Parkinsons disease has long been known to involve the loss of dopaminergic neurons in the substantia nigra and the coincidental appearance of Lewy bodies containing oligomerized forms of alpha-synuclein. The catecholaldehyde hypothesis posits a causal link between these two central pathologies mediated by 3,4-dihydroxyphenylacetaldehyde (DOPAL), the most toxic dopamine metabolite. We have determined the structure of the dominant product formed in reactions between DOPAL and synuclein, a dicatechol pyrrole lysine adduct. This novel modification results from the addition of two DOPAL molecules to the Lys sidechain amine through their aldehyde moieties and the formation of a new carbon-carbon bond between their alkyl chains to generate a pyrrole ring. The product, dicatechol-pyrrole-lysine (DCPL), is the only detectable adduct at low concentrations of DOPAL, and therefore is likely to play a key role in DOPAL-induced crosslinking of alpha-synuclein. Our study of the chemistry that leads to cross-linking of DCPL-modified alpha-synuclein proteins showed that autoxidation of DCPL's catechol rings spurs its decomposition, yielding an intermediate dicatechol isoindole lysine (DCIL) product formed by an intramolecular reaction of the two catechol rings to give an unstable tetracyclic structure. DCIL then reacts with a second DCIL to give a dimeric, di-DCIL. This product is formed by an intermolecular carbon-carbon bond between the isoindole rings of the two DCILs that generates two structurally nonequivalent and separable atropisomers. Using alpha-synuclein, we demonstrated that the DOPAL-catalyzed formation of oligomers can be separated into two steps. The initial adduct formation occurs robustly within an hour, with DCPL as the main product, and the second step cross-links alpha-synuclein molecules. Exploiting this two-stage reaction, we used an isotopic labeling approach to show the predominant cross-linking mechanism is an interadduct reaction. We confirmed by mass spectrometry that a mass consistent with a di-DCIL linkage can be observed in a DOPAL-generated dimeric alpha-synuclein . Our work elucidates previously unknown pathways of catechol-based oxidative protein damage and should facilitate efforts to detect DOPAL-based cross-links in disease-state neurons.
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