Calorie restriction effects on the proteasome of yeast Saccharomyces cerevisiae

Objetivo: Investigar os efeitos da restrição calórica sobre as modificações pós-traducionais e conformação espacial do proteassomo 20S de Saccharomyces cerevisiae. Métodos: O proteassomo 20S foi purificado de células de Saccharomyces cerevisiae cultivadas por 7,5 h em meio YP suplementado com: 2% de glicose (condição controle) e 0,5% de glicose (restrição calórica). A purificação foi realizada por uma etapa de cromatografia de afinidade a níquel seguida de uma cromatografia de troca aniônica. A atividade do proteassomo foi estudada por método fluorimétrico. Para explorar as modificações pós-traducionais, o proteassomo 20S foi submetido a eletroforese bidimensional e espectrometria de massas. Para avaliar sua conformação espacial e estrutura, foram empregados microscopia eletrônica de transmissão, espalhamento de raios X a baixos ângulos e dicroísmo circular. Resultados: O proteassomo isolado de células caloricamente restritas tem maior atividade dos tipos quimiotripsina e tripsina, além de ser mais capaz de degradar a proteína β-caseína, quando comparado ao grupo controle. Pela análise por espectrometria de massas, observou-se que a restrição calórica induz fosforilação da treonina 55 ou serina 56 da subunidade α5 do proteassomo 20S. Modelagens in silico indicaram que a fosforilação de um desses resíduos aumentam a interação entre a proteína α-sinucleína e o anel α do proteassomo 20S. Os resultados dos experimentos de microscopia eletrônica de transmissão, espalhamento de raios X a baixos ângulos e dicroísmo circular não revelaram diferenças entres os grupos analisados. Conclusões: A restrição calórica aumenta a capacidade do proteassomo de degradar substratos modelo e proteínas inteiras. Além disso, induz uma modificação pós-traducional em ao menos um resíduo da subunidade α5 do proteassomo 20S que pode estar associada à sua maior capacidade proteolítica. Ainda que esses resultados possam estar associados a mudanças estruturais do proteassomo, não foi possível observar diferenças nesse contexto usando as técnicas apresentadas.Objective: To investigate the effects of calorie restriction on post-translational modifications and spatial conformation of the Saccharomyces cerevisiae 20S proteasome. Methods: The proteasome 20S was purified from Saccharomyces cerevisiae cells cultured for 7.5 hours in YP medium supplemented with: 2% glucose (control condition) and 0.5% glucose (caloric restriction; CR). Proteasome purification was performed by nickel affinity chromatography followed by anion exchange chromatography. Proteasome activity was studied by fluorimetry. To explore post-translational modifications, the 20S proteasome was subjected to two-dimensional electrophoresis and mass spectrometry. Proteasome structure and spatial conformation were analyzed by transmission electron microscopy, small angle X-ray scattering and circular dichroism. Results: The 20S proteasomes isolated from CR cells presented increased chymotrypsin- and trypsin-like activities and were more capable of degrading β-casein protein when compared to 20S proteasomes isolated from control cells. Mass spectrometry analysis revealed that CR induced the phosphorylation of threonine 55 or serine 56 on α5 proteasome subunit. In silico modelling indicated that CR-induced Thr 55 or Ser 56 phosphorylation facilitates the interaction of α-synuclein with the proteasome α ring. Those findings were not associated with significant alterations in proteasome spatial conformation since no difference was detected by transmission electron microscopy, small angle X-ray scattering or circular dichroism assays between the experimental groups. Conclusions: CR increases the ability of the yeast 20S proteasome to degrade model substrates and whole proteins. In addition, it induces a post-translational modification in at least one residue of the α5 subunit, which may be involved in the increased proteolysis capacity of CR proteasomes. These results were not found to be associated with structural changes in the 20S proteasome.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)2016/11724-82019/27044-

Aggregation limiting cell-penetrating peptides derived from protein signal sequences

Alzheimer’s disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-β (Aβ) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aβ. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aβ interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aβ aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aβ-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-72022/3056-