Tyrosine-to-cysteine modification of human alpha-synuclein enhances protein aggregation and cellular toxicity.
The deposition of alpha-synuclein and other cellular proteins in Lewy bodies in midbrain dopamine neurons is a pathological hallmark of Parkinson's disease. Nitrative and oxidative stress can induce alpha-synuclein protein aggregation, possibly initiated by the formation of stable cross-linking dimers. To determine whether enhanced dimer formation can accelerate protein aggregation and increase cellular toxicity, we have substituted cysteine for tyrosine at positions 39, 125, 133, and 136 in human wild-type (WT) alpha-synuclein, and in A53T and A30P mutant alpha-synuclein. To reduce the likelihood of cross-linking, phenylalanine was substituted for tyrosine at the same sites. We have found that overexpression of Y39C or Y125C mutant proteins leads to increased intracellular inclusions and apoptosis in a rat dopaminergic cell line (N27 cells) and in human embryonic kidney 293 cells. Expression of Y133C, Y136C, and all four Tyr-to-Phe mutations were not more cytotoxic than WT control. Exposure to oxidative stress increased Y39C and Y125C alpha-synuclein aggregation and toxicity. Dimers and oligomers were found in Triton X-100-soluble fractions from adenovirus-mediated overexpression of Y39C and Y125C in N27 cells. In contrast, WT beta-synuclein and all four Tyr-to-Cys mutant beta-synucleins did not cause protein aggregation and cell death. We conclude that cysteine substitution at critical positions in the alpha-synuclein molecule can increase dimer formation and accelerate protein aggregation and cellular toxicity of alpha-synuclein.
Zhou
Wenbo
W
Division of Clinical Pharmacology, Department of Medicine, and the Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Freed
Curt R
CR
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009474:Neurons / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry; D017830:Octoxynol / Q000494:pharmacology; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D051843:Synucleins; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry*; D051844:alpha-Synuclein; D051846:beta-Synuclein
D003902:Detergents; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; C497604:SNCA protein, human; C497607:SNCB protein, human; C497605:Snca protein, mouse; C497606:Snca protein, rat; C497608:Sncb protein, mouse; C497609:Sncb protein, rat; D051843:Synucleins; D051844:alpha-Synuclein; D051846:beta-Synuclein; D014443:Tyrosine; D010649:Phenylalanine; D017830:Octoxynol; D003545:Cysteine; D009584:Nitrogen; D010100:Oxygen