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Abstract
Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.
Highlights
Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson’s disease
We find that endogenous cellular enzymes efficiently process modified Met[1] and Met[5] of a-Syn, whereas Met[116] and Met[127] remain oxidized
We used NMR spectroscopy to verify that complete oxidation of Met[1], Met[5], Met[116] and Met[127] did not alter the overall monomeric, disordered conformation of isolated a-Syn in vitro (Fig. 1c), which we independently confirmed using size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy and dynamic light scattering (DLS; Supplementary Fig. 1a–d)
Summary
Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson’s disease. We investigated the cross-peak positions of Ac-Met[1], Asp[2], Met[5], Lys[6], Leu[8] and Ser[9] in the in-cell NMR spectra of (U)-15N enriched a-Syn, which collectively serve as excellent indicators of the oxidation states of Met[1] and Met[5]
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