A Hydrophobic Stretch of 12 Amino Acid Residues in the Middle of α-Synuclein Is Essential for Filament Assembly
Neuronal and oligodendrocytic aggregates of fibrillar α-synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise vialibity of affected cells. Here, we report that a 12-amino acid stretch (71VTGVTAVAQKTV82) in the middle of the hydrophobic domain of human α-synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human β-synuclein is highly homologous to α-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of α-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of α-synuclein filaments, because it is resistant to proteolytic digestion in α-synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human α-synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human α-synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit α-synuclein fibrillization and might arrest disease progression. Neuronal and oligodendrocytic aggregates of fibrillar α-synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise vialibity of affected cells. Here, we report that a 12-amino acid stretch (71VTGVTAVAQKTV82) in the middle of the hydrophobic domain of human α-synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human β-synuclein is highly homologous to α-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of α-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of α-synuclein filaments, because it is resistant to proteolytic digestion in α-synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human α-synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human α-synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit α-synuclein fibrillization and might arrest disease progression. synuclein electron microscopy Lewy body nonamyloid component of amyloid plaques polyacrylamide gel electrophoresis α-synuclein (α-syn)1is the major component of several pathological lesions diagnostic of specific neurological disorders, including Parkinson's disease, the Lewy body (LB) variant of Alzheimer's disease, dementia with LB, multiple system atrophy, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease; Refs. 1Baba M. Nakajo S. Tu P. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 2Wakabayashi K. Matsumoto K. Takayama K. Yoshimoto M. Takahashi H. Neurosci. 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Lett. 1998; 251: 205-208Crossref PubMed Scopus (820) Google Scholar). Furthermore, in vitro, α-syn has an intrinsic propensity to polymerize into fibrils that resemble authentic filaments in pathological lesions (12Giasson 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 (452) Google Scholar, 13Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 11: 1318-1320Crossref Scopus (1255) Google Scholar, 14El-Agnaf O.M.A. Jakes R. Curran M.D. Wallace A. FEBS Lett. 1998; 440: 67-70Crossref PubMed Scopus (235) Google Scholar, 15Hashimoto M. Hsu L.J. Sisk A. Xia Y. Takeda A. Sundsmo M. Masliah E. Brain Res. 1998; 799: 301-306Crossref PubMed Scopus (248) Google Scholar, 16Narhi L. Wood S.J. Steavenson S. Jiang Y. 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 (622) Google Scholar). α-Syn is a small, 14-kDa protein that has sequence homology to three other proteins termed β-synuclein (β-syn), γ-synuclein (γ-syn), and synoretin (17Surguchov A. Surgucheva I. Solessio E. Baehr W. Mol. Cell. Neurosci. 1999; 13: 95-103Crossref PubMed Scopus (80) Google Scholar, 18Clayton D.F. George J.M. Trends Neurosci. 1998; 21: 249-254Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). These proteins are more highly homologous within the amino-terminal half, which encompasses 5–6 degenerate KTKEGV repeats. Of these proteins, α-syn has the greatest sequence homology to β-syn. Furthermore, the expression and intracellular localization of α- and β-syn are very similar in that both proteins are predominantly expressed in neurons of the central nervous system and are concentrated at the presynaptic terminal (19Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (899) Google Scholar, 20Shibayama-Imazu T. Okahashi I. Omata K. Nakajo S. Ochiai H. Nakai Y. Hama T. Nakamura Y. Nakaya K. Brain Res. 1993; 622: 17-25Crossref PubMed Scopus (109) Google Scholar, 21George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (725) Google Scholar, 22Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. Rohan de Silva H.A. Kittel A. Saitoh T. Neuron. 1995; 14: 467-475Abstract Full Text PDF PubMed Scopus (1122) Google Scholar). In contrast, γ-syn is predominantly expressed in neurons of the peripheral nervous system, although it is also found at low levels in brain but diffusely distributed throughout the cytoplasm (23Buchman V.L. Hunter H.J.A. Pinõn L.G.P. Thompson J. Privalova E.M. Ninkina N.N. Davies A.M. J. Neurosci. 1998; 18: 9335-9341Crossref PubMed Google Scholar). Synoretin is similar to γ-syn with respect to amino acid sequence and intracellular distribution, but synoretin is mainly expressed in the retina (17Surguchov A. Surgucheva I. Solessio E. Baehr W. Mol. Cell. Neurosci. 1999; 13: 95-103Crossref PubMed Scopus (80) Google Scholar). Intriguingly, although α- and β-syn have sequence homology and very similar biological properties, α-syn, but not β-syn, is found in fibrillar pathological lesions (1Baba M. Nakajo S. Tu P. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar, 4Arima K. Uéda K. Sunohara N. Hirai S. Izumiyama Y. Tonozuka-Uehara H. Kawai M. Brain Res. 1998; 808: 93-100Crossref PubMed Scopus (217) Google Scholar, 5Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2396) Google Scholar, 7Spillantini M.G. Schmidt M.L. Lee V.M.-Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6111) Google Scholar, 8Spillantini M.G. Crowther R.A. Jakes R. Cairns N.J. Lantos P.L. Goedert M. Neurosci. Lett. 1998; 251: 205-208Crossref PubMed Scopus (820) Google Scholar, 9Tu P. Galvin J.E. Baba M. Giasson B. Tomita T. Leigth S. Nakajo S. Iwatsubo T. Trojanowski J.Q. Lee V.M.-Y. Ann. Neurol. 1998; 44: 415-422Crossref PubMed Scopus (581) Google Scholar, 24Lippa C.F. Schmidt M.L. Lee V.-M.-Y. Trojanowski J.Q. Ann. Neurol. 1999; 45: 353-357Crossref PubMed Scopus (274) Google Scholar). To determine the reason for this difference, we compared the primary sequences of human α- and β-syn, and we recognized that a hydrophobic stretch of amino acids within the middle hydrophobic region of α-syn is lacking in β-syn and that there is a significant sequence divergence within the carboxyl-terminal region of these two proteins (Fig. 1). Deletion of amino acids 71–82 within the hydrophobic region abrogated the ability of human α-syn to polymerize, whereas the introduction of charged residues within the region significantly reduced the rate of filament formation. Conversely, substituting the carboxyl-terminal region of β-syn for that of α-syn did not affect polymerization. Proteolytic digestion of assembled α-syn as well as coassembly experiments with a synthetic peptide corresponding to the hydrophobic region further confirmed the key role of this region in fibrillogenesis. All synuclein cDNAs were subcloned into the NdeI andHindIII restriction sites of the bacterial expression vector pRK172, and the respective proteins were expressed in Escherichia coli BL21 (DE3). Bacterial pellets were resuspended in high-salt buffer (0.75 m NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA) containing a mixture of protease inhibitors, heated to 100 °C for 10 min, and centrifuged at 70,000 ×g for 30 min. The supernatants were applied onto a Superdex 200 gel filtration column (Amersham Pharmacia Biotech) and separated by size exclusion using high-salt buffer. The fractions were assayed for the presence of the synuclein proteins by SDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie Blue R-250 staining. The proteins were concentrated using Centriprep-10 (Millipore Corp., Bedford, MA), dialyzed against 10 mm Tris, pH 7.5, applied to a Mono Q column (Amersham Pharmacia Biotech), and eluted with a 0–0.5 m NaCl gradient. Protein concentration was determined using the bicinchoninic acid protein assay (Pierce) and bovine serum albumin as a standard. Proteins were resolved on slab gels by SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) and electrophoretically transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH) in buffer containing 48 mm Tris, 39 mm glycine, and 10% methanol. Membranes were with a of in mm Tris, pH mm NaCl, with antibodies to α-syn and β-syn, followed by antibodies to with and onto Blue and synuclein proteins were assembled into filaments by at °C in 100 mm pH with were centrifuged at ×g for min, and buffer mm Tris, pH 1 mm mm 10% was to pellets and which were heated to 100 °C for min. proteins were resolved by with Coomassie Blue and by synuclein filaments were to with and with a electron were with a using the of filaments was using of synuclein proteins in 30 of 100 mm pH were with of at the was by buffer and to 100 °C for 10 min. from were resolved on SDS-polyacrylamide proteins at a concentration of in 100 mm pH were by after at °C for with Proteins were in and transferred to a and the and were with a The and of antibodies and and were B.I. Jakes R. Goedert M. J.E. S. Trojanowski J.Q. Lee V.M.-Y. J. Neurosci. Res. 274: Scopus Google Scholar, R. Crowther R.A. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Goedert M. Neurosci. Lett. 1999; PubMed Scopus Google Scholar). The was to a synthetic acids in human to The peptide corresponding to amino acids 71–82 in human α-syn was and on by the at The and α-syn were by the respective in the using of synthetic containing the sequence and and the The deletion of the sequence for residues 71–82 in α-syn (Fig. 1) was using the and that to the sequence to the sequence and The the protein which of amino acid residues of α-syn and of β-syn (Fig. was by with that the two of α- and β-syn, and β-syn, and The and 3) sequences that were to both by with 1 and The sequences of the were using and an as a by the at the of To human β-syn is of into filaments, we compared the ability of α- and β-syn to form filaments using for the assembly of α-syn (12Giasson 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 (452) Google Scholar). and electron microscopy were to determine the assembly of human α- and β-syn. proteins were found in the for assembly but the of α-syn was in the after of whereas β-syn and and that human α-syn filaments were within (Fig. but fibrils be with β-syn after to not to assemble after in vitro, as by of β-syn (Fig. of with β-syn assembly for to did not the presence of that both proteins but α-syn a assembly (Fig. and of synuclein filament assembly as by Proteins in 100 mm pH were for and at °C with followed by at for min. and pellets were resolved by SDS-PAGE and after Coomassie Blue R-250 by The of protein in the is expressed on the electron microscopy of synuclein human α-syn and human α-syn were for assembly as and at a concentration of for the filaments of human α-syn were after The peptide corresponding to residues 71–82 in human α-syn also assembled into filaments and at the concentration it with human α-syn The of filament is respective of synuclein protein and and after and assembly for at °C with as and of the primary sequences of human α- and β-syn two major the stretch of hydrophobic amino acids in the middle of α-syn is in β-syn (Fig. 1). the of the two proteins are compared with the amino To the of the hydrophobic region and the carboxyl-terminal region in filament two of synuclein protein were amino acid residues 71–82 in human α-syn were to a protein termed a the amino acids of human α-syn and the 48 amino acids of human β-syn was termed (Fig. 1). both proteins were mainly in the after (Fig. and a predominantly (Fig. and the assembly rate of that of α-syn (Fig. and it formed filaments of similar and (Fig. with The fibrillization of also was with a into a (Fig. the other did not assemble similar assembly as by and and and did not the presence of filaments after for to not the of assembly was by a of as by (Fig. The that β-syn with α-syn inhibit the assembly of α-syn was determined by coassembly β-syn on the assembly of α-syn, as assayed by (Fig. and by not Furthermore, protein with α-syn. α-syn can be separated from β-syn by SDS-PAGE because of its the of the was also confirmed by with an specific to human α-syn and an specific to β-syn To further the of this hydrophobic region in a single in the middle of this was with a a the and in α-syn proteins did not the of human α-syn in and both proteins were in the by not The reduced the rate of filament compared with α-syn at after but by the of the protein was assembled and α-syn (Fig. were similar to assembled of the protein (Fig. contrast, the a on the rate of filament after of the protein was predominantly found in the after and and very and fibrils were by not after of the α-syn protein polymerized into fibrils that were from from the protein (Fig. with The presence of the in coassembly experiments did not to inhibit the assembly of as assayed by (Fig. and by of with of both proteins for assembly not of α-syn fibrils with was to that are within and are core elements of these of human α-syn in the of the protein within 30 Conversely, several resistant to were assembled α-syn filaments were with for (Fig. The to was specific to assembled α-syn filaments, because two other proteins that are assembly β-syn and were after assembly To the proteolytic resistant was using antibodies with throughout the α-syn (Fig. and the sequences of the three major were on the of with the antibodies and on SDS-PAGE gels (Fig. All three peptides amino acid residues 71–82 within the middle hydrophobic region of α-syn. To further the of amino acids 71–82 in human α-syn for we synthetic peptides corresponding to this sequence into that peptide 71–82 into filaments, but these filaments are assembled from the full-length α-syn (Fig. with full-length α-syn was assembled in the presence of peptide the more of full-length α-syn, were that this peptide was into the fibrils as full-length α-syn (Fig. a peptide 71–82 is assembled full-length its ability to can also be by (Fig. the of peptide 71–82 was found to the assembly of full-length α-syn. a with to the rate of the of α-syn assembled was in the whereas in the presence of peptide α-syn was found in the after (Fig. a of to that amino acid residues 71–82 within the middle of the hydrophobic domain of α-syn are necessary and sufficient for α-syn to polymerize into The of residues 71–82 for filament was we found that human α-syn an intrinsic ability to assemble into filaments, whereas human β-syn polymerize into filaments after to assembly These are with a report A. S. F. M. L. and Biol. Chem. Scholar). it be that β-syn form filaments because that a of β-syn filaments were after of J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). we did a in the of β-syn after we this to because of the of filaments by these that β-syn is to polymerize into fibrils to α-syn. to the in assembly of human α- and β-syn by which two major 1) β-syn lacks a hydrophobic amino acid stretch found the middle region of and 2) the of α- and β-syn are significantly The ability of a protein containing the amino of α-syn and the of β-syn to assemble into filaments that within the carboxyl-terminal region of α- and β-syn not for the of filament by β-syn. Conversely, a 12-amino acid deletion of the middle hydrophobic region within α-syn polymerization. The of the hydrophobic region is further supported by the of the of a single amino acid to a charged The introduction of of these single did not inhibit but it did the rate of filament the Thus, these that the introduction of a single charged is not sufficient to the hydrophobic filament formation. of and assembled α-syn from digestion was to for of the protein in the because they are likely to be in filament proteolytic resistant were These to the middle hydrophobic region and amino acids which has also termed the nonamyloid component of amyloid plaques K. Fukushima H. Masliah E. Xia Y. A. Yoshimoto M. J. Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar). The peptide was from fractions in amyloid from Alzheimer's disease and it was to be the component of plaques after this peptide is an intrinsic component of amyloid plaques is a of K. Fukushima H. Masliah E. Xia Y. A. Yoshimoto M. J. Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar, E. A. Mallory M. Uéda K. Saitoh T. Am. J. Pathol. Google Scholar, S. F. T. K. Am. J. Pathol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, P. T. L. P. K. Neurosci. Lett. 1999; PubMed Scopus Google Scholar). It may have as a of amyloid brains from Alzheimer's disease of α-syn in and in Lewy The proteolytic of this region within fibrillar α-syn in may the peptide was with amyloid both and may be to by purification to the protease that was in the of To further the role of the hydrophobic region in the polymerization of α-syn, a synthetic peptide corresponding to amino acids 71–82 in human α-syn was assayed for its ability to filaments and to with full-length α-syn. 71–82 the for α-syn, and it further the assembly of α-syn. with these and the notion that the hydrophobic region is for it was that a peptide corresponding to amino acids in in can also form filaments, but not a peptide corresponding to amino acids in in O.M.A. Jakes R. Curran M.D. D. R. E. A. D. Wallace A. FEBS Lett. 1998; 440: PubMed Scopus Google Scholar). Furthermore, a carboxyl-terminal α-syn not formed filaments, it assembled full-length α-syn J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). these the that of α-syn and the accumulation of a the hydrophobic region may as a for the of from dementia with LB brains α-syn (1Baba M. Nakajo S. Tu P. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar). The of β-syn to form filaments likely the of β-syn within pathological lesions by α-syn The is a J.E. Uryu K. Lee V.M.-Y. Trojanowski J.Q. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar) in which an accumulation of both α- and β-syn was in that on neurons in with Parkinson's disease dementia with this is from other α-syn because it is not by the presence of fibrils as in and glial cytoplasmic The accumulation of α- and β-syn in is likely to the of presynaptic of α-syn a notion that is with the accumulation of other proteins as and J.E. Uryu K. Lee V.M.-Y. Trojanowski J.Q. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). of the protein in the and of pathological inclusions is to at the of pathological α-syn for may that with the assembly by to the hydrophobic as by the peptide corresponding to amino acids 71–82 in human α-syn, an to that by that α-syn from into a may be this is with filament in vitro, and α-syn is also likely to be in a as by the of pathological lesions with J.E. Lee V.M.-Y. Trojanowski J.Q. J. Neurosci. Res. PubMed Scopus Google Scholar). of in vitro assembly and the of in are key to and for diseases by α-syn K. S. and for with and the of the of for with the