Cells are constantly exposed to different stresses – one being redox stress, which is induced by metal, reactive oxygen species and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR) helps modulate redox stress by two different mechanisms – either by reducing S-nitrosoglutathione (GSNO) to oxidized glutathione (GSSG) or by oxidizing hydroxymethyl glutathione (HMGSH), a biproduct of glutathione and formaldehyde, to formic acid. GSNO has the potential to posttranslational modify proteins in two different manners, either by S-nitrosation or by S-glutathionylation. Interestingly, GSNOR can be modified by its substrate GSNO, either by S-nitrosation, which has previously been reported, or, as discussed in this thesis, by S-glutathionylation. As S-glutathionylation has been reported to occur through intermediate species, the S-glutathionylation of GSNOR appears to occur though the S-nitrosated intermediate, instead of the most common route of an oxidation pathway. It is hypothesized that the S-glutathionylation, and the overall presence of glutathione, can act as a buffer to regulate the amount of nitrosation that GSNOR experiences, and thus the enzymatic activity. It is has reported that the S-nitrosation occurs on three different non-structural, non-catalytic, solvent-accessible cysteine residues. Experimentation was conducted using Saccharomyces cerevisiae as a model organism to determine how those three cysteine residues of the GSNOR homolog Sensitive to Formaldehyde 1 (SFA1) participate in the indirect detoxification of formaldehyde, through the hydroxymethyl glutathione pathway. It has been determined that cysteine 370 is not as important as previously thought, but the other one or two cysteines (either cysteine 10 or 271) do indeed play a role in the detoxification, but further analysis needs to be conducted.

Orientador: Ione Salgado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:34:18Z (GMT). No. of bitstreams: 1 Frungillo_Lucas_M.pdf: 2003193 bytes, checksum: 004553c47da2f38ee16eaf4e64b34cda (MD5) Previous issue date: 2011 / Resumo: Embora o radical óxido nítrico (NO) seja um importante sinalizador em plantas, pouco se conhece sobre os mecanismos que controlam sua homeostase na célula. Acreditase que a enzima S-nitrosoglutationa redutase (GSNOR) tenha um papel relevante no metabolismo de S-nitrosotióis (SNO), e consequentemente na homeostase do NO, através do catabolismo da S-nitrosoglutationa (GSNO). Apesar de a mitocôndria ser um importante alvo do NO, o papel da GSNOR na funcionalidade de mitocôndrias vegetais ainda não foi descrito. Este trabalho teve como objetivo caracterizar mitocôndrias isoladas a partir de cultura celular liquida de Arabidopsis thaliana transgênicas com maior (L1) e menor (L5) expressão da GSNOR em relação ao tipo selvagem. O conteúdo de S-nitrosotióis e peróxido de hidrogênio e a emissão de NO, determinados espectrofotometricamente e fluorimetricamente com DAF-2, respectivamente, foram comparados entre células nas fases de crescimento linear (5 dias de cultivo) e estacionária (10 dias de cultivo; estresse nutricional). O consumo de oxigênio e a degradação de NO por mitocôndrias isoladas nas diferentes fases de cultivo celular foram determinados com eletrodos específicos. Na fase linear o L1 apresentou menor (81%) e o L5 maior (162%) conteúdo de S-nitrosotióis, em relação ao tipo selvagem. Na fase estacionária o conteúdo de S-nitrosotióis foi reduzido e o padrão foi invertido. A emissão de NO pelas células após 5 dias de cultivo foi maior no L5 e não diferiu estatisticamente entre o L1 e o selvagem. Após 10 dias de cultivo os três genótipos apresentaram incremento na emissão de NO, porém o L5 apresentou menor emissão que os outros genótipos. Após 5 dias de cultivo microcalos dos transgênicos L1 e L5 apresentaram menor conteúdo de peróxido de hidrogênio que o tipo selvagem. Porém, em uma condição de estresse nutricional o conteúdo de peróxido de hidrogênio foi estatisticamente igual para todos os genótipos. Ensaios com mitocôndrias isoladas mostraram que o transgênico L1 foi o único incapaz de aumentar a atividade da oxidase alternativa (AOX) e teve as atividades do complexo I e da NADH desidrogenase externa inibidas na situação de estresse. O L5 apresentou maior atividade da NADH desidrogenase externa de modo constitutivo e da proteína desacopladora (UCP) no décimo dia. Ainda, na situação de estresse a capacidade de degradação de NO foi aumentada nos transgênicos L1 e L5. Entretanto, o L5 apresentou maior resistência à inibição da respiração provocada pelo NO, provavelmente devido a maior atividade da AOX. O conjunto dos resultados sugere um importante papel da GSNOR em controlar as alterações funcionais de mitocôndrias de A. thaliana mediadas por NO / Abstract: Although the radical nitric oxide (NO) is an important sign in plants, little is known about the mechanisms that control it's homeostasis in cell. It is believed that the enzyme Snitrosoglutathione reductase (GSNOR) has an important role in the metabolism of Snitrosothiols (SNO), and consequently of NO homeostasis through catabolism of Snitrosoglutathione (GSNO). Although mitochondria are an important target of NO, the role of GSNOR on plant mitochondria functionality has not been described yet. This study aimed to characterize mitochondria isolated from liquid cell culture of transgenic Arabidopsis thaliana with higher (L1) and lower (L5) GSNOR expression relative to wild type. The content of S-nitrosothiols and hydrogen peroxide and the NO emissions, determined spectrophotometrically and fluorimetric with DAF-2, respectively, were compared between cells in the linear (5 days culture) and stationary (10 days culture, nutritional stress) growth phases. Oxygen uptake and NO degradation by mitochondria isolated at different stages of cell culture were determined with specific electrodes. In the linear phase L1 showed lower (81%) and L5 increased (162%) content of S-nitrosothiols compared to wild type. At stationary phase S-nitrosothiols contents has been reduced and the pattern was reversed. The emission of NO by the cells after 5 days of culture was higher in L5 and do not statistically different between the L1 and wild type. At 10 days culture the genotypes showed an increase in the NO emission, but L5 showed lower emissions than the other genotypes. At 5 culture transgenic lines L1 and L5 showed a lower content of hydrogen peroxide than the wild type. However, in a condition of nutritional stress, the content of hydrogen peroxide was statistically the same for all genotypes. Tests with isolated mitochondria showed that transgenic L1 was the only one unable to increase the activity of alternative oxidase (AOX) and had the activities of complex I and NADH dehydrogenase at stress. The L5 showed a constitutive higher activity of the external NADH dehydrogenase and uncoupling protein (UCP) activity at the tenth day. Furthermore, NO degradation capability by mitochondria at nutritional stress situation of NO was increased in transgenic L1 and L5. However, L5 mitochondria showed greater resistance to respiration inhibition caused by NO, probably due to increased activity of AOX. The overall results suggest an important GSNOR role in controlling the mitochondria functional changes of A. thaliana mediated by NO / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Indiana University-Purdue University Indianapolis (IUPUI) / S-nitrosoglutathione reductase (GSNOR) also known as glutathione- dependent formaldehyde dehydrogenase (FDH), is a zinc-dependent dehydrogenase. GSNOR oxidizes long chain alcohols to an aldehyde with the help of a molecule of NAD+. GSNOR was initially identified as FDH because of its role in the formaldehyde detoxification pathway. The only S-nitrosothiol (SNO) substrate recognized by GSNOR is GSNO. A transnitrosation reaction transfers NO from nitrosylated proteins or S-nitrosothiols (RSNO) to glutathione to form S-nitrosoglutathione. This GSNO is finally converted to glutathione disulfide (GSSG) by a two step mechanism. Cellular GSNO is a nitric oxide reservoir that can either transfer to or remove from the proteins a NO group. Reduction of GSNO by GSNOR depletes this reservoir and therefore indirectly regulates protein nitrosylation. GSNOR inhibitors which can increase the basal GSNO levels will be another potential therapy. Several GSNOR inhibitors were identified in our laboratory and the aim of this study was to understand their cellular effects. One of the experiments studied the effect of the compound on protein-SNO. The role of nitric oxide in regulation of NF-κB pathway is reviewed by Bove and van der Vliet. We focused on identification of nitrosylated proteins using protein specific antibodies. We identified nitrosylation of IKKβ. So the question raised was whether nitrosylation of IKKβ affects its activity. IKKβ is responsible for phosphorylation of IκBα and phosphorylation of IκBα results in its degradation and activation of NF-κB pathway. Therefore, we studied the phosphorylation of IκBα in the presence of inhibitor C3. Results showed a dose-dependent decrease of pIκB. So the next question was whether the phosphorylation of IKKβ was affected by nitrosylation. We did not detect any change in pIKKβ with different concentrations of C3. The decreased degradation of IκBα caused by C3 translated into decreased NF-κB activity as seen by a dose-dependent decrease in amounts of ICAM-1 with increasing C3 concentration. This data supports the premise that the activity of transcription factor NF-κB is suppressed by inhibiting GSNOR with compound C3