Orientador: Felix Guillermo Reyes Reyes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T15:46:40Z (GMT). No. of bitstreams: 1 Maciel_RaquelApratto_M.pdf: 938949 bytes, checksum: 248a32d15aed7abd02f0ac036eb29872 (MD5) Previous issue date: 2011 / Resumo: As doenças cardiovasculares ocupam o primeiro lugar em causas de morte no mundo e, segundo pesquisas, estas podem estar associadas ao estresse oxidativo - desequilíbrio entre produção de radicais livres e defesas antioxidantes. O tratamento e prevenção não farmacológico, dessas doenças, preconizado pela Organização Mundial da Saúde (OMS), considerando os hábitos alimentares, consiste em aumentar o consumo de frutas, hortaliças, cereais integrais e grãos, devido as suas propriedades antioxidantes, assim como aos efeitos fisiológicos das fibras alimentares também presentes nessas fontes alimentares. Por outro lado, estudos mostram que algumas substâncias xenobióticas podem estar presentes nos alimentos, como por exemplo, os inseticidas piretróides, aumentando o risco do desenvolvimento ou agravamento de alterações cardiovasculares e, ao mesmo tempo, desestruturando as defesas antioxidantes e produzindo radicais livres nocivos às células, colocando em risco a saúde humana. É inquestionável a necessidade do uso de agrotóxicos na produção agrícola, em programas de saúde pública, na saúde animal, bem como na silvicultura. O não cumprimento da legislação em vigor tem sido uma realidade preocupante, pois coloca em risco a saúde dos trabalhadores que manipulam essas substâncias, as pessoas que consomem os alimentos e o ecossistema. Assim, pesquisas que dimensionem os efeitos adversos que os agrotóxicos podem causar na saúde humana, contribuem com o cumprimento da legislação específica e, até mesmo, na atualização da mesma. Este trabalho teve por objetivo avaliar a exposição aguda, por via oral, de deltametrina, agrotóxico da classe dos piretróides, na excitação e condução cardíaca e no estresse oxidativo em ratos Wistar machos adultos, através de parâmetros eletrocardiográficos e da atividade de enzimas antioxidantes nos tecidos cardíacos e hepáticos. O plano de trabalho consistiu na administração aguda, por gavagem, de deltametrina (DMT), com os animais distribuídos em 4 grupos, com 10 animais,pesando em média 200 ± 10g cada, assim constituídos: 1) Grupo controle (5 mL de óleo de milho); 2) Grupo DMT10 [10% do valor da DL50 da deltametrina (3,2 mg/5mL de óleo de milho)]; 3) Grupo DMT25 [25% do valor da DL50 da deltametrina (8 mg/5mL de óleo de milho)]; 4) Grupo DMT100 [100% do valor da DL50 de deltametrina (32mg/mL de óleo de milho)]. No início do ensaio, foi introduzida uma sonda de gavagem nos ratos. Logo após, os animais foram submetidos à anestesia para realização do eletrocardiograma (ECG). Após 5 min de início dos registros do ECG, a DMT foi administrada através de gavagem, conforme o planejamento experimental de cada grupo, registrando-se o ECG por mais 30 min. Após, fez-se o sacrifício dos animais por aprofundamento da anestesia. Foram retirados o fígado e o coração para mensurar a atividade das enzimas: catalase, superóxido dismutase, fosfatase alcalina, glutationa S-transferase e quantificação da glutationa reduzida e do produto da peroxidação lipídica malondialdeído. A deltametrina alterou a condutividade elétrica do coração. O eletrocardiograma mostrou redução significativa da frequência cardíaca e aumento do intervalo RR, indicando bradicardia. Também foi constatado aumento da duração do complexo QRS e redução da amplitude da onda R, o que sugere alterações na excitação ventricular. A amplitude P não foi significativamente diferente, mostrando que a excitação e a condução elétrica atrial não foram prejudicadas. Da mesma forma, os intervalos QT e QTc não foram alterados significativamente, embora o grupo DMT100 para o intervalo QT tenha se mostrado elevado, descartando-se assim, o risco de morte súbita. O segmento ST apresentou infradesnivelamento progressivo, embora não significativo. A onda T mostrou-se positiva e sem alterações significativas, não podendo assim sugerir possibilidade de isquemia miocárdica induzida por deltametrina. As análises bioquímicas mostraram que a deltametrina provocou estresse oxidativo tanto no fígado como no coração. A lipoperoxidação ocorreu nos hepatócitos e não parece ter ocorrido nos miócitos. No fígado, o estresse oxidativo foi confirmado pelo aumento significativo da atividade das enzimas glutationa S-transferase e fosfatase alcalina, aumento da concentração da glutationa reduzida, e significativa redução da atividade das enzimas superóxido dismutase e catalase. Já no coração, o estresse oxidativo foi evidenciado pelo significativo aumento da atividade da catalase e redução significativa da superóxido dismutase e glutationa S-transferase, assim como, pelo aumento da concentração da enzima glutationa reduzida. Em conclusão, este trabalho constatou que a deltametrina, administrada por via oral, diferentes dosagens, em ratos Wistar, causou aumento do estresse oxidativo e este pode ter ocasionado alterações na condução e excitação cardíaca verificadas no presente estudo / Abstract: Cardiovascular diseases are in the first rank among causes of death in the world and, according to researches, they can be associated with oxidative stressimbalance between free radicals productions and antioxidant defenses. The treatment and non-pharmacological prevention of these diseases, advocated by the World Health Organization (WHO), consist in to increase consumption of fruits, vegetables, whole grains and beans, because of their antioxidant properties and the physiological effects of dietary fiber also present on those foods. On the other hand, studies have shown that some xenobiotic substances may be present in food, such as pyrethroid insecticides, increasing the risk of development or aggravation of cardiovascular alterations and, at the same time, destabilizing the antioxidant defenses and producing free radicals which are harmful to cells, endangering human health. There is no doubt about the necessity of the use of pesticides in agricultural production, in public health programs, animal health, as well as in forestry. The non-compliance with the legislation in force has been a troubling reality because it puts in risk the health of workers who handle those substances, the people who consume foods and the ecosystem. Researches that evaluate the harmful effects that pesticides can cause to the human health, contribute to the fulfilling of the specific legislation and, even, in the update of it. This work aims to evaluate the acute oral exposure of deltamethrin, a pyrethroid pesticide, on the excitation system of the heart and on the oxidative stress in adult male Wistar rats, by means of electrocardiographic parameters, and antioxidant enzymes in liver and heart tissues, respectively. The work consisted on the acute administration, by gavagem, of deltamethrin (DMT), to animals distributed in 4 groups, containing 10 animals each, as follows: 1) Control Group (5 mL of corn oil); 2) DMT10 Group [10% of the LD50 value of deltamethrin (3,2mg/5mL corn oil)]; 3) DMT25 Group [25% of the LD50 value of deltamethrin (8 mg/5mL corn oil)]; 4) DMT100 Group [100% of the LD50 value of deltamethrin (32mg/mL corn oil)]. At the beginning of the assay, it was introduced in the rats a probe of gavagem. Soon after, the animals were subjected to anesthesia for conduction of the electrocardiogram (EGC). After 5 minutes of the beginning of the ECG record, the deltamethrin was administered via gavagem, accordingly to the experimental planning of each group, and the ECG recorded for additional 30 minutes. Upon completion of the ECG recording, the rats were killed by deepening of anesthesia. The liver and heart were withdrawn to measure the activity of the enzymes: catalase, superoxide dismutase, glutathione transferase, alkaline phosphatase, as well as of the quantification of the malondialdehyde biomarker and reduced glutathione. Deltamethrin was capable of changing the electrical conductivity of heart. The electrocardiogram showed significant reduction of the heart rate and increase of the RR interval, indicating bradycardia. It was also found to increase of the duration QRS complex and reduction of the R wave amplitude, which means that there were alterations in ventricular excitation. The P amplitude was not significantly different, showing that arousal and atrial electrical conduction was not impaired. Similarly, the QT and QTc intervals were not altered significantly, although has been shown high of the group DMT100, discarding the risk of sudden death. The ST segment depression showed a progressive, though not significant. The T wave was positive and no significant changes and therefore can not suggest the possibility of myocardial ischemia induced by deltamethrin. Biochemical analysis showed that deltamethrin causes oxidative stress in both liver and heart. Lipid peroxidation occurred in hepatocytes and not observed in myocytes and this is confirmed by checking the increased activity of the alkaline phosphatase, glutathione s-transferase and reduced activity of the superoxide dismutase and catalase and increased of concentration of the reduced glutathione in the liver. In the heart, oxidative stress has been shown by increased activity of the catalase, increased concentration of the glutathione reduced and reduced activity of the superoxide dismutase and glutathione s-transferase. In conclusion, this study found that deltamethrin administered orally at different doses in rats, caused increased oxidative stress and this may have caused changes in cardiac excitation conduction and verified / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

Vitamin C is used in the diets in order to improve growth, resistance to stress and disease, as well as the survival of fish. It can also be effective in the conservation of fish during processing and storage, inhibiting the degradation of lipids by oxidation. This study evaluated the stocking density and the effects of supplementation of vitamin C in the final quality of the fillet tilapia (Oreochromis nilotius, Linneaus, 1875). We used one hundred and twenty fingerlings for each trial, the study of fish stocking density had an initial weight of 4.0 g and evaluation of supplementation with vitamin C, 11.0 g. Both studies had a randomized design, comprising four treatments of different stocking densities (50, 75, 100 and 125 fish/m3) with five replications. Already working with vitamin C (ascorbic acid 2 - sulfate acid - protected form) was carried out in four treatments with six replicates, characterized by four levels of supplementation of vitamin C in the diets (250, 500 and 750 mg/kg diet) and control group (zero mg/kg diet) with two different stocking densities (50 and 100 fish/m3). The ration given to the first study contained 36% crude protein and 3100 kcal/DE/kg feed and the second was 28% crude protein and 3400 kcal/DE/kg. After ninety days, the fish were slaughtered and evaluated the growth performance parameters for the study of stocking density, chemical composition, cholesterol content, fatty acid profile, quality of lipid fraction in both studies. The liver of fish from the second stage of labor was frozen at - 80°C for lipid peroxidation analysis and determination of enzyme activity of oxidative stress (catalase and superoxide dismutase). It follows that for the study of stoking significant difference ρ <0.05 between the final average weight and total weight gain between the different densities tested, the highest weight was found in the group density of 75 fish/m3. The body composition analysis, differences were observed ρ <0.05 between the densities on moisture, total lipid content, protein, calories and cholesterol. The ratio polyunsaturated / saturated was higher in density of 11.76 for 100 fish/m3, since the density of 50 fish/m3 presented a sum of 6.85 for the levels of EPA + DHA. In assessing the nutritional quality of lipids, the densities studied showed the contents of n-6/n-3, hypocholesterolemic/hypercholesterolemic index of polyunsaturated fatty acids of atherogenicity and index of thrombogenicity as favorable for consumption. Fish reared at a density of 50 fish/m3 can be seen as a good food from a nutritional standpoint, they presented a good level of fatty acids, especially omega-3 fatty acid and its value in hypocholesterolemic/hypercholesterolemic. Regarding the second stage of labor was observed difference ρ<0.05 between the levels of vitamin C supplementation levels in total lipid content, crude protein, calories and cholesterol. The ratio polyunsaturated/saturated was higher in the supplementation level of 250 mg of vitamin C (1.26), since the level of 500 mg of vitamin C showed a sum of 6.48 for the levels of EPA + DHA. In assessing the nutritional quality of lipids, the levels of vitamin C study showed rates of n-6/n-3, hypocholesterolemic / hypercholesterolemic index of atherogenicity and index of thrombogenicity for subsequent food consumption. Fish reared with diets supplemented with 750 mg of vitamin C in the density of 50 fish/m3 can be seen as a good food from a nutritional standpoint, they presented a good level of fatty acids, especially omega-3 fatty acid and its value in hypocholesterolemic/hypercholesterolemic. In the study of redox balance results showed that there was an increase in lipid peroxidation, suggesting that there may have been damage to the liver of animals and the antioxidant defenses were not sufficient to prevent oxidative stress. Density of 50 fish/m3, the dietary supplementation with vitamin C can be pro-oxidant. Since the density 100 fish/m3, vitamin C supplementation may be an antioxidant. The catalase activity of the enzyme showed low activity, suggesting an impairment in hepatic redox balance for this enzyme in animals receiving diets containing different levels of vitamin C. Since superoxide dismutase activity of the enzyme showed high activity, suggesting a redox balance in protecting liver for this enzyme in animals receiving diets containing different levels of vitamin C. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A vitamina C é usada nas dietas com a finalidade de melhorar o crescimento, a resistência ao estresse e às doenças, assim como a sobrevivência de peixes. Também pode ser eficiente na conservação do pescado durante o processamento e estocagem, inibindo a degradação dos lipídeos pela oxidação. O presente trabalho avaliou a densidade de estocagem e os efeitos da suplementação de vitamina C na qualidade final do filé de Tilápia-do-Nilo (Oreochromis nilotius, Linneaus, 1875). Foram utilizados cento e vinte alevinos para cada estudo, sendo que os peixes do estudo da densidade de estocagem tinham peso inicial de 4,0 g e os da avaliação com a suplementação de vitamina C de 11,0 g. O delineamento inteiramente casualizado foi utilizado em ambos os estudos perfazendo quatro tratamentos de diferentes densidades de estocagem (50, 75, 100 e 125 peixes/m3) com cinco repetições. Já o trabalho com a vitamina C (ácido 2 - sulfato ascórbico - forma protegida) foi realizado em quatro tratamentos com seis repetições, caracterizado pela suplementação de quatro níveis de vitamina C nas rações (250, 500 e 750 mg/kg de ração) e o grupo controle (zero mg/kg de ração) com duas diferentes densidades de estocagem (50 e 100 peixes/m3). A ração fornecida para o primeiro estudo continha 36% de proteína bruta e 3100 kcal/energia digestiva/kg de ração e a segunda tinha 28% de proteína bruta e 3400 kcal/energia digestiva/kg. Após noventa dias, os peixes foram abatidos e foram avaliados os parâmetros de desempenho zootécnico para o estudo de densidade de estocagem, composição centesimal, teor de colesterol, perfil de ácidos graxos, qualidade da fração lipídica para ambos os estudos. O fígado dos peixes da segunda etapa do trabalho foi congelado a – 80ºC para análises de peroxidação lipídica e determinação da atividade das enzimas do estresse oxidativo (catalase e superóxido dismutase). Conclui-se que para o estudo de densidade de estocagem houve diferença significativa ρ<0,05 entre o peso médio final e no ganho de peso total entre as diferentes densidades populacionais testadas; o maior peso foi encontrado no grupo de densidade de 75 peixes/m3. Quanto à análise da composição corporal observou-se diferença ρ<0,05 entre as densidades nos teores de umidade, lipídeos totais, teor de proteína bruta, valor calórico e de colesterol. A relação poliinsaturados/saturados foi mais elevada na densidade de 100 peixes/m3 de 11,76, já a densidade de 50 peixes/m3 apresentou uma somatória de 6,85 para os teores de ácido eicosapentaenóico + ácido docosahexaenóico. Na avaliação da qualidade nutricional dos lipídeos, as densidades estudadas mostraram os índices de n-6/n-3, hipocolesterolêmicos/hipercolesterolêmicos, índice de ácidos graxos poliinsaturados de aterogenicidade e índice de trombogenicidade como favoráveis para consumo alimentar. Os peixes criados na densidade de 50 peixes/m3 podem ser considerados como um bom alimento do ponto de vista nutricional, pois apresentaram um bom teor de ácidos graxos, principalmente de ácido ômega-3 e pelo seu valor em hipocolesterolêmicos/hipercolesterolêmicos. Com relação à segunda etapa do trabalho foi observado diferença ρ<0,05 entre os níveis de suplementação da vitamina C nos teores de lipídeos totais; teor de proteína bruta; valor calórico e de colesterol. A relação poliinsaturados/saturados foi mais elevada no nível de suplementação de 250 mg de vitamina C (1,26), já o nível de 500 mg de vitamina C apresentou um somatório de 6,48 para os teores de ácido eicosapentaenóico + ácido docosahexaenóico. Na avaliação da qualidade nutricional dos lipídeos, os níveis de suplementação de vitamina C estudados mostraram os índices de n-6/n-3, hipocolesterolêmicos/hipercolesterolêmicos, índice de aterogenicidade e índice de trombogenicidade favoráveis quanto ao consumo alimentar. Os peixes criados com ração suplementada com 750 mg de vitamina C na densidade de 50 peixes/m3 podem ser considerados como um bom alimento do ponto de vista nutricional, pois apresentaram um bom teor de ácidos graxos, principalmente de ácido ômega-3 e pelo seu valor em hipocolesterolêmicos/hipercolesterolêmicos. Já no estudo do balanço redox os resultados mostraram que houve aumento na peroxidação lipídica, sugerindo que podem ter existido danos ao fígado dos animais e as defesas antioxidantes não foram suficientes para evitar o estresse oxidativo. Na densidade de 50 peixes/m3, a ração suplementada com vitamina C pode ser pró-oxidante. Já na densidade 100 peixes/m3, a vitamina C suplementada pode ser antioxidante. A atividade da enzima catalase apresentou uma baixa atividade, sugerindo um comprometimento no balanço redox hepático para esta enzima nos animais submetidos à ração com diferentes níveis de suplementação de vitamina C. Já atividade da enzima superóxido dismutase apresentou elevada atividade, sugerindo uma proteção no balanço redox hepático para esta enzima nos animais submetidos à ração com diferentes níveis de suplementação de vitamina C.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 4 uppsatser.

Contamination of perchlorate and chlorate in nature is primarily the result of various industrial processes. The microbial respiration of these oxyanions of chlorine plays a major role in reducing the society’s impact on the environment. The focus with this thesis is to investigate the oxygen-dependent regulation of key components involved in the chlorate respiration in the gram‑negative bacterium Ideonella dechloratans. Chlorate metabolism is based on the action of the enzymes chlorate reductase and chlorite dismutase and results in the end products molecular oxygen and chloride ion. Up‑regulation of chlorite dismutase activity in the absence of oxygen is demonstrated to occur at the transcriptional level, with the participation of the transcriptional fumarate and nitrate reduction regulator (FNR). Also, the chlorate reductase enzyme was shown to be regulated at the transcriptional level with the possible involvement of additional regulating mechanisms as well. Interestingly, the corresponding chlorate reductase operon was found to be part of a polycistronic mRNA which also comprises the gene for a cytochrome c and a putative transcriptional regulator protein.

In the stamen (male reproductive tissue) of petunia 'Hurrah' flowers, the occurrence of SOD (superoxide dismutase) provided an effective anti-oxidative mechanism against superoxide production. Superoxide production and SOD activities at five developmental stages showed a positive correlation. The highest superoxide production and SOD activity in different parts of the stamen (anther, filament and pollen) were at stages with high metabolic activity: (i) during growing buds (in anthers and filaments) (ii) when flowers with predehiscent anthers were fully open (in pollen). In all parts of the stamen, SOD activity was the lowest at stage five (fully open flowers with dehiscent anthers), superoxide production was also lower at this stage with the exception of the pollen. The highest SOD activity was localized in anthers with the pollen, suggesting that the filaments only have a structural support function. SOD was examined on a native PAGE with regard to the isozymes present within the stamen of five developmental stages. Three isozymes, which were identified as Mn SOD, Fe SOD and Cu/Zn SOD by reactions with inhibitors, were commonly found at five developmental stages in crude extracts of anthers, filaments and pollen. The developmental stages with stronger isozyme bands on the native PAGE were consistent with the stages with higher SOD activities, and the Mn SOD and Fe SOD isozyme bands were more intense than Cu/Zn SOD bands, suggesting the activities of Mn SOD and Fe SOD in the crude extracts were much higher than Cu/Zn SOD. SOD from 1,000 stamens of dehiscent mature flowers was partially purified using ammonium sulphate fractionation and DEAE cellulose column chromatography. The purified bound fraction contained only one SOD isozyme on a native PAGE, which was shown to be a Mn SOD, as it is sensitive to neither hydrogen peroxide nor cyanide. The specific activity of the purified SOD was 66.5 U/mg and the yield of total activity was 3.0%. The progress of enzyme purification was monitored using SDS-PAGE and the bound fraction contained two major polypeptide bands. The purified enzyme activity was optimal in the range of neutral pH, but it was the highest at pH 7.8. Through incubation at various pH levels for 24 hours, favourable stability of the purified fraction was confirmed around a pH range of 7 to 8.5. The purified enzyme retained 87% of its initial activity at -20 ? after one month of storage, but at 4 ? only 38% of the initial activity remained after the same period of storage.

Récemment, notre laboratoire a démontré pour la première fois, que la protéine Damaged DNA Binding 2 (DDB2) possédait une activité régulatrice négative de l’expression basale de la superoxyde dismutase mitochondriale (SOD Mn) en se fixant sur un élément de réponse dans la région promotrice de son gène. Cette protéine était connue jusque là pour sa participation dans le système de réparation de l’ADN par excision de nucléotides. L’objectif de ce travail a été de définir précisément l’implication de ces deux protéines dans la croissance des cellules d’adénocarcinome mammaire, en développant des modèles cellulaires dont l’expression de la SOD Mn ou de la DDB2 est modulée expérimentalement. Nos résultats montrent pour la 1ère fois, que la SOD Mn est surexprimée dans les cellules tumorales mammaires insensibles aux oestrogènes (ER-) et ayant un pouvoir métastatique, et non dans les cellules épithéliales mammaires normales et les cellules ER+. L’inhibition de l’expression de la SOD Mn entraîne une stimulation de la croissance et une diminution de l’invasivité cellulaires, associées à une activité réduite de la métalloprotéinase 9. L’addition d’antioxydants, éliminant spécifiquement l’H2O2 issu de l’activité élevée de la SOD Mn, entraîne à la fois une inhibition de la croissance et du pouvoir invasif des cellules ER-. Ces résultats révèlent que la SOD Mn participe aux capacités invasives des cellules ER- via la production d’H2O2. Nous avons également montré pour la 1ère fois, que la DDB2 présente une activité oncogénique dans les cellules tumorales mammaires sensibles aux oestrogènes (ER+), non seulement parce que son gène est surexprimé, mais également parce qu’elle active leur prolifération en agissant sur la phase de transition G1/S et sur la progression dans la phase S du cycle cellulaire. Contrairement à la SOD Mn, l’expression de la DDB2 n’est pas observée dans les cellules tumorales mammaires ER-. De même à partir de biopsies provenant de patientes atteintes d’un cancer du sein, nous avons montré que la DDB2 est significativement plus exprimée dans les tumeurs les moins agressives et exprimant le récepteur aux oestrogènes. En montrant l’importance de la SOD Mn et la DDB2 dans la croissance et l’invasion des cellules tumorales mammaires, l’ensemble de ce travail révèle ainsi ces deux protéines comme des marqueurs prédictifs potentiels de la progression tumorale, et ouvre de nombreuses perspectives en cancérologie mammaire. / Recently, our laboratory demonstrated for the first time, that Damaged DNA Binding 2 (DDB2) played a role as a negative transcriptional regulator on the mitochondrial superoxide dismutase (MnSOD) expression through its binding to a specific DNA sequence located into the promoter of MnSOD gene. DDB2 was known as a protein which participates in the nucleotide excision repair of DNA. The goal of this study was to define precisely the involvement of the both proteins in the growth of mammary adenocarcinoma cells, using experimental procedures to modulate their expression in the breast cancer cell lines. Our results show for the first time that MnSOD is overexpressed in the estrogen receptor (ER) negative and metastatic breast tumor cells, but not in normal epithelial mammary cells and ER-positive tumor cells. Inhibition of MnSOD expression stimulates proliferation but decreases the invasive ability and the metalloproteinase 9 activity of tumor cells. Elimination of H2O2 from the elevated MnSOD activity by addition of specific antioxidants decreases proliferation as well as invasive ability of tumor cells, suggesting that the role of MnSOD in the invasive ability of tumor cells is mediated by H2O2. We have shown too for the first time that DDB2 has an oncogenic activity in the ER-positive breast tumor cells, because its gene is overexpressed and stimulates the proliferation by activating the entry of cells in the G1/S transition phase and the S phase progression. In contrast to MnSOD, DDB2 expression is not observed in ER-negative breast tumor cells, but is higher in ER-positive than in ER-negative tumor samples from patients with breast carcinoma. Taken together, our findings demonstrate that both MnSOD and DDB2 play a role in the growth and invasiveness of tumor cells and may become a promising candidate as a predictive markers in breast cancer. More studies will be need to define molecular mechanism controlling this activity of these both proteins.

Cu/Zn Superoxide Dismutase (SOD1), is a ubiquitous antioxidant enzyme with several emerging roles outside of its canonical function. SOD1 is also emerging in central roles in cancer and neurodegenerative pathologies. Little is known about SOD1 regulation, particularly at a post-translational level. Post-translational modifications (PTMs) play an important role in enabling proteins to rapidly respond to their environment. Therefore, identifying specific PTMs involved in protein regulation represents a powerful opportunity to interfere with any associated pathologies. This work employs proteomics to identify mechanisms of post-translation regulation on cell survival signaling proteins. We focused on SOD1, which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Interestingly, we also found that K122 acyl mutants were sufficient to prevent mitochondrial accumulation of the G93A SOD1 clinical mutant. We observed increased autophagic activity in G93A expressing cells compared to WT or G93A/K122-acyl mimic double mutants, and found that this double mutant was just as prone to aggregate as G93A SOD1—suggesting that SOD1 aggregation is more toxic when in the mitochondria. We observed increased protein turnover rates in cells expressing SOD1 G93A, in support of increased autophagy. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a new interplay between SOD1 acylation, metabolic regulation, SOD1 aggregate toxicity, and SOD1-mediated cell survival.

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and the mitochondrial localized fraction of Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by a growing number of metallochaperone proteins. Here we describe two novel copper chaperones of COX, Cmc1 and Cmc2. In Saccharomyces cerevisiae, both Cmc1 and Cmc2 localize to the mitochondrial inner membrane facing the intermembrane space. Cmc1 and Cmc2 are essential for full expression of COX and cellular respiration, contain a twin Cx9C domain, and are conserved from yeast to humans. Additionally, the presence or absence of these proteins not only determines full assembly of functional COX but also affects metallation of Sod1 suggesting these proteins might play a role on co-modulation of copper transfer to COX and Sod1. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vise versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. We conclude that Cmc1 and Cmc2 have cooperative but non-overlapping functions in cytochrome c oxidase biogenesis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease and is characterized by progressive paralysis leading to death, typically, within 3-5 years of the onset of symptoms. The majority of ALS cases are sporadic with no known causative agent; however, 5-10% of ALS cases are genetically inherited and termed familial ALS (fALS). Approximately, 15-20% of these fALS cases have been linked to mutations in the gene encoding human Cu/Zn superoxide dismutase (hSOD). To date, over 140 hSOD mutations have been discovered. The mechanisms by which mutant hSOD confers toxicity in fALS patients are still unknown. However, there is growing evidence that ALS is a type of protein conformational disease whereby cell damage or death is caused by the accumulation of protein aggregates in the cell. It is hypothesized that mutations destabilise hSOD and increase its propensity to aggregate. There is some controversy as to which hSOD species contributes to aggregation. Many believe that only apo or mismetallated forms of hSOD are able to aggregate. Due to the abundance of fully metallated or holo hSOD in the cell, we hypothesize that holo hSOD can also lead to aggregation. Holo dimer interface mutants A4S, A4T and I113T as well as G41D were found to be destabilized compared to holo pseudo wildtype (pWT) while zinc binding mutant H80R was shown to form fragments via an unknown mechanism. Holo dimer interface mutants A4S and A4T were also shown to have an increased propensity to aggregate compared to pWT, which correlates to their decreased stability as well a short disease durations.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease and is characterized by progressive paralysis leading to death, typically, within 3-5 years of the onset of symptoms. The majority of ALS cases are sporadic with no known causative agent; however, 5-10% of ALS cases are genetically inherited and termed familial ALS (fALS). Approximately, 15-20% of these fALS cases have been linked to mutations in the gene encoding human Cu/Zn superoxide dismutase (hSOD). To date, over 140 hSOD mutations have been discovered. The mechanisms by which mutant hSOD confers toxicity in fALS patients are still unknown. However, there is growing evidence that ALS is a type of protein conformational disease whereby cell damage or death is caused by the accumulation of protein aggregates in the cell. It is hypothesized that mutations destabilise hSOD and increase its propensity to aggregate. There is some controversy as to which hSOD species contributes to aggregation. Many believe that only apo or mismetallated forms of hSOD are able to aggregate. Due to the abundance of fully metallated or holo hSOD in the cell, we hypothesize that holo hSOD can also lead to aggregation. Holo dimer interface mutants A4S, A4T and I113T as well as G41D were found to be destabilized compared to holo pseudo wildtype (pWT) while zinc binding mutant H80R was shown to form fragments via an unknown mechanism. Holo dimer interface mutants A4S and A4T were also shown to have an increased propensity to aggregate compared to pWT, which correlates to their decreased stability as well a short disease durations.