The aggregation of alpha-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson’s disease. Amyloid fibrils of alpha-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of alpha-synuclein. During this process, alpha-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of alpha-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of alpha-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1-104 and 29-140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo alpha-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the alpha-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with alpha-synuclein fibrils. This study demonstrates that although alpha-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.
Covalent alfa-synuclein dimers: chemico-physical and aggregation properties
PIVATO, MICAELA;DE FRANCESCHI, GIORGIA;TESSARI, ISABELLA;BISAGLIA, MARCO;POLVERINO DE LAURETO, PATRIZIA;BUBACCO, LUIGI
2012
Abstract
The aggregation of alpha-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson’s disease. Amyloid fibrils of alpha-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of alpha-synuclein. During this process, alpha-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of alpha-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of alpha-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1-104 and 29-140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo alpha-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the alpha-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with alpha-synuclein fibrils. This study demonstrates that although alpha-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.
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