α-Synuclein Aggregation Is Triggered by Oligomeric Amyloid-ß 42 via Heterogeneous Primary Nucleation.

An increasing number of cases where amyloids of different proteins are found in the same patient are being reported. This observation complicates diagnosis and clinical intervention. Amyloids of the amyloid-ß peptide or the protein α-synuclein are traditionally considered hallmarks of Alzheimer's and Parkinson's diseases, respectively. However, the co-occurrence of amyloids of these proteins has also been reported in patients diagnosed with either disease. Here, we show that soluble species containing amyloid-ß can induce the aggregation of α-synuclein. Fibrils formed under these conditions are solely composed of α-synuclein to which amyloid-ß can be found associated but not as part of the core of the fibrils. Importantly, by global kinetic analysis, we found that the aggregation of α-synuclein under these conditions occurs via heterogeneous primary nucleation, triggered by soluble aggregates containing amyloid-ß.

N-Terminal Acetylation of α-Synuclein Slows down Its Aggregation Process and Alters the Morphology of the Resulting Aggregates.

Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibril-catalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein exhibit a lower ß-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates.

A Potent Sybody Selectively Inhibits α-Synuclein Amyloid Formation by Binding to the P1 Region.

Increasing research efforts focus on exploiting antibodies to inhibit the amyloid formation of neurodegenerative proteins. Nevertheless, it is challenging to discover antibodies that inhibit this process in a specific manner. Using ribosome display, we screened for synthetic single-domain antibodies, i.e., sybodies, of the P1 region of α-synuclein (residues 36-42), a protein that forms amyloid in Parkinson's disease and multiple-system atrophy. Hits were assessed for direct binding to a P1 peptide and the inhibition of amyloid formation. We discovered a sybody, named αSP1, that inhibits amyloid formation of α-synuclein at substoichiometric concentrations in a specific manner, even within highly crowded heterogeneous mixtures. Fluorescence resonance energy transfer-based binding assays and seeding experiments with and without αSP1 further demonstrate the importance of the P1 region for both primary and secondary nucleation mechanisms of amyloid assembly.

Effects of N-terminal Acetylation on the Aggregation of Disease-related α-synuclein Variants.

Mutations in the SNCA gene, which encodes the protein α-synuclein, have been linked with early onset Parkinson's disease. The exact nature of this association, however, is still poorly understood. To investigate this problem, we started from the observation that α-synuclein is constitutively N-terminally acetylated, a post-translational modification that alters the charge and structure of α-synuclein molecules and affects their interaction with lipid membranes, as well as their aggregation process. We thus studied five N-terminal acetylated familial variants (A30P, E46K, H50Q, G51D and A53T) of α-synuclein through a wide range of biophysical assays to probe the microscopic steps in their aggregation process and the structures of the resulting aggregates. Our results reveal a great complexity in the combined effects of the disease-related mutations with N-terminal acetylation on the aggregation of α-synuclein, which underscores the great sensitivity to even relatively small perturbations of the behaviour of this protein.

A Facile Method to Produce N-Terminally Truncated α-Synuclein.

α-Synuclein is a key protein of the nervous system, which regulates the release and recycling of neurotransmitters in the synapses. It is also involved in several neurodegenerative conditions, including Parkinson's disease and Multiple System Atrophy, where it forms toxic aggregates. The N-terminus of α-synuclein is of particular interest as it has been linked to both the physiological and pathological functions of the protein and undergoes post-translational modification. One such modification, N-terminal truncation, affects the aggregation propensity of the protein in vitro and is also found in aggregates from patients' brains. To date, our understanding of the role of this modification has been limited by the many challenges of introducing biologically relevant N-terminal truncations with no overhanging starting methionine. Here, we present a method to produce N-terminally truncated variants of α-synuclein that do not carry extra terminal residues. We show that our method can generate highly pure protein to facilitate the study of this modification and its role in physiology and disease. Thanks to this method, we have determined that the first six residues of α-synuclein play an important role in the formation of the amyloids.