Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

Abstract

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

Crucial role of protein oligomerization in the pathogenesis of Alzheimer's and Parkinson's diseases

“…Overproduction of ROS has been well established in neurodegenerative diseases [80]. In terms of oligomeric-mediated toxicity, there has been accumulating data showing oligomeric species of aggregating proteins produce high levels of ROS [65,81]. Acute application of recombinant b-sheet-rich oligomers dramatically induced higher production of ROS in neuron and astrocytes co-cultures, while monomers, unstructured oligomers, and insoluble fibrils did not [4,23,82,83].…”

N-terminal ubiquitination of amyloidogenic proteins triggers removal of their oligomers by the proteasome holoenzyme

“…As tight assembly and packing are key features of amyloid aggregates, presence of a Ub moiety at the N-terminus could potentially induce soluble oligomers into a conformation distinct from unmodified protein aggregates. In another of our previous work, we found that arachidonic acid could induce a conformational change in soluble α S oligomers that could subsequently be targeted by the proteasome [12], suggesting that the proteasome acted more effectively on modified than unmodified oligomers, which contained more compact structures.…”

“…Our previous work using the same confocal single-molecule technique found that the proteasome did not target oligomers that are not modified with Ub [12] and that these oligomers are not affected by active chaperones [33]. We therefore attribute the observed reduction in aggregate level specifically to the proteasome.…”

“…To study the degradation of Ub-modified amyloidogenic proteins, we purified proteasome holoenzyme from an established mammalian cell line [32] It has been reported that non-ubiquitinated aggregates resist degradation [12][13][14][15], and that tau and α S oligomers have recently been shown to impede proteasome activity [18]. In addition, we recently demonstrated that the product of proteasome-7 catalyzed fragmenting of tau and α S fibrils was small aggregate entities, supporting our previous report that proteasomes had no effect on soluble α S oligomers without ubiquitin modification [39].…”

“…Fluorescent protein samples were diluted to 100 pM (Ub-αS) or 40 pM (Ub-tau K18 ) final concentration and measured under flow according to previously established methods [30,38,39]. The bin time and flow rates for Ub-αS and Ub-tau K18 constructs were individually optimized to achieve the highest value of Q as described previously [12,30,38]. Ub-αS aggregates were measured at 100 µl/hr flow rate and the fluorescence signals were collected with 0. measurements is shown in Figure 3d and e.…”