Inhibition of alpha-synuclein fibril assembly by small molecules: analysis using epitope-specific antibodies.

The conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases. Existing treatments are at best symptomatic. Accordingly, small molecule inhibitors of amyloid fibril formation and their mechanisms are of great interest. Here we report that the conformational changes undergone by alpha -synuclein as it assembles into amyloid fibrils can be detected by epitope-specific antibodies. We show that the conformations of polyphenol-bound alpha-synuclein monomers and dimers differ from those of unbound monomers and resemble amyloid fibrils. This strongly suggests that small molecule inhibitors bind and stabilize intermediates of amyloid fibril formation, consistent with the view that inhibitor-bound molecular species are on-pathway intermediates.

Conversion of wild-type alpha-synuclein into mutant-type fibrils and its propagation in the presence of A30P mutant.

Fibrillization or conformational change of alpha-synuclein is central in the pathogenesis of alpha-synucleinopathies, such as Parkinson disease. We found that the A30P mutant accelerates nucleation-dependent fibrillization of wild type (WT) alpha-synuclein. Electron microscopy observation and ultracentrifugation experiments revealed that shedding of fragments occurs from A30P fibrils and that these fragments accelerate fibrillization by serving as seeds. Immunochemical analysis using epitope-specific antibodies and biochemical analyses of protease-resistant cores demonstrated that A30P fibrils have a distinct conformation. Interestingly, WT fibrils formed with A30P seeds exhibited the same character as A30P fibrils, as did A30P fibrils formed with WT seeds, indicating that the A30P mutation affects the conformation and fibrillization of both WT and A30P. These effects of A30P mutation may explain the apparent conflict between the association of A30P with Parkinson disease and the slow fibrillization of A30P itself and therefore provide new insight into the molecular mechanisms of alpha-synucleinopathies.

Death effector domain-only polypeptides of caspase-8 and -10 specifically inhibit death receptor-induced cell death.

Caspase-8 and -10 are thought to be involved in a signaling pathway leading to death receptor-mediated apoptosis. The prodomains of these caspases are known to form fibrous structures in the perinuclear region when overexpressed, though the meaning of the structures remains unclear. In a previous study we showed that the overexpressed caspase-8 or -10 prodomain (PDCasp8 or PDCasp10) did not induce cell death, and we hypothesized that these prodomains interfere with the receptor-mediated cell death signaling pathway. Indeed, in 293, HeLa and Jurkat cells, cell death mediated by agonistic anti-Fas antibody, TRAIL or overexpression of full-length caspase-8 was significantly inhibited by overexpression of PDCasp8 or PDCasp10 which colocalized with the Golgi complex and with overexpressed FADD. However, when about 20 amino acid residues were deleted from either terminus of the caspase-10 prodomain (amino acid residue 1 to 219), the ability to inhibit Fas-mediated cell death was lost. Interestingly, these deletion mutants also lost the ability to make fibrous structures and to bind FADD, suggesting that FADD binding is important for their function, and that PDCasp8 and PDCasp10 act as dominant-negative inhibitors.