Publicação
Production and Characterization of Recombinant AlphaSynuclein Protein Species
| Resumo: | Parkinson’s disease (PD) is the second-most common neurodegenerative disorder, affecting 2–3% of the population aged over 65. Its pathological hallmarks are dopaminergic neuron loss and accumulation of Lewy bodies, intracellular proteinaceous aggregates containing alpha-synuclein (aSyn). Familial PD, caused by genetic mutations, constitutes only 5-10% of all cases, but the study of genes associated with familial forms provides important insight into disease mechanisms, also relevant for the idiopathic forms. aSyn can exhibit a range of conformations. Encompassing various species from monomers to fibrils, aSyn is also subject to posttranslational modifications and point mutations causative of familial PD. The E46K aSyn variant, found in familial PD cases, consistently demonstrates an increased aggregation propensity. Recently, our group was involved in the discovery of two novel aSyn mutations in PD patients, K58N and G14R, whose effect on aggregation is still poorly understood. In this context, this project focused on a comparative study of different forms of aSyn: recombinant human WT and mouse WT aSyn, as well as human aSyn carrying the K58N, G14R or E46K mutations. Using ThT fluorescence assays, gel electrophoresis, and microscopy, it was concluded that the mutant variants have a higher propensity for aggregation. In parallel, it was also investigated the impact of glycation, a non-enzymatic reaction between a sugar and a protein, on aSyn aggregation. Methylglyoxal and ribose were found to glycate aSyn, causing significant biochemical alterations. Mutant aSyn fibril formation was increased by glycation, yet it was reduced for WT aSyn, aligning with previous results from our group. Interestingly, aggregates formed with or without glycation seed endogenous aSyn aggregation, leading to the formation of similar types of inclusions upon microscopic evaluation. Future studies will benefit from using a combination of biochemical techniques, considering the inherent variability in the behaviour of aSyn, and a larger sample size to improve statistical significance. |
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| Autores principais: | Quadros Ramón, Maria Ángel de |
| Assunto: | alfa-sinucleína doença de Parkinson neurodegeneração agregação de proteínas glicação Teses de mestrado - 2023 |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Parkinson’s disease (PD) is the second-most common neurodegenerative disorder, affecting 2–3% of the population aged over 65. Its pathological hallmarks are dopaminergic neuron loss and accumulation of Lewy bodies, intracellular proteinaceous aggregates containing alpha-synuclein (aSyn). Familial PD, caused by genetic mutations, constitutes only 5-10% of all cases, but the study of genes associated with familial forms provides important insight into disease mechanisms, also relevant for the idiopathic forms. aSyn can exhibit a range of conformations. Encompassing various species from monomers to fibrils, aSyn is also subject to posttranslational modifications and point mutations causative of familial PD. The E46K aSyn variant, found in familial PD cases, consistently demonstrates an increased aggregation propensity. Recently, our group was involved in the discovery of two novel aSyn mutations in PD patients, K58N and G14R, whose effect on aggregation is still poorly understood. In this context, this project focused on a comparative study of different forms of aSyn: recombinant human WT and mouse WT aSyn, as well as human aSyn carrying the K58N, G14R or E46K mutations. Using ThT fluorescence assays, gel electrophoresis, and microscopy, it was concluded that the mutant variants have a higher propensity for aggregation. In parallel, it was also investigated the impact of glycation, a non-enzymatic reaction between a sugar and a protein, on aSyn aggregation. Methylglyoxal and ribose were found to glycate aSyn, causing significant biochemical alterations. Mutant aSyn fibril formation was increased by glycation, yet it was reduced for WT aSyn, aligning with previous results from our group. Interestingly, aggregates formed with or without glycation seed endogenous aSyn aggregation, leading to the formation of similar types of inclusions upon microscopic evaluation. Future studies will benefit from using a combination of biochemical techniques, considering the inherent variability in the behaviour of aSyn, and a larger sample size to improve statistical significance. |
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