RESUMO
Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
Assuntos
Glutamato-Amônia Ligase , Ácido Peroxinitroso , Processamento de Proteína Pós-Traducional , Humanos , Cromatografia Líquida , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/farmacologia , Espectrometria de Massas em Tandem , Tirosina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Oxirredução , Mutação , Agregação Patológica de Proteínas/induzido quimicamenteRESUMO
Cell physiology is impaired before protein aggregation and this may be more relevant than inclusions themselves for neurodegeneration. The present study aimed to characterize an animal model to enable the analysis of the cell biology before and after protein aggregation. Ten-month-old Lewis rats were exposed either to 1 or 2 mg/kg/day of rotenone, delivered subcutaneously through mini-pumps, for one month. Hyperphosphorylated TAU, alpha-synuclein, amyloid-beta peptide and protein carbonylation (indicative of oxidative stress) were evaluated in the hippocampus, substantia nigra and locus coeruleus through immunohistochemistry or western blot. It was found that 2 mg/kg/day rotenone increased amyloid-beta peptide, hyperphosphorylation of TAU and alpha-synuclein. Rotenone at 1mg/kg/day did not alter protein levels. Protein carbonylation remained unchanged. This study demonstrated that aged Lewis rats exposed to a low dose of rotenone is a useful model to study cellular processes before protein aggregation, while the higher dose makes a good model to study the effects of protein inclusions.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Agregação Patológica de Proteínas/induzido quimicamente , Agregação Patológica de Proteínas/patologia , Rotenona/administração & dosagem , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Western Blotting , Sistema Nervoso Central/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Locus Cerúleo/efeitos dos fármacos , Locus Cerúleo/metabolismo , Locus Cerúleo/patologia , Masculino , Estresse Oxidativo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/metabolismo , Carbonilação Proteica , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/efeitos dos fármacos , alfa-Sinucleína/metabolismoRESUMO
ABSTRACT Cell physiology is impaired before protein aggregation and this may be more relevant than inclusions themselves for neurodegeneration. The present study aimed to characterize an animal model to enable the analysis of the cell biology before and after protein aggregation. Ten-month-old Lewis rats were exposed either to 1 or 2 mg/kg/day of rotenone, delivered subcutaneously through mini-pumps, for one month. Hyperphosphorylated TAU, alpha-synuclein, amyloid-beta peptide and protein carbonylation (indicative of oxidative stress) were evaluated in the hippocampus, substantia nigra and locus coeruleus through immunohistochemistry or western blot. It was found that 2 mg/kg/day rotenone increased amyloid-beta peptide, hyperphosphorylation of TAU and alpha-synuclein. Rotenone at 1mg/kg/day did not alter protein levels. Protein carbonylation remained unchanged. This study demonstrated that aged Lewis rats exposed to a low dose of rotenone is a useful model to study cellular processes before protein aggregation, while the higher dose makes a good model to study the effects of protein inclusions.
RESUMO A fisiologia celular está prejudicada antes da agregação proteica podendo ser mais importante para a neurodegeneração do que as próprias inclusões. Assim, o objetivo deste estudo é caracterizar um modelo animal para analisar os mecanismos e efeitos da agregação proteica. Ratos Lewis com 10 meses de idade foram expostos a rotenona (1 ou 2 mg/kg/dia), administrada subcutaneamente, utilizando minibombas osmóticas. Os níveis de peptídeo beta-amiloide, TAU hiperfosforilada, alfa-sinucleína e proteínas carboniladas (indicativo de estresse oxidativo) foram avaliados por imunohistoquímica e western blot no hipocampo, substância negra e locus coeruleus. Foi demonstrado que 2 mg/kg/dia de rotenona promoveu aumento do peptídeo beta-amiloide, hiperfosforilação da TAU e alfa-sinucleína. Já 1 mg/kg/dia de rotenona não alterou os níveis dessas proteína nessas regiões. As proteínas carboniladas não se alteraram. Foi demonstrado que ratos Lewis idosos expostos a baixas doses de rotenona são modelo de estudo dos processos celulares antes da agregação proteica, enquanto 2 mg/kg/dia de rotenona permite estudos sobre os efeitos da agregação proteica.