Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications.
Filamentous inclusions of alpha-synuclein protein are hallmarks of neurodegenerative diseases collectively known as synucleinopathies. Previous studies have shown that exposure to oxidative and nitrative species stabilizes alpha-synuclein filaments in vitro, and this stabilization may be due to dityrosine cross-linking. To test this hypothesis, we mutated tyrosine residues to phenylalanine and generated recombinant wild type and mutant alpha-synuclein proteins. alpha-Synuclein proteins lacking some or all tyrosine residues form fibrils to the same extent as the wild type protein. Tyrosine residues are not required for protein cross-linking or filament stabilization resulting from transition metal-mediated oxidation, because higher Mr SDS-resistant oligomers and filaments stable to chaotropic agents are detected using all Tyr --> Phe alpha-synuclein mutants. By contrast, cross-linking resulting from exposure to nitrating agents required the presence of one or more tyrosine residues. Furthermore, tyrosine cross-linking is involved in filament stabilization, because nitrating agent-exposed assembled wild type, but not mutant alpha-synuclein lacking all tyrosine residues, was stable to chaotropic treatment. In addition, the formation of stable alpha-synuclein inclusions in intact cells after exposure to oxidizing and nitrating species requires tyrosine residues. These findings demonstrate that nitrative and/or oxidative stress results in distinct mechanisms of alpha-synuclein protein modifications that can influence the formation of stable alpha-synuclein fibrils.
Norris
Erin H
EH
Center for Neurodegenerative Disease Research and the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
Giasson
Benoit I
BI
Ischiropoulos
Harry
H
Lee
Virginia M-Y
VM
D002460:Cell Line; D003432:Cross-Linking Reagents; D004355:Drug Stability; D006801:Humans; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051843:Synucleins; D014443:Tyrosine / Q000737:chemistry; D051844:alpha-Synuclein
D003432:Cross-Linking Reagents; D009419:Nerve Tissue Proteins; D009566:Nitrates; D011994:Recombinant Proteins; C497604:SNCA protein, human; D051843:Synucleins; D051844:alpha-Synuclein; D014443:Tyrosine