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Danos em biomoléculas podem ocorrer a partir de uma interação direta entre as biomoléculas e espécies reativas de oxigênio e nitrogênio como também, pela reação de produtos secundários dessas espécies como eletrófilos gerados na peroxidação lipídica. Alguns desses produtos secundários possuem estabilidade química maior que as espécies reativas das quais foram derivadas e podem se ligar covalentemente as biomoléculas comprometendo o funcionamento normal das mesmas. Portanto, modificações em proteínas por aldeídos gerados na lipoperoxidação têm sido investigadas por suas implicações com desordens patológicas relacionadas à agregação proteica, e modificações em diversas vias de sinalização amplificando os efeitos deletérios em sistemas biológicos. Os objetivos desse trabalho foi contribuir na elucidação dos mecanismos moleculares associados ao desenvolvimento da esclerose lateral amiotrófica (ELA) através da identificação, caracterização e quantificação de modificações póstraducionais em proteínas pelos aldeídos 4-hidroxi-2-hexenal (HHE) e trans-4-hidroxi-2-nonenal (HNE) in vitro (citocromo c) e in vivo (modelo ELA) a partir de técnicas de Western blot, imunoprecipitação e espectrometria de massa com abordagem proteômica de \"shotgun\" em ratosSOD1G93A modelo de esclerose lateral amiotrófica (ELA). Estudos com citocromo c mostraram a ligação dos aldeídos ao citocromo c e mecanismos de reação foram propostos. Foram encontrados seis peptídeos modificados por HHE e um para o HNE, e o peptídeo TGPNLHGLFGR se mostrou modificado pelos dois aldeídos paralelamente. Foi demonstrado que a histidina 33 é um \"hot spot\" frente as adições pelos aldeídos. Nas análises por western blot das proteínas ligadas a aldeídos foi possível observar uma tendência de aumento na concentração de proteínas ligadas ao HNE nos animais ELA, mais acentuada nas amostras de 70 dias comparadas ao controle. Com relação aos resultados obtidos com HHE tanto os animais pré-sintomáticos quanto os sintomáticos não apresentaram diferenças de HHE-proteína, tantonos controles quanto nos animais ELA. Nas amostras dos animais sintomáticos não detectamos diferença significativa na concentração de aldeído-proteína entre os grupos. Já as análises proteômicas revelaram 24 proteínas diferencialmente expressas, com destaque para proteínas com os maiores valores de significância (p-value), como a ubiquitina no grupo dos pré- sintomáticos e a neurogranina, no grupo dos animais sintomáticos e várias proteínas de metabolismo energético, de neurofilamentos, proteínas de processos redox e proteínas ligadas o metabolismo de cálcio (fundamentais na fisiopatologia em ELA). Algumas proteínas importantes foram encontradas com exclusividades nos grupos pré-sintomáticos e sintomáticos pelo diagrama de Venn. Com relação a proteínas modificadas pelos aldeídos, foram encontradas algumas relevantes como a proteína 2 de interação com a polimerase delta que foi modificada por HNE via adição de Michael encontrada nos animais ELA pré-sintomáticos e sintomáticos, a catalase que foi encontrada modificada por HNE via base de Schiff apenas nos ELA pré- sintomáticos, e a tiol redutase induzível por interferon gama no grupo dos animais ELA sintomáticos. Com relação a proteínas modificadas por HHE, foram encontradas a Janus quinase e proteína 3 de interação com microtúbulo, modificadas tanto por adição de Michael quanto via base de Schiff nos animais ELA sintomáticos. É interessante ressaltar que algumas modificações encontradas em proteínas não caracterizadas podem indicar proteínas novas ainda não descritas como modificadas por esses aldeídos. Os resultados mostram que algumas das proteínas modificadas por HNE e HHE encontradas neste trabalho, estão relacionadas ao estresse redox, vias metabólicas energéticas, proteínas envolvidas na resposta a danos oxidativos, e processos inflamatórios. Tais modificações ocorrem não só no modelo de neurodegeneração, mas foram previamente descritas em outros processos patológicos, como doença cardiovascular, lesão hepática por uso crônico de álcool. / Damage to biomolecules can occur from a direct interaction between biomolecules and reactive of oxygen and nitrogen species as well as from the reaction of secondary products of these species as electrophiles generated in lipid peroxidation. Some of these by-products have greater chemical stability than the derived reactive species and can bind to biomolecules compromising their normal function. Therefore, protein modifications by aldehydes generated during lipoperoxidation have been investigated for their implications with pathological disorders related to protein aggregation and modifications in signaling pathways amplifying the deleterious effects in biological systems. The aim of this work was to contribute to the elucidation of the molecular mechanisms associated with the development of amyotrophic lateral sclerosis (ALS) through the identification, characterization and quantification of posttranslational modifications in proteins by 4-hydroxy-2-hexenal (HHE) and trans-4-hydroxy-2- nonenal (HNE) in vitro, cytochrome c, and in vivo, rat model (SOD1G93A) of amyotrophic lateral sclerosis (ALS), throught Western blot techniques, and mass spectrometry with shotgun proteomics approach. The results showed the binding of aldehydes to cytochrome c. Six peptides were modified by HHE and one by HNE. The peptide TGPNLHGLFGR was modified by the two aldehydes. Histidine 33 has been shown to be a hot spot against aldehydes additions. By western blot analysis of the aldehyde-bound proteins, it was possible to observe a tendency of increase in the concentration of HNE-bound proteins in the ALS animals, more pronounced in the samples of 70 days compared to control samples. Regarding the results obtained with HHE, both pre-symptomatic and symptomatic animals did not show HHE-protein differences, both in controls and in ALS animals. We did not detect a significant difference in the aldehyde-protein concentration between the groups in the samples of the symptomatic animals. Proteomic analysis revealed 24 differentially expressed proteins, with emphasis on proteins with thehighest values of significance (p-value), such as the ubiquitin in the pre-symptomatic group and neurogranin in the group of the symptomatic animals and several proteins of the energetic metabolism pathways, neurofilaments, proteins of redox processes and proteins linked to calcium metabolism (fundamental in the pathophysiology of ALS). Some important proteins were found exclusivity in the pre-symptomatic and symptomatic groups by the Venn diagram. With regard to aldehyde-modified proteins, some relevant ones such as Delta-2 polymerase interaction protein, that was modified by HNE via the addition of Michael found in presymptomatic and symptomatic ELA animals, catalase that was found to be modified by HNE via Schiff\'s base only in pre-symptomatic ALS, and gamma interferon-inducible thiol reductase in the group of symptomatic ALS animals. Janus kinase and microtubule interaction protein 3, were found to be modified by Michael addition and Schiff base pathway respectively in symptomatic ALS animals. It is interesting to note that some modifications found in uncharacterized proteins may indicate new proteins not yet described as modified by these aldehydes. The results show that some of the proteins modified by HNE and HHE found in this work are related to redox stress, energetic metabolic pathways, proteins involved in the response to oxidative damage, and inflammatory processes. Such modifications occur not only in the neurodegeneration model, but were previously described in other pathological processes, such as cardiovascular disease, liver injury due to chronic alcohol use

Danos em biomoléculas podem ocorrer a partir de uma interação direta entre as biomoléculas e espécies reativas de oxigênio e nitrogênio como também, pela reação de produtos secundários dessas espécies como eletrófilos gerados na peroxidação lipídica. Alguns desses produtos secundários possuem estabilidade química maior que as espécies reativas das quais foram derivadas e podem se ligar covalentemente as biomoléculas comprometendo o funcionamento normal das mesmas. Portanto, modificações em proteínas por aldeídos gerados na lipoperoxidação têm sido investigadas por suas implicações com desordens patológicas relacionadas à agregação proteica, e modificações em diversas vias de sinalização amplificando os efeitos deletérios em sistemas biológicos. Os objetivos desse trabalho foi contribuir na elucidação dos mecanismos moleculares associados ao desenvolvimento da esclerose lateral amiotrófica (ELA) através da identificação, caracterização e quantificação de modificações póstraducionais em proteínas pelos aldeídos 4-hidroxi-2-hexenal (HHE) e trans-4-hidroxi-2-nonenal (HNE) in vitro (citocromo c) e in vivo (modelo ELA) a partir de técnicas de Western blot, imunoprecipitação e espectrometria de massa com abordagem proteômica de \"shotgun\" em ratosSOD1G93A modelo de esclerose lateral amiotrófica (ELA). Estudos com citocromo c mostraram a ligação dos aldeídos ao citocromo c e mecanismos de reação foram propostos. Foram encontrados seis peptídeos modificados por HHE e um para o HNE, e o peptídeo TGPNLHGLFGR se mostrou modificado pelos dois aldeídos paralelamente. Foi demonstrado que a histidina 33 é um \"hot spot\" frente as adições pelos aldeídos. Nas análises por western blot das proteínas ligadas a aldeídos foi possível observar uma tendência de aumento na concentração de proteínas ligadas ao HNE nos animais ELA, mais acentuada nas amostras de 70 dias comparadas ao controle. Com relação aos resultados obtidos com HHE tanto os animais pré-sintomáticos quanto os sintomáticos não apresentaram diferenças de HHE-proteína, tantonos controles quanto nos animais ELA. Nas amostras dos animais sintomáticos não detectamos diferença significativa na concentração de aldeído-proteína entre os grupos. Já as análises proteômicas revelaram 24 proteínas diferencialmente expressas, com destaque para proteínas com os maiores valores de significância (p-value), como a ubiquitina no grupo dos pré- sintomáticos e a neurogranina, no grupo dos animais sintomáticos e várias proteínas de metabolismo energético, de neurofilamentos, proteínas de processos redox e proteínas ligadas o metabolismo de cálcio (fundamentais na fisiopatologia em ELA). Algumas proteínas importantes foram encontradas com exclusividades nos grupos pré-sintomáticos e sintomáticos pelo diagrama de Venn. Com relação a proteínas modificadas pelos aldeídos, foram encontradas algumas relevantes como a proteína 2 de interação com a polimerase delta que foi modificada por HNE via adição de Michael encontrada nos animais ELA pré-sintomáticos e sintomáticos, a catalase que foi encontrada modificada por HNE via base de Schiff apenas nos ELA pré- sintomáticos, e a tiol redutase induzível por interferon gama no grupo dos animais ELA sintomáticos. Com relação a proteínas modificadas por HHE, foram encontradas a Janus quinase e proteína 3 de interação com microtúbulo, modificadas tanto por adição de Michael quanto via base de Schiff nos animais ELA sintomáticos. É interessante ressaltar que algumas modificações encontradas em proteínas não caracterizadas podem indicar proteínas novas ainda não descritas como modificadas por esses aldeídos. Os resultados mostram que algumas das proteínas modificadas por HNE e HHE encontradas neste trabalho, estão relacionadas ao estresse redox, vias metabólicas energéticas, proteínas envolvidas na resposta a danos oxidativos, e processos inflamatórios. Tais modificações ocorrem não só no modelo de neurodegeneração, mas foram previamente descritas em outros processos patológicos, como doença cardiovascular, lesão hepática por uso crônico de álcool. / Damage to biomolecules can occur from a direct interaction between biomolecules and reactive of oxygen and nitrogen species as well as from the reaction of secondary products of these species as electrophiles generated in lipid peroxidation. Some of these by-products have greater chemical stability than the derived reactive species and can bind to biomolecules compromising their normal function. Therefore, protein modifications by aldehydes generated during lipoperoxidation have been investigated for their implications with pathological disorders related to protein aggregation and modifications in signaling pathways amplifying the deleterious effects in biological systems. The aim of this work was to contribute to the elucidation of the molecular mechanisms associated with the development of amyotrophic lateral sclerosis (ALS) through the identification, characterization and quantification of posttranslational modifications in proteins by 4-hydroxy-2-hexenal (HHE) and trans-4-hydroxy-2- nonenal (HNE) in vitro, cytochrome c, and in vivo, rat model (SOD1G93A) of amyotrophic lateral sclerosis (ALS), throught Western blot techniques, and mass spectrometry with shotgun proteomics approach. The results showed the binding of aldehydes to cytochrome c. Six peptides were modified by HHE and one by HNE. The peptide TGPNLHGLFGR was modified by the two aldehydes. Histidine 33 has been shown to be a hot spot against aldehydes additions. By western blot analysis of the aldehyde-bound proteins, it was possible to observe a tendency of increase in the concentration of HNE-bound proteins in the ALS animals, more pronounced in the samples of 70 days compared to control samples. Regarding the results obtained with HHE, both pre-symptomatic and symptomatic animals did not show HHE-protein differences, both in controls and in ALS animals. We did not detect a significant difference in the aldehyde-protein concentration between the groups in the samples of the symptomatic animals. Proteomic analysis revealed 24 differentially expressed proteins, with emphasis on proteins with thehighest values of significance (p-value), such as the ubiquitin in the pre-symptomatic group and neurogranin in the group of the symptomatic animals and several proteins of the energetic metabolism pathways, neurofilaments, proteins of redox processes and proteins linked to calcium metabolism (fundamental in the pathophysiology of ALS). Some important proteins were found exclusivity in the pre-symptomatic and symptomatic groups by the Venn diagram. With regard to aldehyde-modified proteins, some relevant ones such as Delta-2 polymerase interaction protein, that was modified by HNE via the addition of Michael found in presymptomatic and symptomatic ELA animals, catalase that was found to be modified by HNE via Schiff\'s base only in pre-symptomatic ALS, and gamma interferon-inducible thiol reductase in the group of symptomatic ALS animals. Janus kinase and microtubule interaction protein 3, were found to be modified by Michael addition and Schiff base pathway respectively in symptomatic ALS animals. It is interesting to note that some modifications found in uncharacterized proteins may indicate new proteins not yet described as modified by these aldehydes. The results show that some of the proteins modified by HNE and HHE found in this work are related to redox stress, energetic metabolic pathways, proteins involved in the response to oxidative damage, and inflammatory processes. Such modifications occur not only in the neurodegeneration model, but were previously described in other pathological processes, such as cardiovascular disease, liver injury due to chronic alcohol use

Activation of mammalian stress responsive pathways by plant secondary metabolites may contribute to the protection against certain chronic diseases afforded by fruit and vegetable consumption. This work focuses on the identification of plant compounds that activate the stress-responsive enzyme quinone reductase (QR) by stabilizing the transcription factor NF-E2 related factor-2 (Nrf2). Screening methanolic extracts of plants from Western Canada for QR induction in a mouse hepatoma cell line (Hepa-1c1c7) led to the identification of twenty-one extracts capable of doubling the activity of QR. Bioassay-guided fractionation of six extracts led to the identification of novel classes of compounds with QR-inducing activity including fatty-acid derived polyacetylenes, phthalides, and cannabinoids. Studies using low molecular weight thiols and the recombinantly expressed protein Keap1, the principal negative regulator of Nrf2, supported a mechanism of QR activation involving covalent modification of Keap1 cysteines for the polyacetylenes and phthalides. Analysis of transcriptional changes in response to treatment with a panel of QR-inducing compounds provided strong support for Nrf2 activation by the polyacetylene (3S,8S)-falcarindiol and the isothiocyanate (R)-sulforaphane and weaker support for the compounds (3R,8S)-falcarindiol, 6-isovaleryl-umbelliferone (6-IVU) and (Z)-ligustilide. Additionally, transcript level analyses supported a role for the aryl-hydrocarbon receptor in QR-activation by (3R,8S)-falcarindiol, (Z)-ligustilide, (R)-sulforaphane, 6-IVU and cannabidiol and suggested that treatment with polyacetylenes with a (3R)-configuration, (Z)-ligustilide and 6-IVU causes substantial changes in the expression of genes associated with lipid homeostasis and energy metabolism. As a whole, this work provides evidence that compounds that activate QR (and Nrf2) are widely distributed in the Canadian flora. However, of these QR activators, few are active at concentrations that are expected to be achieved through dietary consumption. Nevertheless, the most exceptional compounds isolated in this work, the compounds (3S,8S)-falcarindiol and epoxyfalcarindiol are highly potent and appear to be or are expected to be specific for activating Nrf2 and thus warrant attention with respect to dietary implications and as drug candidate leads.

The available antiretroviral compounds can effectively suppress the replication of HIV-1 and block the disease progression. However it is impossible to eradicate the virus from the organism as the HIV-1 integrated in the genome is not affected by the existing anti-HIV-1 drugs. Therefore, new latency reversing agents are being actively developed as part of "shock and kill" therapy to reactivate the provirus and clear the reservoir. Normosang (heme arginate; HA) is a human hemin- containing compound used to treat acute porphyria. Heme is physiologically catabolised by heme oxygenases to form iron (Fe2+ ), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. In this study, we have demonstrated that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA synergised with phorbol myristyl acetate (PMA) and reactivated the HIV-1 provirus in ACH-2 cells and the HIV-1 "mini-virus" in Jurkat cell clones A2 and H12. HIV-1 ''mini-virus'' was reactivated also by HA-alone. Further, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually. We employed addition of ascorbate to generate Fe2+ , resulting in an increased...

The available antiretroviral compounds can effectively suppress the replication of HIV-1 and block the disease progression. However it is impossible to eradicate the virus from the organism as the HIV-1 integrated in the genome is not affected by the existing anti-HIV-1 drugs. Therefore, new latency reversing agents are being actively developed as part of "shock and kill" therapy to reactivate the provirus and clear the reservoir. Normosang (heme arginate; HA) is a human hemin- containing compound used to treat acute porphyria. Heme is physiologically catabolised by heme oxygenases to form iron (Fe2+ ), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. In this study, we have demonstrated that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA synergised with phorbol myristyl acetate (PMA) and reactivated the HIV-1 provirus in ACH-2 cells and the HIV-1 "mini-virus" in Jurkat cell clones A2 and H12. HIV-1 ''mini-virus'' was reactivated also by HA-alone. Further, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually. We employed addition of ascorbate to generate Fe2+ , resulting in an increased...

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