Creation of Aggregation‐Defective α‐Synuclein Variants by Engineering the Sequence Connecting β‐Strand‐Forming Domains

Abstract

The aggregation of α-synuclein (αS), which is implicated in the pathology of Parkinson's disease, produces fibrils in which layers of parallel, in-register β-sheet-loop-β-sheets are formed. The effects of sequence variation in the loop-forming region (referred to as the linker region) on αS aggregation have yet to be systematically studied. In the study described here, we created and characterized αS variants containing mutations in the linker regions. Our results indicate that although the physicochemical properties of the linker region, evaluated based on an intrinsic property of a single amino acid, still play a significant role in aggregation, additional factors can also determine aggregation of αS linker mutants. Our analyses suggest that these factors include a pairwise potential for parallel in-register β-sheet formation. A linker variant displaying significantly reduced self-aggregation interfered with αS aggregation by inhibiting the conversion of αS soluble species to αS insoluble fibrils. We anticipate that linker mutations could serve as a novel method of creating αS variants that are aggregation-defective and/or inhibit αS aggregation.

Effects of Intramolecular Distance between Amyloidogenic Domains on Amyloid Aggregation

“…Previous biochemical and biophysical studies revealed several physicochemical factors determining aggregation [4–8]. However, our results demonstrate that an additional factor may as well determine amyloid aggregation behaviors.…”

“…Amyloid aggregation involves two major steps, early stage aggregation into soluble oligomers and their subsequent conversion to amyloid fibrils [1]. Significant efforts have been put into identifying the physicochemical factors important in amyloid aggregation in order to better understand the molecular basis of amyloid diseases [4–8]. These factors include hydrophobicity, secondary structure propensity and charge state of amino acids contained in sequences [4–8].…”

“…Significant efforts have been put into identifying the physicochemical factors important in amyloid aggregation in order to better understand the molecular basis of amyloid diseases [4–8]. These factors include hydrophobicity, secondary structure propensity and charge state of amino acids contained in sequences [4–8]. Amyloid aggregation is a generic property of peptides and proteins [9], and amyloid aggregation of proteins and peptides of different primary sequences share similar molecular mechanisms and aggregate morphologies [10].…”

“…For example, β-amyloid (Aβ) implicated in AD is composed of two hydrophobic amyloidogenic domains connected by a hydrophilic stretch of amino acids displaying a high propensity to form turn structures [7,16]. Similar placement of a turn-forming sequence between hydrophobic amyloidogenic domains is also found in α-synuclein (αS) implicated in PD [8]. The influences of mutations in amino acid sequences on amyloid aggregation have been extensively studied [4–8].…”

“…Similar placement of a turn-forming sequence between hydrophobic amyloidogenic domains is also found in α-synuclein (αS) implicated in PD [8]. The influences of mutations in amino acid sequences on amyloid aggregation have been extensively studied [4–8]. However, effects of intramolecular distance between amyloidogenic domains, which may be determined by the length of a linker region ( i.e.…”