Elucidating the Role of Site-Specific Nitration of alpha-Synuclein in the Pathogenesis of Parkinson's Disease via Protein Semisynthesis and Mutagenesis

Parkinsons disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of intraneuronal inclusions consisting of aggregated and post-translationally modified a-synuclein (a-syn). Despite advances in the chemical synthesis of a-syn and other proteins, the generation of site-specifically nitrated synthetic proteins has not been reported. Consequently, it has not been possible to determine the roles of nitration at specific residues in regulating the physiological and pathogenic properties of a-syn. Here we report, for the first time, the site-specific incorporation of 3-nitrotyrosine at different regions of a-syn using native chemical ligation combined with a novel desulfurization strategy. This strategy enabled us to investigate the role of nitration at single or multiple tyrosine residues in regulating a-syn structure, membrane binding, oligomerization, and fibrils formation. We demonstrate that different site-specifically nitrated a-syn species exhibit distinct structural and aggregation properties and exhibit reduced affinity to negatively charged vesicle membranes. We provide evidence that intermolecular interactions between the N- and C-terminal regions of a-syn play critical roles in mediating nitration-induced a-syn oligomerization. For example, when Y39 is not available for nitration (Y39F and Y39/125F), the extent of cross-linking is limited mostly to dimer formation, whereas mutants in which Y39 along with one or multiple C-terminal tyrosines (Y125F, Y133F, Y136F and Y133/136F) can still undergo nitration readily to form higher-order oligomers. Our semisynthetic strategy for generating site-specifically nitrated proteins opens up new possibilities for investigating the role of nitration in regulating protein structure and function in health and disease.

Published in:
Journal Of The American Chemical Society, 137, 15, 5041-5052
Washington, Amer Chemical Soc

 Record created 2015-05-29, last modified 2019-12-05

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