichlorophenolindophenol (DCPIP) and sulfenic acid to ascorbate. DCPIP is a redox sensor, whose blue color is lost when reduced. Firstly, we confirmed that in our conditions DCPIP was reduced by ascorbate with a second-order rate constant of 718 M-1s-1, similar to values previously described. This procedure validated our assay conditions and allowed us to proceed in the competitive method for the determination of the second order rate constant of the reaction of AfPrxA-SOH with ascorbate (1,5 x 103M-1s-1) at pH 7.44, 25ºC by pseudo first order approach. Therefore, by two independent approaches, we showed that ascorbate reduced AfPrxA-SOH with rate constants in the 103 M-1s-1 range. It also allowed us to determine the second order rate constants for the reduction 1-Cys Prxs from other organisms such as bacteria, yeast, plant and mammal. In all cases, the rate constants were in the 103 M-1s-1 range. Subsequently, we analyzed whether a recombinant yeast 2-Cys Prx (Tsa1), whose resolving Cys (Cysr) was mutated to a serine residue (Tsa1-C170S) acquired the ascorbate dependent activity. Again, the sulfenic acid form of Tsa1-C170S was reduced by ascorbate in the same 103 M-1s-1 range. In addition, we decided to investigate the reduction of Cys-SOH by ascorbate in other proteins like glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Papain that also have a sulfenic acid as product of their oxidation. The second order rate constants obtained for the reaction with ascorbate were again in the same range (103 M-1s-1). In conclusion, the reduction of sulfenic acids present in Prx by ascorbate might be relevant in the subcellular compartments in which this reductant is present at high levels, capable to compete with other reductants. Furthermore, ascorbate can reduce sulfenic acid in other proteins, and this interaction may represent a new route in redox biology that has yet be explored in vivo