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• W2076163606 abstract "Detergent-stable multimers of α-synuclein have been found specifically in the brains of patients with Parkinson’s disease and other neurodegenerative diseases. Here we show that recombinant α-synuclein forms multimers in vitro upon exposure to vesicles containing certain polyunsaturated fatty acid (PUFA) acyl groups, including arachidonoyl and docosahexaenoyl. This process occurs at physiological concentrations and much faster than in aqueous solution. PUFA-induced aggregation involves physical association with the vesicle surface via the large apolipoprotein-like lipid-binding domain that constitutes the majority of the protein. β- and γ-synucleins, as well as the Parkinson’s disease-associated α-synuclein variants A30P and A53T, show similar tendencies to multimerize in the presence of PUFAs. Multimerization does not require the presence of any tyrosine residues in the sequence. The membrane-based interaction of the synucleins with specific long chain polyunsaturated phospholipids may be relevant to the protein family’s physiological functions and may also contribute to the aggregation of α-synuclein observed in neurodegenerative disease. Detergent-stable multimers of α-synuclein have been found specifically in the brains of patients with Parkinson’s disease and other neurodegenerative diseases. Here we show that recombinant α-synuclein forms multimers in vitro upon exposure to vesicles containing certain polyunsaturated fatty acid (PUFA) acyl groups, including arachidonoyl and docosahexaenoyl. This process occurs at physiological concentrations and much faster than in aqueous solution. PUFA-induced aggregation involves physical association with the vesicle surface via the large apolipoprotein-like lipid-binding domain that constitutes the majority of the protein. β- and γ-synucleins, as well as the Parkinson’s disease-associated α-synuclein variants A30P and A53T, show similar tendencies to multimerize in the presence of PUFAs. Multimerization does not require the presence of any tyrosine residues in the sequence. The membrane-based interaction of the synucleins with specific long chain polyunsaturated phospholipids may be relevant to the protein family’s physiological functions and may also contribute to the aggregation of α-synuclein observed in neurodegenerative disease. Parkinson's disease 1-palmitoyl-2-oleoyl phosphatidylcholine 1-stearoyl-2-oleoyl phosphatidylcholine 1-stearoyl-2-arachidonoyl phosphatidylcholine 1-palmitoyl-2-arachidonoyl phosphatidylcholine 1-stearoyl-2-docosahexaenoyl phosphatidylcholine 1-palmitoyl-2-oleoyl phosphatidic acid, PI, phosphatidylinositol phosphatidylinositol 4,5-bisphosphate phosphatidic acid phosphatidylcholine polyunsaturated fatty acid arachidonic acid small unilamellar vesicles monoclonal antibody The synucleins are small highly conserved proteins of uncertain function, abundant in the vertebrate nervous system (1Clayton D.F. George J.M. Trends Neurosci. 1998; 21: 249-254Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). Their dominant structural feature is the presence of a long apolipoprotein-like domain capable of binding reversibly to specific phospholipids (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar). Increased expression of α-, β-, and γ-synucleins has been detected in breast, ovarian, and bladder cancers (3Harvey S. Zhang Y. Landry F. Miller C. Smith J.W. Physiol. Genomics. 2001; 5: 129-136Crossref PubMed Scopus (31) Google Scholar, 4Bruening W. Giasson B.I. Klein-Szanto A.J. Lee V.M. Trojanowski J.Q. Godwin A.K. Cancer. 2000; 88: 2154-2163Crossref PubMed Scopus (159) Google Scholar, 5Ji H. Liu Y.E. Jia T. Wang M. Liu J. Xiao G. Joseph B.K. Rosen C. Shi Y.E. Cancer Res. 1997; 57: 759-764PubMed Google Scholar, 6Celis A. Rasmussen H.H. Celis P. Basse B. Lauridsen J.B. Ratz G. Hein B. Ostergaard M. Wolf H. Orntoft T. Celis J.E. Electrophoresis. 1999; 20: 355-361Crossref PubMed Scopus (91) Google Scholar) and has been mechanistically linked to increased potential for tumor growth (7Liu J.W. Spence M.J. Zhang Y.L. Jiang Y.F. Liu Y.L.E. Shi Y.E. Breast Cancer Res. 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Bereznai B. Gasser T. Bonifati V. Demichele G. Fabrizio E. Volpe G. Bandmann O. Johnson W.G. Golbe L.I. Breteler M. Meco G. Agid Y. Brice A. Marsden C.D. Wood N.W. Ann. Neurol. 1998; 44: 270-273Crossref PubMed Scopus (80) Google Scholar, 36Nagar S. Juyal R.C. Chaudhary S. Behari M. Gupta M. Rao S.N. Thelma B.K. Acta Neurol. Scand. 2001; 103: 120-122Crossref PubMed Scopus (26) Google Scholar). Purified synucleins will aggregate in vitro into fibrils resembling those found in Lewy bodies, following an initial nucleation step in which smaller synuclein multimers are formed (37Wood S.J. Wypych J. Steavenson S. Louis J.C. Citron M. Biere A.L. J. Biol. Chem. 1999; 274: 19509-19512Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 38Narhi L. Wood S.J. Steavenson S. Jiang Y.J. Wu G.M. Anafi D. Kaufman S.A. Martin F. Sitney K. Denis P. Louis J.C. Wypych J. Biere A.L. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 39Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1347) Google Scholar, 40Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Crossref PubMed Scopus (1271) Google Scholar, 41Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M.Y. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). In healthy brain tissue, multimeric forms of synuclein are not typically observed, but multimers are prominent in brain tissue from individuals afflicted with Lewy body diseases (15Lippa C.F. Fujiwara H. Mann D.M.A. Giasson B. Baba M. Schmidt M.L. Nee L.E. O'Connell B. Pollen D.A. George-Hyslop P.S. Ghetti B. Nochlin D. Bird T.D. Cairns N.J. Lee V.M.Y. Iwatsubo T. Trojanowski J.Q. Am. J. Pathol. 1998; 153: 1365-1370Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 17Dickson D.W. Liu W. Hardy J. Farrer M. Mehta N. Uitti R. Mark M. Zimmerman T. Golbe L. Sage J. Sima A. D'Amato C. Albin R. Gilman S. Yen S.H. Am. J. Pathol. 1999; 155: 1241-1251Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 42Culvenor J.G. McLean C.A. Cutt S. Campbell B.C. Maher F. Jakala P. Hartmann T. Beyreuther K. Masters C.L. Li Q.X. Am. J. Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar,43Langston J.W. Sastry S. Chan P. Forno L.S. Bolin L.M. Di Monte D.A. Exp. Neurol. 1998; 154: 684-690Crossref PubMed Scopus (46) Google Scholar). In a previous study of α-synuclein’s interaction with phospholipids, we incidentally observed that exposure of recombinant α-synuclein to phosphatidylinositol stimulates the formation of stable apparent multimers that are maintained throughout SDS gel electrophoresis (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar). Phosphatidylinositols play a critical role in vesicle cycling at presynaptic terminals, where α-synuclein is particularly enriched. Moreover, factors that stimulate multimerization of α-synuclein may have a direct role in promoting neurodegenerative disease. Therefore, we set out to characterize more thoroughly the basis for the enhancement of synuclein multimerization by specific phospholipids. The human α-synuclein cDNA used in this study was the generous gift of M. Irizarry. Human β- and γ-synuclein cDNAs were cloned from an adult human brain cDNA library (CLONTECH). The canary α-synuclein cDNA was originally cloned from mRNA representing a portion of the canary telencephalon (9George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (732) Google Scholar). For bacterial expression, these cDNAs were subcloned into pET28(a) (Novagen), which directs inducible expression under a T7 lac promoter. All mutant forms of human and canary α-synuclein were constructed by long polymerase chain reaction-based techniques using Pfupolymerase (Stratagene), 5′-phosphorylated primers, and subsequent re-circularization of polymerase chain reaction products by T4 ligase. The correct DNA sequences of all constructs were confirmed by DNA sequencing. Relative to wild type human α-synuclein, the mutant constructs are described as follows: Δ3, residues 9–43 deleted (retains SYN303 epitope at N terminus); Δ3′, residues 2–42 replaced by a single residue, Ala; Δ4, residues 43–56 deleted; Δ5, residues 56–102 replaced by a single residue, Glu; Δ6, residues 103–130 deleted; Δ7, residues 130–140 deleted; A30P, residue Ala30 replaced by a single residue, Pro; A53T, residue Ala53 replaced by a single residue, Thr. Relative to canary α-synuclein, in mutant construct Y4F, residues Tyr39, Tyr128, Tyr136, and Tyr139 were each replaced by a single residue, Phe. Recombinant proteins were purified from bacteria as described (44Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2000; 275: 34393-34398Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar) with minor modifications for mutant proteins and for β- and γ-synucleins. The molecular mass of each protein was confirmed by electrospray mass spectrometry ± 2 Da. 1-Palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC), 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), 1-stearoyl-2-arachidonoyl phosphatidylcholine (SAPC), 1-stearoyl-2-docosahexaenoyl phosphatidylcholine (SDPC), 1-palmitoyl-2- oleoyl phosphatidic acid (POPA), soy and liver-derived phosphatidylinositols (PI), and bovine brain-derived phosphatidylinositol (4Bruening W. Giasson B.I. Klein-Szanto A.J. Lee V.M. Trojanowski J.Q. Godwin A.K. Cancer. 2000; 88: 2154-2163Crossref PubMed Scopus (159) Google Scholar, 5Ji H. Liu Y.E. Jia T. Wang M. Liu J. Xiao G. Joseph B.K. Rosen C. Shi Y.E. Cancer Res. 1997; 57: 759-764PubMed Google Scholar) bisphosphate (PIP2), were purchased from Avanti Polar Lipids. Small unilamellar vesicles (SUV) of various lipid compositions were prepared by 20 min of probe sonication at 4 °C (45Barenholz Y. Gibbes D. Litman B.J. Goll J. Thompson T.E. Carlson R.D. Biochemistry. 1977; 16: 2806-2810Crossref PubMed Scopus (730) Google Scholar). The SUV were isolated, and phospholipid concentrations determined as described in Ref. 44Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2000; 275: 34393-34398Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar. Arachidonic acid (AA) was purchased from Sigma and solubilized in ethanol. For SDS-polyacrylamide gel electrophoresis studies, protein concentrations were always 1 μm. Lipids of interest and proteins were incubated at molar ratios of 5:1 (Figs. 2 A and3) or 50:1 (Figs. 2 B, 4, 5, and 6). Assays were performed at 37 °C in 100-μl volumes of 10 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.02% NaN3(excepting the assays shown in Figs. 2 and 3, which were performed in 10 mm HEPES, pH 7.5, and those in Fig. 1, which are described separately). Arachidonic acid incubations were performed at 0, 10, 100, or 1000 μm. Samples were mixed with Laemmli SDS-loading buffer, heated 5–10 min at 95 °C, subjected to SDS-polyacrylamide gel electrophoresis, and immunoblotted with H3C mAb (1:100,000), LB509 mAb (1:10,000; generous gift of T. Iwatsubo), Syn303 mAb (1:2000; generous gift of B. Giasson), HATCAN mAb (1:10,000), or γ-2 polyclonal antibody (1:2000; generous gift of B. Giasson) followed by horseradish peroxidase-anti-mouse or horseradish peroxidase-anti-rabbit secondary antibody (1:3000; Amersham Pharmacia Biotech) as appropriate. Bands were visualized using ECL Western blot detection reagents (Amersham Pharmacia Biotech). Molecular weights were estimated using Bio-Rad broad range prestained ladders.Figure 3Membrane recruitment stimulates PUFA-induced synuclein multimerization. α-Synuclein was incubated with POPC vesicles containing 10% POPA (PA), 10% SAPC (Arach), or 10% of each (PA + Arach) for the times indicated and then immunoblotted as in Fig. 2. Size markers are as in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4PUFA-dependent multimerization is a conserved feature of the synuclein protein family. Recombinant synucleins were incubated with POPC vesicles containing 10% POPA and 10% PAPC and then immunoblotted as in Fig. 2. Duplicate samples were processed in parallel. Size markers are as in Fig. 1. A, full-length human β-synuclein (β); clostripain fragment of human β-synuclein (βc); full-length canary α-synuclein (α); clostripain fragment of canary α-synuclein (αc); full-length human γ-synuclein (γ). B, common form of human α-synuclein (AS); A30P, PD-associated point mutations (A53T).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Deletion mutant analysis of domains for PUFA-induced multimerization. Purified, recombinant versions of human α-synuclein, each lacking one exon (44Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2000; 275: 34393-34398Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar), were incubated with 10% POPA, 10% PAPC, 80% POPC vesicles for 18 h and immunoblotted as in Fig. 2. Size markers are as in Fig. 1.AS, full-length α-synuclein. Δ3, Δ4, Δ5, Δ6, and Δ7 are described under “Experimental Procedures.” Construct Δ3′ also multimerized under these conditions (data not shown). mAbs SYN303 and LB509 bind residues 2–12 and 115–122, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Tyrosine residues are not required for PUFA-induced multimerization. Recombinant wild-type canary synuclein (AS) and canary mutant Y4F, devoid of tyrosines, were incubated with 80% POPC, 10% POPA, 10% PAPC vesicles and probed with HATCAN mAb, raised against residues 108–120 of canary α-synuclein. Size markers are as in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Multimerization of α-synuclein associated with binding to brain PIP2 vesicles. A mixture of PIP2 vesicles (Table I) and recombinant human α-synuclein was separated by size exclusion chromatography (see “Experimental Procedures”). Shown is an H3C immunoblot of consecutive fractions (lanes from left), with elution profiles of multilamellar vesicles (MLV), SUV, and unbound protein (free) indicated below. Markers on the left indicate the migration of prestained protein standards (in kilodaltons). Free monomeric α-synuclein migrates with a mass of 21 kDa (not shown), but incubation with PIP2 results in a small retardation of the monomer’s electrophoretic mobility and the formation of apparent α-synuclein dimers (∼45 kDa) and trimers (∼70 kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Vesicles containing 92% POPC, 8% PIP2 (Fig. 1) were incubated with recombinant human α-synuclein (1.0 μm) at 37 °C for 30 min (lipid/protein mass ratio of 50:1, PIP2/protein molar ratio of 80:1) in 15 mm HEPES (pH 7.5), 6 mm Tris (pH 7.5), 0.6 mm EGTA, 0.3 mm EDTA, 16 mm KCl, 0.5 mm dithiothreitol, 0.6 mm MgCl2, 0.4 mm CaCl2. Lipid/protein mixtures were separated (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar) by size exclusion chromatography on a calibrated Superose 6 FPLC column (Amersham Pharmacia Biotech) into 1-ml fractions, which were then mixed with Laemmli SDS-loading buffer and boiled for 5 min prior to SDS-polyacrylamide gel electrophoresis and transblotting. Immunoblots were probed with H3C monoclonal antibody (1:100,000) and horseradish peroxidase-anti-mouse secondary (1:3000; Amersham Pharmacia Biotech). Elution profiles of multilamellar vesicles, SUV, and unbound protein were monitored by light scattering (vesicles) and absorbance at 273 nm (protein) (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 44Perrin R.J. Woods W.S. Clayton D.F. George J.M. J. Biol. Chem. 2000; 275: 34393-34398Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Clostripain (Sigma) was activated in 2 mm dithiothreitol, 1 mm CaCl2 at 22 °C for 3 h and then incubated with purified, recombinant canary α-synuclein or human β-synuclein at 37 °C for 2 h. Clostripain and undigested synuclein proteins were removed by passage through a Centricon 10 filter (Amicon). β-Synuclein C-terminal fragment was purified sequentially by reversed-phase chromatography (POROS-R2; PerSeptive Biosystems), anion exchange chromatography (POROS-HQ, PerSeptive Biosystems), and size exclusion chromatography (Superdex 75; Amersham Pharmacia Biotech). Canary C-terminal fragment was purified by size exclusion (Superdex 75; Amersham Pharmacia Biotech). Recombinant human α-synuclein was purified from bacteria and exposed to vesicles prepared from a base of synthetic POPC, following conditions previously described (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar). The protein/vesicle mixtures were then fractionated on SDS-polyacrylamide gels and analyzed by immunoblotting with the H3C monoclonal antibody (9George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (732) Google Scholar). When the vesicles were supplemented with liver-derived PI (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar) or brain-derived PIP2, α-synuclein migrated on SDS-polyacrylamide gels in two predominant forms (Fig. 1): a monomer (∼21 kDa) and a larger apparent dimer (∼45 kDa). Smaller amounts of apparent trimers and even larger multimers were also sometimes evident, especially with prolonged incubations, after which immunoreactivity would appear in the stacking gel. Upon size exclusion chromatography of synuclein PIP2 mixtures, both monomeric and multimeric forms of synuclein eluted with SUV fractions (Fig. 1), indicating a stable and specific physical association with membrane surfaces of high curvature (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar). In parallel incubations using vesicles containing several other synthetic phospholipids, only monomeric synuclein eluted, as free protein with neutral phospholipids and with SUV fractions when acidic phospholipids were used (not shown; see Ref. 2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar). We reasoned that the apparent selectivity of multimerization for PI and PIP2 might be attributable to the lipid head group (phosphoinositol), the lipid acyl chains, or some combination of both. The PI and PIP2 used in these studies were purified from animal sources and thus bear a heterogeneous mix of acyl groups (TableI). To test for sensitivity to acyl chain composition, we compared the response to PI derived from either bovine liver or soy. Liver PI, but not soy PI, induced a large amount of α-synuclein dimer and detectable trimer after 12 h (Fig. 2 A). Soy PI is abundant in polyunsaturated fatty acid (PUFA) tails of 18 carbons (linoleoyl and linolenoyl) but lacks the longer chain PUFAs characteristic of animal-derived phospholipids (Table I). Soy PI is also deficient in the intermediate length saturated acyl chain, stearoyl.Table ICompositions (mol %) of experimental vesicles containing natural source phospholipidsVesicle designation:Brain PIP2Liver PISoy PIHead group PC929292 PI88 PIP28Acyl group Palmitoyl (16:0)4646.148.4 Stearoyl (18:0)2.73.30.7 Oleoyl (18:1)4746.946.5 Linoleoyl (18:2)0.53.8 Linolenoyl (18:3)0.6 Eicosatrienoyl (20:3)0.8 Arachidonoyl (20:4)2.61.4 Docosahexaenoyl (22:6)0.2 Other1.90.80.2Vesicles were prepared (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar) from synthetic POPC with the addition of 8% bovine brain PIP2, bovine liver PI, or soy PI. Numbers in the table indicate molar percentage of lipid head groups and fatty acid tails (acyl groups) in the lipid population, calculated from values provided by the supplier (Avanti). For each acyl group, the number of carbons and unsaturated bonds is given in parentheses, following standard conventions. Open table in a new tab Vesicles were prepared (2Davidson W.S. Jonas A. Clayton D.F. George J.M. J. Biol. Chem. 1998; 273: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar) from synthetic POPC with the addition of 8% bovine brain PIP2, bovine liver PI, or soy PI. Numbers in the table indicate molar percentage of lipid head groups and fatty acid tails (acyl groups) in the lipid population, calculated from values provided by the supplier (Avanti). For each acyl group, the number of carbons and unsaturated bonds is given in parentheses, following standard conventions. Since soy PI was less effective at inducing multimerization and was especially deficient in arachidonoyl and stearoyl (Table I), we tested the hypothesis that the presence of these acyl chains in the phospholipid vesicles is sufficient to induce multimerization. As Fig. 2 B demonstrates, multimerization did not occur when synuclein was exposed to acidic vesicles containing only palmitoyl and oleoyl acyl chains (lanes labeled —). However, when synuclein was incubated with POPC vesicles supplemented with both stearoyl and arachidonoyl (by inclusion of 10% SAPC), robust multimerization occurred. Inclusion of stearoyl chains alone did not induce multimerization (Fig. 2 B, SOPC), whereas arachidonoyl inclusion alone was sufficient (Fig. 2 B,PAPC). We also tested another long chain PUFA acyl group with a different arrangement of unsaturated bonds, docosahexaenoyl (Fig. 2 B, SDPC). This acyl chain was as effective as arachidonoyl at inducing multimerization of α-synuclein, indicating a potentially general role for long chain polyunsaturated acyl groups in triggering α-synuclein multimer formation. In a related experiment (Fig. 2 C), we tested whether PUFAs alone (as free fatty acids) can cause the same effect as PUFA-containing phospholipids. Incubation of α-synuclein with free AA at 10–100 μm (lanes 3–6) caused little if any acceleration of multimerization compared with that observed for α-synuclein in buffer with no AA (lanes 1 and 2). At 1 mm (above the critical micellar concentration for AA), robust multimerization occurred (lanes 7 and 8). This concentration dependence suggests that PUFAs exert their effects on synuclein when organized as either a micellar (free fatty acid) or vesicular (phospholipid) surface. As a further test of the role of surface association in PUFA-dependent multimerization, we compared arachidonoyl-containi" @default.
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• W2076163606 title "Exposure to Long Chain Polyunsaturated Fatty Acids Triggers Rapid Multimerization of Synucleins" @default.
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