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Structural features and environmental conditions regulating α-synuclein aggregation


Hoyer,  W.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Hoyer, W. (2004). Structural features and environmental conditions regulating α-synuclein aggregation. PhD Thesis, Westfälische Wilhelms-Universität, Münster, Germany.

Cite as: http://hdl.handle.net/11858/00-001M-0000-0012-EC89-F
Misfolding and self-association of peptides and proteins resulting in aggregates denoted as amyloid has been implicated in various diseases, including Alzheimer’s and Parkinson’s diseases and the spongiform encephalopathies. Amyloid aggregates are comprised of protein fibrils, typically ~10 nm in width, that exhibit an extended cross-β structure. The major component of the neuronal inclusions linked to Parkinson’s disease (PD) is the 140 amino acid protein a-synuclein. Apart from its importance for the pathogenesis of PD and other neurodegenerative disorders (synucleinopathies), α-synuclein is interesting from a structural perspective as it is natively unfolded in its monomeric state, but can adopt α-helical or β-sheet secondary structure upon membrane binding or aggregation, respectively. The mechanism of α−synuclein aggregation is incompletely understood. In this work, the monomeric state of α-synuclein and its interconversion into amyloid aggregates were investigated by atomic force microscopy (AFM), electron microscopy (EM), fluorescent dye binding (Thioflavin T, ANS), static light scattering, turbidity, circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR), in order to identify structural features and environmental conditions modulating the aggregation process. Alpha-synuclein aggregation was greatly facilitated by biogenic polyamines, metal ions, double-stranded DNA (dsDNA), negatively charged mica surfaces, pH decrease and C-terminal truncation. The complexes of the monomeric protein with several polyamines were characterized by NMR, leading to the identification of the polyamine binding site as the acidic C-terminal region comprising amino acids 109-140 (aa109-140). Greater molecular charge of the polyamine correlated with greater affinity and enhancement of fibrillation of full-length α-synuclein. The kinetic data was analyzed with a simple model involving a dimeric nucleation center, yielding an increase in the extent of nucleation by 103 and in the rate of addition to existing fibrils by a factor of ~40 for the polyamine bound state. Similar to polyamines, metal ions and pH decrease promoted α-synuclein aggregation as a consequence of their interaction with aa109-140. Alpha-synuclein aggregates exhibited various morphologies. Individual, long fibrils prevailed at neutral pH in the absence of additions, whereas large, amorphous structures were formed at low pH or in the presence of polyamines or metal ions. Fibrillar structures were intermediates on the pathway to amorphous aggregates, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. We are able to conclude that the aggregate polymorphism is caused by the C-terminal region aa109-140, which is located on the fibril surface and thus regulates the surface characteristics, e.g. its charge state. In addition to fibrillation and amorphous aggregation, formation of ~1 nm high substrate-oriented sheet-like structures, possibly representing individual b-sheets, and ~6 nm high spheroids, reminiscent of soluble oligomers implicated in PD pathogenesis, could be observed directly by in situ AFM. Spheroid formation was promoted by polyamines, whereas sheet formation preferentially occurred on negatively charged mica substrates. The capability of α-synuclein to self-assemble on negatively charged surfaces was further evidenced by the association of α-synuclein fibrils with dsDNA, which apparently serves as a template for fibril growth. The variability of aggregation kinetics and morphology observed in this study points to a critical impact of the structural plasticity inherent to the natively unfolded protein for α-synuclein self-assembly. The charge state of the C-terminus is a major factor determining the rate and morphology of aggregation, although it does not regulate secondary structure formation in the monomeric protein. The accessibility of the pathways leading to either fast, amorphous aggregation or slow formation of individual fibrils can be controlled by adjusting the extent of screening of negative charges within aa109-140. The C-terminal charge state also affects the formation of spheroidal oligomers. However, further work is required to elucidate the structural and thermodynamic characteristics of these and other early stage intermediates and their role in the aggregation process. The great impact of the C-terminal region aa109-140 on the protein properties, structures and kinetics at all levels of the aggregation process is particularly remarkable inasmuch as it is not incorporated into the aggregate core, but remains on the fibril surface. We hypothesize that there is a regulatory interaction between the C-terminus and the amyloidogenic region. The strong dependence of the aggregation process on solution conditions in vitro suggests a similar sensitivity to slight variations in the intracellular milieu, presumably affecting α-synuclein cytotoxicity. Cellular polyamines, dsDNA and negatively charged surfaces (in vivo represented for example by anionic membrane phospholipid surfaces) significantly promote aggregation, implying possible roles in the pathogenesis of synucleinopathies.