UV-induced selective oxidation of Met5 to Met-sulfoxide leads to the formation of neurotoxic fibril-incompetent α-synuclein oligomers

Abstract

Oxidative stress and the formation of cytotoxic aggregates of the presynaptic protein α-synuclein (AS) are two important events associated with the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative diseases. In this context, extensive efforts have been made to elucidate the molecular basis of the cytotoxic synergy between oxidative stress and AS aggregation. In this study, we demonstrate that the exposure of AS to oxidative stress induced by UV radiation (ASUV) blocks the protein fibrillation, leading to the formation of highly toxic fibril-incompetent oligomers. In addition, ASUV exhibited stronger anti-fibrillogenic properties than H2O2-treated AS, inhibiting the fibrillation of unmodified AS at notably low concentrations. Mass spectrometry indicated that Met5 oxidation to Met-sulfoxide was the only modification promoted by UV exposure, which is reinforced by NMR data indicating that Met5 is the only residue whose amide resonance completely disappeared from the (1)H-(15)N HSQC spectrum after UV exposure. This result is supported by previous data that indicate that C-terminal Met residues (Met116 and Met127) and N-terminal Met1 are less susceptible to oxidation than Met5 because of the residual structure of the disordered AS monomer. Overall, our findings suggest that specific oxidation of Met5 might be sufficient to promote the formation of highly neurotoxic oligomers of AS.

Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL)

“…Fragments b 2 ϩ and y 2 ϩ present in the MS/MS spectrum confirm that both Met-1 and Met-5 are oxidized to sulfoxide in this peptide. NMR experiments indicate that Met-5 is more susceptible to DOPAL-induced oxidation than the C-terminal methionines (Met-116 and Met-127), consistent with previous data that indicate that the C-terminal Met residues (Met-116 and Met-127) are protected by a residual structure of the disordered aS monomer (35,36). This is corroborated by our MS analysis, which indicates the co-existence of non-oxidized and oxidized forms of Met-116 and Met-127, with variable proportions depending on the sample preparation (data not shown).…”

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites

“…Accordingly, intracellular α-Syn aggregates often carry different types of oxidative modifications, such as nitrated tyrosines 45 , di-tyrosines 46 and oxidized methionines 9 . Several in vitro studies further strengthened the notion that methionine oxidation promotes the formation of cytotoxic α-Syn oligomers 17 18 19 . How these oxidative modifications eventually accumulate in α-Syn amyloid fibrils and Lewy bodies remains unknown.…”

“…Given that C-terminal α-Syn sulfoxides are stably preserved in cells, they likely contribute to the accumulation of permanently altered protein species with possible roles in neurotoxicity 17 18 19 . Such effects may be mediated by aberrant interactions with proteins that selectively bind to the C-terminus of α-Syn, such as synaptobrevin 56 and Rab8 (ref.…”

“…In the presence of H 2 O 2 , all four α-Syn methionines are readily converted into sulfoxides 16 . In turn, methionine oxidation triggers the formation of intermediate oligomer species that display different degrees of cytotoxicity 17 18 19 . In PC12 cells, and in cultured primary dopaminergic neurons, exposure to the electron transport chain inhibitor Rotenone 20 results in α-Syn methionine oxidation and the formation of intracellular inclusions 6 21 .…”

Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

“…Classically, the on-pathway oligomers are presumed to be early stage assemblies that nucleate and mature into bsheet rich fibrils, whereas off-pathway oligomers do not feed into the fibrillogenesis cascade. Within the last few years, the importance of targeting oligomers as the pathological species has become forefront in the alphasynucleinopathy research field 9,[62][63][64][65][66] with increasing evidence that both on-and off-pathway oligomers are a heterogenous ensemble of oligomers comprised both toxic and non-toxic species.…”