Identification of rotenone-induced modifications in alpha-synuclein using affinity pull-down and tandem mass spectrometry.
Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of "Lewy body" inclusions enriched with aggregated forms of alpha-synuclein, a presynaptic protein. Although alpha-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged alpha-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of alpha-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that alpha-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of alpha-synuclein contribute to neurologic disorders such as Parkinson's disease.
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Hamid
H
Mirzaei
Jeremy L
JL
Schieler
Jean-Christophe
JC
Rochet
Fred
F
Regnier
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009410:Nerve Degeneration / Q000139:chemically induced* / Q000473:pathology; D009422:Nervous System Diseases / Q000473:pathology; D009474:Neurons / Q000378:metabolism; D018384:Oxidative Stress; D016716:PC12 Cells; D010300:Parkinson Disease / Q000473:pathology; D011499:Protein Processing, Post-Translational; D051381:Rats; D012402:Rotenone; D053719:Tandem Mass Spectrometry / Q000379:methods*; D051844:alpha-Synuclein / Q000032:analysis* / Q000378:metabolism
D051844:alpha-Synuclein; D012402:Rotenone