α-Synuclein Dimers as Potent Inhibitors of Fibrillization

Abstract

Aggregation of the neuronal protein α-synuclein into amyloid fibrils plays a central role in the development of Parkinson’s disease. Growth of fibrils can be suppressed by blocking fibril ends from their interaction with monomeric proteins. In this work, we constructed inhibitors that bind to the ends of α-synuclein amyloid fibrils with very high affinity. They are based on synthetic α-synuclein dimers and interact with fibrils via two monomeric subunits adopting conformation that efficiently blocks fibril elongation. By tuning the charge of dimers, we further enhanced the binding affinity and prepared a construct that inhibits fibril elongation at nanomolar concentration (IC50 ≈ 20 nM). To the best of our knowledge, it is the most efficient inhibitor of α-synuclein fibrillization.

Inhibitor and substrate cooperate to inhibit amyloid fibril elongation of α-synuclein

“…The WT-WT dimer was almost as inhibitory as CC48 alone, a result that is in agreement with what has been observed for similar constructs. 24,49 The CC48-CC48 dimer also exhibited strongly increased inhibition compared to CC48, which could be an avidity effect. As expected, the heterodimeric construct exhibited much less monomer dependency than what was observed for CC48 alone (Fig.…”

“…These inhibitors were based on different design principles, namely transport of steric bulk to the bril-end or direct linkage of two aS subunits at different positions within the aS sequence, and reached IC 50 values of 300 nM, 23,50 or 22 nM. 24 In one of these approaches, the function of a fused WT monomer is to serve as a bril-end-binding domain that brings the fused inhibitor domain close to the second protolament, with the inhibitor acting as steric bulk that impedes incorporation of further WT monomers. 23,50 While this approach is related to the current study with regard to the fusion of a WT monomer domain to an inhibitor domain, there are crucial differences: First, CC48 forms an inhibiting FI complex without requiring fusion to a WT monomer.…”

“…12 This is reminiscent of enzyme kinetics, and elongation can be treated as a two-step enzymatic reaction, in which bril-end and monomer serve as catalyst and substrate, respectively. [19][20][21] Proteins and peptides have been designed to specically inhibit the elongation of aS brils, [22][23][24][25] e.g., by aiming to dock complementary b-strands onto the open b-sheets at the brilends, to prevent the catalytic site from guiding the conformational conversion of further monomers. However, understanding how monomers and inhibitors get incorporated at bril-ends is still a subject of active research.…”

Acrolein and Copper as Competitive Effectors of α‐Synuclein

“…Teno ut of the fifteen Lysr esidues are involved in the ACR modification, althoughe videncef or the scale of reactivity cannot be drawn from the data. It is worth pointingo ut that the ACR-modified tryptic peptides aSyn [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58] and aSyn 46-60 have the same molecular weight and both encompass the His residue. MS/MS characterization of these peptides ( Figures S5 and S6) allowed the correct sequence to be assigned to each LC signal and also revealed that the carbonylated site wast he internal Lysr esidue (K45 and K58).…”

“…[21][22][23] These small oligomers could cause the inhibition of the fibrillation similar to other dimers of aSyn. [44] Instead, the addition of Cu 2 + to a aSyn solution instantly causedt he formation of as mall percentage of aggregates with am ean diameter of 44 AE 3nm( Figure S13). The size and relative population of small oligomers did not change significantly duringt he incubation.…”