ABSTRACT
Parkinson's disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and alpha-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant alpha-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetylcysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant alpha-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD.
Subject(s)
Neurons/metabolism , Neurons/pathology , Oxidoreductases/metabolism , Parkinson Disease/metabolism , Parkinson Disease/pathology , Acetylcysteine/pharmacology , Animals , Antioxidants/pharmacology , Blotting, Western , Cell Death/physiology , Cells, Cultured , Cysteine Proteinase Inhibitors/toxicity , Dopamine/metabolism , Humans , Leupeptins/toxicity , Mesencephalon/drug effects , Mesencephalon/metabolism , Mesencephalon/pathology , Methionine Sulfoxide Reductases , Mice , Neurons/drug effects , Oxidation-Reduction/drug effects , Rats , Rotenone/toxicity , Uncoupling Agents/toxicity , Vitamin E/pharmacology , alpha-Synuclein/metabolismABSTRACT
Lipid bilayers are two-dimensional fluids. Here, the effect of monovalent ion concentration on the mixing, and consequently the organization, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) bilayers has been examined. Epifluorescence microscopy was used to visualize the organization. Fluorescence recovery after photobleaching and attenuated total reflection-Fourier transform infrared spectroscopy were used to assess the fluidity of the lipids. At high ionic strength the DOPC and DOPA lipids appear uniformly mixed. Upon lowering the ionic strength, rapid separation is observed. The DOPA-rich regions appear fractal-like and exhibit hysteresis in their properties. The lipids freely exchange between the two regions. These experiments clearly demonstrate the significant effect that electrostatics can have on membrane organization.
Subject(s)
Dihydroxyphenylalanine/chemistry , Ions , Lipid Bilayers/chemistry , Phosphatidic Acids/chemistry , Phosphatidylcholines/chemistry , Escherichia coli/metabolism , Fluorescence Recovery After Photobleaching , Lipids/chemistry , Microscopy, Fluorescence , Protein Structure, Tertiary , Spectroscopy, Fourier Transform Infrared , Static Electricity , Time Factors , alpha-Synuclein/chemistryABSTRACT
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.
