Abstract
Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.
Highlights
Mutations and copy number variation in the SNCA gene encoding the neuronal protein ␣-synuclein have been linked to familial Parkinson disease
The protein is associated with several neurodegenerative diseases, and mutations and copy number variants in the gene coding for ␣-synuclein have been linked to familial Parkinson disease (PD)1 [2, 44]
A physiological role for vesicle binding by ␣-synuclein is suggested by the observation that in PC12 and chromaffin cells ␣-synuclein appears to be a negative regulator of synaptic vesicle exocytosis and neurotransmitter release [4]
Summary
Obtained during Epilepsy Surgery) was approved by the NINDS Intramural Institutional Review Board For both preparations, synaptosomes were solubilized in a Triton buffer (ϳ1.5% Triton, 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 2 mM EGTA, 50 mM NaF, 0.5 mM sodium vanadate, 1ϫ protease inhibitor mixture (Sigma-Aldrich)) resulting in final concentrations of 1% Triton and ϳ5 mg/ml synaptosome protein and centrifuged at 16,000 ϫ g for 10 min at 4 °C, and the supernatant was used for binding experiments. Assigned probable peptide sequences with Mascot Ion Scores exceeding their Identity Scores were used throughout this study to produce minimal, parsimonious protein lists concatenated for each full gel lane. Summed maximum ion intensities for all peptides mapped to respective proteins were normalized to the total assigned ion current for each experiment For both methods corresponding gel migrations could be used to validate peptide comparisons. Proteins were detected by ECL (Amersham Biosciences) and visualized by autoradiography
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