A simple algorithm locates beta-strands in the amyloid fibril core of alpha-synuclein, Abeta, and tau using the amino acid sequence alone.

Fibrillar inclusions are a characteristic feature of the neuropathology found in the a-synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Familial forms of a-synucleinopathies have also been linked with missense mutations or gene multiplications that result in higher protein expression levels. In order to form these fibrils, the protein, a-synuclein (a-syn), must undergo a process of self-assembly in which its native state is converted from a disordered conformer into a ß-sheet-dominated form. Here, we have developed a novel polypeptide property calculator to locate and quantify relative propensities for ß-strand structure in the sequence of a-syn. The output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of the ß-sheet core in a-syn fibrils. In particular, the plot features three peaks, the largest of which is completely absent for the nonfibrillogenic protein, ß-syn. We also report similar significant correlations for the Alzheimer's disease-related proteins, Aß and tau. A substantial region of a-syn is also of converting from its disordered conformation into a long amphipathic a-helical protein. We have developed the aforementioned algorithm to locate and quantify the a-helical hydrophobic moment in the amino acid sequence of a-syn. As before, the output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of a-helical structure in membrane bilayer-associated a-syn

Synuclein Proteins of the Pufferfish Fugu rubripes: Sequences and Functional Characterization

In humans, three genes encode the related a-, ß-, and ¿-synucleins, which function as lipid-binding proteins in vitro. They are being widely studied, mainly because of the central involvement of a-synuclein in a number of neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In these diseases, the normally soluble a-synuclein assembles into abnormal filaments. Here, we have identified and characterized the synuclein gene family from the pufferfish Fugu rubripes. It consists of four genes, which encode a-, ß-, ¿1-, and ¿2-synucleins. They range from 113 to 127 amino acids in length and share many of the characteristics of human synucleins, including the presence of imperfect amino-terminal repeats of 11 amino acids, a hydrophobic middle region, and a negatively charged carboxy-terminus. All four synucleins are expressed in the Fugu brain. Recombinant Fugu synucleins exhibited differential liposome binding, which was strongest for a-synuclein, followed by ß-, ¿2-, and ¿1-synucleins. In assembly experiments, Fugu a-, ¿1-, and ¿2-synucleins formed filaments more readily than human a-synuclein. Fugu ß-synuclein, by contrast, failed to assemble in bulk. Filament assembly of synucleins was directly proportional to their degree of hydrophobicity and their tendency to form ß-sheet structure, and correlated inversely with their net charge

The involvement of dityrosine crosslinking in a-synuclein assembly and deposition in Lewy Bodies in Parkinson’s disease

Parkinson’s disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable a-synuclein (a-syn) amyloid fibrils. a-synuclein is an intrinsically disordered protein that has the capacity to assemble to form ß-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in a-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of a-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of a-syn in PD

Synuclein Proteins of the Pufferfish <i>Fugu rubripes</i>: Sequences and Functional Characterization^†

In humans, three genes encode the related α-, β-, and γ-synucleins, which function as lipid-binding proteins in vitro. They are being widely studied, mainly because of the central involvement of α-synuclein in a number of neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In these diseases, the normally soluble α-synuclein assembles into abnormal filaments. Here, we have identified and characterized the synuclein gene family from the pufferfish Fugu rubripes. It consists of four genes, which encode α-, β-, γ1-, and γ2-synucleins. They range from 113 to 127 amino acids in length and share many of the characteristics of human synucleins, including the presence of imperfect amino-terminal repeats of 11 amino acids, a hydrophobic middle region, and a negatively charged carboxy-terminus. All four synucleins are expressed in the Fugu brain. Recombinant Fugu synucleins exhibited differential liposome binding, which was strongest for α-synuclein, followed by β-, γ2-, and γ1-synucleins. In assembly experiments, Fugu α-, γ1-, and γ2-synucleins formed filaments more readily than human α-synuclein. Fugu β-synuclein, by contrast, failed to assemble in bulk. Filament assembly of synucleins was directly proportional to their degree of hydrophobicity and their tendency to form β-sheet structure, and correlated inversely with their net charge

The involvement of dityrosine crosslinking in a-synuclein assembly and deposition in Lewy Bodies in Parkinson’s disease

Parkinson’s disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable a-synuclein (a-syn) amyloid fibrils. a-synuclein is an intrinsically disordered protein that has the capacity to assemble to form ß-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in a-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of a-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of a-syn in PD