Supramolecular Modulation of Structural Polymorphism in Pathogenic α‐Synuclein Fibrils Using Copper(II) Coordination

Abstract

Structural variation of α-synuclein (αSyn) fibrils has been linked to the diverse etiologies of synucleinopathies. However, little is known about what specific mechanism provides αSyn fibrils with pathologic features. Herein, we demonstrate Cu(II)-based supramolecular approach for unraveling the formation process of pathogenic αSyn fibrils and its application in a neurotoxic mechanism study. The conformation of αSyn monomer was strained by macrochelation with Cu(II), thereby disrupting the fibril elongation while promoting its nucleation. This non-canonical process formed shortened, β-sheet enriched αSyn fibrils (<0.2 μm) that were rapidly transmitted and accumulated to neuronal cells, causing neuronal cell death, in sharp contrast to typical αSyn fibrils (ca. 1 μm). Our approach provided the supramolecular basis for the formation of pathogenic fibrils through physiological factors, such as brain Cu(II).

Inactivation of USP14 Perturbs Ubiquitin Homeostasis and Delays the Cell Cycle in Mouse Embryonic Fibroblasts and in Fruit Fly Drosophila

“…The noncatalytic inhibition of the proteasome by USP14 is mediated by the direct interaction with the ATPase ring of the proteasome; this process results in strong interference with RPN11 function and with a conformational change of the proteasome for proper substrate translocation [16]. USP14 is also involved in the “gating” system which is controlled by various cellular factors including metal ions [17-19]. Therefore, mammalian proteasomes function under tonic inhibition by USP14, which expected to be crucial as a secondary quality control mechanism for intracellular proteins.…”

Acrolein and Copper as Competitive Effectors of α‐Synuclein

“…The spectro- scopic studies of Cu 2 + complexes with model peptides encompassing the binding sites of aSyn revealed that the modification by ACR decreases His affinity for Cu 2 + in species 1'.T his speciesm ay arise from either an intramolecular macro-chelate or Cu 2 + -bridged oligomers. [45] In both scenarios, the decreased affinity of species1' in ACR-modified aSyn might impact the metal-induced protein aggregation.I ndeed, it could influence the morphology of aSyn aggregates,a sH is50-dependent macrochelation has been shown to modulate aSyn aggregates polymorphism, [46] and it could interferew itht he formation of Cu 2 + -bridged oligomers. In this study,wes how that ACR modification of aSyn inhibits the protein aggregation process, both in the absence and presence of Cu 2 + ions.…”

“…Moreover, their formationw as not significantly affected by the ACR reaction, thus confirming the low percentage of aSyn modification. aSyn 46-58 carbonylated at the His residue is the only tryptic peptidew ithout missed cleavages whose formation significantly increases in at ime-dependent manner.T he chromatogram of the ACR-modified aSyn [46][47][48][49][50][51][52][53][54][55][56][57][58] shows two distinguished peaks (Table S2, R t = 14.73 and1 4.92 min), related to isobaric peptides (m/z 677.3724). The fragmentation pattern depicted by the MS/MS spectra of both the isomers ( Figure S3) confirmst hat, in both cases, the His residue is covalently linked to ACR.…”

“…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).…”

“…To evaluate whether the modificationo fH is influencest he Cu 2 + -binding, we resorted to short peptides that are considered suitable modelso faSyn Cu 2 + -binding sites. [10,14] Hence, we synthesized two minimal model peptides, aSyn 1-5 (NH 2 -MDVFM-NH 2 ), and Ac-aSyn [46][47][48][49][50] (Ac-EGVVH-NH 2 ), hereafter called P1 and P2, respectively,s ot hat species1 is modeled by Cu-P1, species2 by Cu-P2, and species 1' by the ternary complex Cu-P1-P2. Cu-P1 (species 1), Cu-P2 (species 2), and Cu-P1-P2 (species 1')s how CD and EPR signatures analogous to those reported in the literature (FigureS8), [14] making them suitable modelso faSyn Cu 2 + -binding sites.…”