The involvement of dityrosine crosslinking in α-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 α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically disordered protein that has the capacity to assemble to form β-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 α-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 α-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of α-syn in PD

Sequence Determinants for Amyloid Fibrillogenesis of Human a-Synuclein

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the presence of filamentous inclusions in nerve cells. These filaments are amyloid fibrils that are made of the protein a-synuclein, which is genetically linked to rare cases of PD and DLB. ß-Synuclein, which shares 60% identity with a-synuclein, is not found in the inclusions. Furthermore, while recombinant a-synuclein readily assembles into amyloid fibrils, ß-synuclein fails to do so. It has been suggested that this may be due to the lack in ß-synuclein of a hydrophobic region that spans residues 73¿83 of a-synuclein. Here, fibril assembly of recombinant human a-synuclein, a-synuclein deletion mutants, ß-synuclein and ß/a-synuclein chimeras was assayed quantitatively by thioflavin T fluorescence and semi-quantitatively by transmission electron microscopy. Deletion of residues 73¿83 from a-synuclein did not abolish filament formation. Furthermore, a chimera of ß-synuclein with a-synuclein(73¿83) inserted was significantly less fibrillogenic than wild-type a-synuclein. These findings, together with results obtained using a number of recombinant synucleins, showed a correlation between fibrillogenesis and mean ß-strand propensity, hydrophilicity and charge of the amino acid sequences. The combination of these simple physicochemical properties with a previously described calculation of ß-strand contiguity allowed us to design mutations that changed the fibrillogenic propensity of a-synuclein in predictable way

Sequence Determinants for Amyloid Fibrillogenesis of Human a-Synuclein

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the presence of filamentous inclusions in nerve cells. These filaments are amyloid fibrils that are made of the protein a-synuclein, which is genetically linked to rare cases of PD and DLB. ß-Synuclein, which shares 60% identity with a-synuclein, is not found in the inclusions. Furthermore, while recombinant a-synuclein readily assembles into amyloid fibrils, ß-synuclein fails to do so. It has been suggested that this may be due to the lack in ß-synuclein of a hydrophobic region that spans residues 73¿83 of a-synuclein. Here, fibril assembly of recombinant human a-synuclein, a-synuclein deletion mutants, ß-synuclein and ß/a-synuclein chimeras was assayed quantitatively by thioflavin T fluorescence and semi-quantitatively by transmission electron microscopy. Deletion of residues 73¿83 from a-synuclein did not abolish filament formation. Furthermore, a chimera of ß-synuclein with a-synuclein(73¿83) inserted was significantly less fibrillogenic than wild-type a-synuclein. These findings, together with results obtained using a number of recombinant synucleins, showed a correlation between fibrillogenesis and mean ß-strand propensity, hydrophilicity and charge of the amino acid sequences. The combination of these simple physicochemical properties with a previously described calculation of ß-strand contiguity allowed us to design mutations that changed the fibrillogenic propensity of a-synuclein in predictable way

The structure of cross-ß tapes and tubes formed by an octapeptide, aSß1

Elaborate morphology: The aSß1 peptide, a fragment of a-synuclein, assembles into flat tapes consisting of a peptide bilayer, which can be modeled based on the cross-ß structure found in amyloid proteins. The tapes are stabilized by hydrogen bonding, whilst the amphiphilic nature of the peptide results in the thin bilayer structure. To further stabilize the structure, these tapes may twist to form helical tapes, which subsequently close into nanotubes

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

A simple algorithm locates β-strands in the amyloid fibril core of α-synuclein, Aβ, and tau using the amino acid sequence alone

Fibrillar inclusions are a characteristic feature of the neuropathology found in the α-synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Familial forms of α-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, α-synuclein (α-syn), must undergo a process of self-assembly in which its native state is converted from a disordered conformer into a β-sheet-dominated form. Here, we have developed a novel polypeptide property calculator to locate and quantify relative propensities for β-strand structure in the sequence of α-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 β-sheet core in α-syn fibrils. In particular, the plot features three peaks, the largest of which is completely absent for the nonfibrillogenic protein, β-syn. We also report similar significant correlations for the Alzheimer's disease-related proteins, Aβ and tau. A substantial region of α-syn is also of converting from its disordered conformation into a long amphipathic α-helical protein. We have developed the aforementioned algorithm to locate and quantify the α-helical hydrophobic moment in the amino acid sequence of α-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 α-helical structure in membrane bilayer-associated α-syn