Proteomics Analysis Identifies Phosphorylation-dependent α-Synuclein Protein Interactions

Abstract

Mutations and copy number variation in the SNCA gene encoding the neuronal protein α-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 α-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 α-synuclein. J. Biol. Chem. 275, 390–397). Under pathological conditions, such as in Parkinson disease, α-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 α-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739–29752). Controversy exists over the extent to which phosphorylation of α-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 α-synuclein. We showed that non-phosphorylated α-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas α-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated α-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 α-synuclein is phosphorylated. Mutations and copy number variation in the SNCA gene encoding the neuronal protein α-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 α-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 α-synuclein. J. Biol. Chem. 275, 390–397). Under pathological conditions, such as in Parkinson disease, α-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 α-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739–29752). Controversy exists over the extent to which phosphorylation of α-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 α-synuclein. We showed that non-phosphorylated α-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas α-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated α-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 α-synuclein is phosphorylated. α-Synuclein is a 140-amino acid protein that is highly expressed in the brain and localizes at presynaptic terminals (1George J.M. The synucleins.Genome Biol. 2002; 3 (REVIEWS3002)Google Scholar). The protein is associated with several neurodegenerative diseases, and mutations and copy number variants in the gene coding for α-synuclein have been linked to familial Parkinson disease (PD) 1The abbreviations used are: PD, Parkinson disease; NP, peptide consisting of carboxyl-terminal 40 amino acids of α-synuclein; pS129, peptide consisting of carboxyl-terminal 40 amino acids of α-synuclein that is phosphorylated at serine position 129; pY125, peptide consisting of carboxyl-terminal 40 amino acids of α-synuclein that is phosphorylated at tyrosine position 125; VDAC, voltage-dependent anion-selective channel; MAP, microtubule-associated protein; AP-2, adaptor protein complex 2; CaM, calmodulin. 1The abbreviations used are: PD, Parkinson disease; NP, peptide consisting of carboxyl-terminal 40 amino acids of α-synuclein; pS129, peptide consisting of carboxyl-terminal 40 amino acids of α-synuclein that is phosphorylated at serine position 129; pY125, peptide consisting of carboxyl-terminal 40 amino acids of α-synuclein that is phosphorylated at tyrosine position 125; VDAC, voltage-dependent anion-selective channel; MAP, microtubule-associated protein; AP-2, adaptor protein complex 2; CaM, calmodulin. (2Beal M.F. Experimental models of Parkinson's disease.Nat. Rev. Neurosci. 2001; 2: 325-334Crossref PubMed Scopus (506) Google Scholar, 44Thomas B. Beal M.F. Parkinson's disease.Hum. Mol. Genet. 2007; 16: R183-R194Crossref PubMed Scopus (665) Google Scholar). The presence of large amounts of α-synuclein in the insoluble aggregates known as Lewy bodies, a pathological hallmark of all PD including sporadic disease, also suggests a connection between the protein and disease. The protein is predominantly natively unfolded in solution but can bind to phospholipid membranes by adopting an amphipathic helical conformation in its amino-terminal 100 amino acids (3Bennett M.C. The role of α-synuclein in neurodegenerative diseases.Pharmacol. Ther. 2005; 105: 311-331Crossref PubMed Scopus (151) Google Scholar). The remainder of the protein, an acidic, hydrophilic 40-amino acid carboxyl-terminal tail, is thought unlikely to participate directly in membrane binding. A physiological role for vesicle binding by α-synuclein is suggested by the observation that in PC12 and chromaffin cells α-synuclein appears to be a negative regulator of synaptic vesicle exocytosis and neurotransmitter release (4Larsen K.E. Schmitz Y. Troyer M.D. Mosharov E. Dietrich P. Quazi A.Z. Savalle M. Nemani V. Chaudhry F.A. Edwards R.H. Stefanis L. Sulzer D. α-Synuclein overexpression in PC12 and chromaffin cells impairs catecholamine release by interfering with a late step in exocytosis.J. Neurosci. 2006; 26: 11915-11922Crossref PubMed Scopus (321) Google Scholar). In addition, loss of α-synuclein in cell culture or in mice results in a significant decrease in the population of presynaptic vesicles in the resting or reserve pool (5Murphy D.D. Rueter S.M. Trojanowski J.Q. Lee V.M. Synucleins are developmentally expressed, and α-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons.J. Neurosci. 2000; 20: 3214-3220Crossref PubMed Google Scholar, 6Cabin D.E. Shimazu K. Murphy D. Cole N.B. Gottschalk W. McIlwain K.L. Orrison B. Chen R. B. vesicle with synaptic to in mice Neurosci. 2002; PubMed Google Scholar, K. of α-synuclein in synaptic 2007; PubMed Scopus Google Scholar). for a role of α-synuclein in vesicular trafficking in that the that of α-synuclein be by proteins involved in vesicle trafficking B. K. K. K.E. F. J. α-Synuclein and loss in Parkinson's 2006; PubMed Scopus Google Scholar, T. T. T. A of in neuronal in α-synuclein C. Mol. Genet. PubMed Scopus Google Scholar). The carboxyl-terminal of α-synuclein can be phosphorylated at tyrosine at position 125 and at serine at position by and α-Synuclein is phosphorylated by of the of Biol. Chem. 2001; PubMed Scopus Google Scholar, T. T. α-synuclein at tyrosine 2001; PubMed Scopus Google Scholar, M. J. M. T. L. C. Constitutive phosphorylation of the Parkinson's disease associated Biol. Chem. 2000; PubMed Scopus Google Scholar). α-Synuclein phosphorylated at Ser-129 is the of α-synuclein in Lewy bodies but only a small fraction of α-synuclein in the D.E. J.M. de R. K. R. K. Lee M. L. T. P. D. Phosphorylation of Ser-129 is the dominant pathological modification of α-synuclein in familial and sporadic Lewy body Biol. Chem. 2006; PubMed Scopus Google Scholar, M. E. J. K. T. α-Synuclein is phosphorylated in Biol. 2002; PubMed Scopus Google Scholar). over the extent to which phosphorylation of α-synuclein at Ser-129 is in the of A of α-synuclein with acid serine at position was to the of α-synuclein in a for PD F. in and culture of the a Genet. PubMed Scopus Google Scholar). In overexpression of α-synuclein directly whereas the with serine 129, was highly Chen W. The phosphorylation of Ser-129 in α-synuclein in a of Parkinson 105: PubMed Scopus Google Scholar). identification of protein-protein interactions of α-synuclein that on the phosphorylation of α-synuclein at Ser-129 or Tyr-125 is to on the normal physiological of α-synuclein phosphorylation as as identify for Lewy body and α-synuclein We that the hydrophilic of α-synuclein a that in phosphorylation protein-protein interactions to the normal of α-synuclein. of protein-protein interactions a unbiased for the of protein We to a mass of protein complexes that be pulled down in with a peptide the hydrophilic the carboxyl-terminal 40 amino acids of α-synuclein. We used a targeted mass functional proteomics approach to identify and in protein-protein interactions of phosphorylated non-phosphorylated α-synuclein. We showed that a phosphorylated peptide the carboxyl-terminal 40 amino acids of α-synuclein with cytoskeletal proteins, vesicular trafficking proteins involved in and enzymes involved in protein serine whereas the non-phosphorylated peptide with mitochondrial electron transport This suggests that phosphorylation a on the and/or of α-synuclein. was to and by the and brain were as Murphy F. and electron transport in mice Biol. 2005; PubMed Scopus Google and at brain normal brain was by a in in and at The was the at in a by and were and at as Murphy F. and electron transport in mice Biol. 2005; PubMed Scopus Google Scholar). was the with as a The for of was by the were in a 100 in of and protein and at for at and the was used for binding of The used were as the non-phosphorylated carboxyl-terminal 40 amino acids of α-synuclein carboxyl-terminal 40-amino acid phosphorylated either at Ser-129 or at Tyr-125 and a peptide with the amino acid as but with the in were by a and the of peptide or by the with as a used for pulldown in a binding of were at in 100 with of brain protein and of binding conditions and were in and proteins were in by at for for peptide were in and to The proteins were at for and by and the was with and mass were was to 40 and peptide a of a M. M. of proteins Chem. PubMed Scopus Google Scholar). were with in 100 with by and were and with at by 100 at were with by and were with of in was to at were the with acid and was with and were and to were in acid and a to an mass were a A was to over The was at a of J. M. T. and or for spectrometry with for 2005; PubMed Scopus Google Scholar). The was in and were for in a with the in the for by were for were with or The mass were with the the and protein were as mass of and mass of assays were with in of for The of of the 40 were by in when the all were peptide with were used to protein for results a pulldown are the by the in the on and for and V. R. J. Chen for PubMed Scopus Google Scholar, E. X. of the synaptic 2007; PubMed Scopus Google Scholar, X. in the on and for Scholar). and were also used for peptide and protein were to were only all was by In the was on the peptide or protein number of by to a protein and with was to peptide for a L. M. complexes proteomics and 2006; PubMed Scopus Google Scholar, C. L. of complexes 2006; PubMed Scopus Google Scholar). The number of peptide was by to the of the number of of a protein in the on and for Scholar). in the of a peptide complex for a protein were as the of peptide of α-synuclein to In the an of of the peptide was by of primary mass In were mass and the were for associated with peptide with V. R. J. Chen for PubMed Scopus Google Scholar, E. X. of the synaptic 2007; PubMed Scopus Google Scholar, X. in the on and for Scholar). for all to proteins were to the for also be used to peptide of a protein in a pulldown with either the phosphorylated or non-phosphorylated peptide was the of peptide or for the of that protein either the phosphorylated or the non-phosphorylated was as a number of peptide or for the phosphorylated non-phosphorylated peptide or an to mass the proteins the were to membrane membranes were in with and was used complex and and complex and and were by and by We to identify protein that with the carboxyl-terminal of α-synuclein in a The was to down brain proteins the carboxyl-terminal of α-synuclein. The were non-phosphorylated phosphorylated on serine or phosphorylated on tyrosine 125 A peptide with the amino acids as the peptide but with the was used as a for binding. The visible and binding. 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PubMed Google Scholar). we the for proteins pulled down by and NP, was in with as with the peptide The to was the of interactions with a phosphorylation neuronal in protein protein mitochondrial protein in a A of proteins that were pulled down by as with and The was that of proteins of of the mitochondrial electron transport complex proteins, we of the and of We also of the complex proteins and of complex proteins but complex and but complex a the de R. M.F. C. of for the of complex Biol. Chem. PubMed Scopus Google with for the non-phosphorylated α-synuclein complex complex protein protein, mitochondrial complex protein complex complex membrane vesicle membrane protein membrane protein transport membrane protein of mitochondrial membrane 40 protein in a A major were proteins pulled down by the carboxyl-terminal of α-synuclein as with the but the was of the phosphorylation of the peptide The proteins in microtubule-associated proteins and as as mitochondrial membrane the and the of voltage-dependent was that showed a targeted such as to We used of the of proteins pulled down by phosphorylated and non-phosphorylated as an of the of the mass spectrometry We a of proteins for which were for of brain proteins pulled down by the in we that all of the proteins showed a of in complexes pulled down by and or the peptide as we with the mass were all α-synuclein to down proteins, we to that the in of proteins on the phosphorylation of the peptide was with We a of proteins pulled down by the brain and with and brain we the protein interactions with the carboxyl-terminal of α-synuclein be when the was of the α-synuclein at to was to the pulldown of electron transport proteins as as the mitochondrial the pulldown α-synuclein amino acids the amino to when at the with the protein was a of an of protein or we the extent of the between and 125 the the protein a peptide the amino including the Ser-129 was to α-synuclein and were to with peptide in the whereas α-synuclein was α-synuclein and are highly to α-synuclein at carboxyl and phosphorylation on serine K. 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M. is the major in brain that of for 2007; PubMed Scopus Google Scholar). the we for and with the phosphorylated peptide suggests either that are the of phosphorylation or that are with α-synuclein but are of a complex that phosphorylated α-synuclein is that the of for phosphorylated α-synuclein is when the phosphorylation is at Tyr-125 at the that Tyr-125 phosphorylation be involved in a with serine phosphorylation at The tyrosine at position 125 can be phosphorylated by in cell but we that physiological phosphorylation at that in the brain (2Beal M.F. Experimental models of Parkinson's disease.Nat. Rev. Neurosci. 2001; 2: 325-334Crossref PubMed Scopus (506) Google Scholar, M.C. The role of α-synuclein in neurodegenerative diseases.Pharmacol. 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M. of the for and complex Biol. 2007; PubMed Scopus Google Scholar, M. M. of mitochondrial complexes 2007; PubMed Scopus Google Scholar). The of complex in are known of complex and proteins that complex such as the proteins, that the complex but to of the and proteins that the complex late and are in the hydrophilic such as and M. M. of the for and complex Biol. 2007; PubMed Scopus Google Scholar). A proteomics of complex showed that of the complex and proteins as M. M.F. T. proteomics for the of of mitochondrial complex 2006; PubMed Scopus Google the of a consisting of complexes and to as the de R. M.F. C. of for the of complex Biol. Chem. PubMed Scopus Google Scholar). we that the for electron transport proteins pulled down with the non-phosphorylated carboxyl terminus of α-synuclein is with a of α-synuclein with or of the are a number of to the The is that of the proteins pulled down in a are are to interactions with peptide and the on the results with we of is A of is of proteins are to be in protein complexes in which only a small number of proteins are whereas the are pulled down in the which proteins are directly to and for We have such the to for interactions and have in of the cytoskeletal proteins in that can directly with the carboxyl-terminal of α-synuclein. and R. L. A is that the were with only a of α-synuclein. the that the terminus when of the protein was to with a of the that all the in conformation and interactions of the protein by phosphorylation. the results are all in and the of of interactions an to be in such as in which the α-synuclein is either phosphorylated or is The results are only the steps that for to be in complex and are a number of proteomics interactions of α-synuclein with have involved cells or with mitochondrial such as or and a proteomics of the in the and of proteins to with α-synuclein on an Y. T. C. R.H. J. of proteins by Biol. Chem. PubMed Scopus Google Scholar, J. J. D. Y. C. J. of proteins associated with α-synuclein and 2007; PubMed Scopus Google Scholar, Chen L. Y. J. P. 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V. and of α-synuclein complex in neuronal and Parkinson disease Biol. Chem. PubMed Scopus Google and R. of α-synuclein to of 2007; PubMed Scopus Google on to that the large number of in the in the amino terminus of the protein can as a mitochondrial that α-synuclein to can that with of of non-phosphorylated α-synuclein the protein is to cytoskeletal and is to can with complex of the electron transport the to α-synuclein is overexpression of the protein or mutations that its the of non-phosphorylated protein mitochondrial results are with the results of Chen W. The phosphorylation of Ser-129 in α-synuclein in a of Parkinson 105: PubMed Scopus Google and a that non-phosphorylated α-synuclein in the to mitochondrial and the of The enzymes involved in and α-synuclein be in We and J. for with and for on and and for in and the We Cole for and to with