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alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinson's disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors.

The Rad51 protein, which catalyzes homologous-pairing and strand-exchange reactions, is an essential enzyme for homologous recombinational repair (HRR) and meiotic homologous recombination in eukaryotes. In humans, the conventional Rad51 (HsRad51) protein has a Lys residue at position 313; however, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by Gln, was reported as an isoform, probably corresponding to a polymorphic variant. In this study, we purified the HsRad51-K313 and HsRad51-Q313 isoforms and analyzed their biochemical activities in vitro. Compared to the conventional HsRad51-K313 protein, the HsRad51-Q313 protein exhibited significantly enhanced strand-exchange activity under conditions with Ca2+, although the difference was not observed without Ca2+. A double-stranded DNA (dsDNA) unwinding assay revealed that the HsRad51-Q313 protein clearly showed enhanced DNA unwinding activity, probably due to its enhanced filament-formation ability. Mutational analyses of the HsRad51-Lys313 residue revealed that positively charged residues (Lys and Arg), but not negatively charged, polar and hydrophobic residues (Glu, Gln and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These results suggest that the electrostatic environment around position 313 is important for the regulation of the HsRad51 recombinase activity.

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn-HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn-HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0-3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn-HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.