| Oxidative stress and aggregation of the protein alpha-synuclein are thought to be key factors in Parkinson's disease. Previous work shows that cytochrome c plus H2O2 causes tyrosine-dependent in vitro peroxidative aggregation of proteins, including alpha-synuclein. Herein, I detail a method for monitoring alpha-synuclein and cytochrome c in a variety of experiments. Using this system, I examine the role of each of alpha-synuclein's four tyrosine residues and how the protein's conformation affects covalent oxidative aggregation. When alpha-synuclein adopts a collapsed conformation, tyrosine 39 is essential for wild-type-like covalent aggregation. This lone N-terminal tyrosine, however, is not required for wild type-like covalent aggregation in the presence of a denaturant or when alpha-synuclein is present in non-covalent fibrils. I also show that pre-formed oxidative aggregates are not incorporated into non-covalent fibrils. These data provide insight as to how dityrosine may be formed in Lewy bodies seen in Parkinson's disease. Additionally, I detail the progress made toward studying the toxicity of alpha-synuclein aggregates using neuronal microinjection. |
