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Elucidation of Primary Structure Elements Controlling Early Amyloid β-Protein Oligomerization

2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês

10.1074/jbc.m300825200

ISSN

1083-351X

Autores

Gal Bitan, Sabrina S. Vollers, David B. Teplow,

Tópico(s)

Prion Diseases and Protein Misfolding

Resumo

Assembly of monomeric amyloid β-protein (Aβ) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight Aβ40 and Aβ42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight Aβ40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight Aβ42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant Aβ alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in Aβ42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus self-association. Aβ40 oligomerization was particularly sensitive to substitutions of Glu22 or Asp23 and to truncation of the N terminus, but not to substitutions of Phe19 or Ala21. Aβ42 oligomerization, in contrast, was largely unaffected by substitutions at positions 22 or 23 or by N-terminal truncations, but was affected significantly by substitutions of Phe19 or Ala21. These results reveal how specific regions and residues control Aβ oligomerization and show that these controlling elements differ between Aβ40 and Aβ42. Assembly of monomeric amyloid β-protein (Aβ) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight Aβ40 and Aβ42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight Aβ40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight Aβ42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant Aβ alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in Aβ42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus self-association. Aβ40 oligomerization was particularly sensitive to substitutions of Glu22 or Asp23 and to truncation of the N terminus, but not to substitutions of Phe19 or Ala21. Aβ42 oligomerization, in contrast, was largely unaffected by substitutions at positions 22 or 23 or by N-terminal truncations, but was affected significantly by substitutions of Phe19 or Ala21. These results reveal how specific regions and residues control Aβ oligomerization and show that these controlling elements differ between Aβ40 and Aβ42. 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However, the biophysical and structural characterization of oligomeric Aβ assemblies has been difficult due to their metastable nature. Previously, we demonstrated that the size distribution of Aβ oligomers could be determined quantitatively (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 27Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335Crossref PubMed Scopus (1106) Google Scholar) using the technique photo-induced cross-linking of unmodified proteins (PICUP) (28Fancy D.A. Kodadek T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6020-6024Crossref PubMed Scopus (442) Google Scholar, 29Fancy D.A. Denison C. Kim K. Xie Y.Q. Holdeman T. Amini F. Kodadek T. Chem. Biol. 2000; 7: 697-708Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). This approach revealed that low molecular weight (LMW) Aβ40, rather than existing in a stable monomeric or dimeric state, as previously suggested (30Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 31Garzon-Rodriguez W. Sepulveda-Becerra M. Milton S. Glabe C.G. J. Biol. Chem. 1997; 272: 21037-21044Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), comprised a mixture of monomer, dimer, trimer, and tetramer in rapid equilibrium. In contrast, LMW Aβ42 produced a distinct oligomer distribution in which the main components were pentamer/hexamer units (paranuclei), which then formed larger oligomers through self-association (27Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335Crossref PubMed Scopus (1106) Google Scholar). Here, PICUP was used to determine systematically the effects of primary structure modifications on early Aβ oligomerization. We studied Aβ42 analogues bearing modifications at the C-terminal dipeptide, Aβ40 and Aβ42 analogues containing clinically relevant mutations at or near the central hydrophobic cluster (CHC), N-terminally truncated alloforms of Aβ40 and Aβ42 found in amyloid plaques, and Aβ40 analogues containing substitutions that alter the net charge of the peptide. The results advance our understanding of early Aβ assembly, provide deeper mechanistic insight into the distinct oligomerization behaviors of Aβ40 and Aβ42, and suggest new targets for AD therapy. Peptides and Reagents—Aβ40, Aβ42, and analogues thereof (Table I) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, purified by reversed-phase high performance liquid chromatography, and characterized by mass spectroscopy and amino acid analysis, as described (32Lomakin A. Chung D.S. Benedek G.B. Kirschner D.A. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1125-1129Crossref PubMed Scopus (736) Google Scholar). Tris(2,2′-bipyridyl)dichlororuthenium(II) (Ru(Bpy)) and ammonium persulfate were purchased from Aldrich. Polyacrylamide gels, buffers, stains, standards, and equipment for SDS-PAGE were from Invitrogen.Table IAβ peptides: primary structure and isoelectric point (pI)aThe WT Aβ42 sequence is: (H)-DAEFRHDSGYEVHHQKLVFFAEDVSGNKGAIIGLMVGGVVIA-(OH).GroupPositionModificationbThe symbols Δ and < signify amino acid deletion and pyroglutamyl N terminus, respectively. The common name for each mutation is listed parenthetically.Aβ40cThe columns indicate on which backbone the primary structure changes were made. The symbols + and - signify whether the specific peptide was or was not studied.Aβ42cThe columns indicate on which backbone the primary structure changes were made. The symbols + and - signify whether the specific peptide was or was not studied.pIdIsoelectric point (pI) values were estimated using MacVector, v. 7.1 (Accelrys, San Diego, CA).N terminus1-2Δ++5.771-2Δ, <Glu3+-6.031-4Δ++6.301-9Δ++6.031-10Δ++6.031-10Δ, <Glu11+-6.03Central regionPhe19Pro++5.22Ala21Gly (Flemish)++5.22Glu22Gly (Arctic)++5.76Glu22Gln (Dutch)++5.76Glu22Lys (Italian)++6.31Asp23Asn (Iowa)++5.77C terminusIle41Gly-+5.22Ile41Ala-+5.22Ile41Val-+5.22Ile41Leu-+5.22Ala42Gly-+5.22Ala42Val-+5.22-COOH-CONH2-+5.77Asp/HisAsp1Asn+-5.77Asp7Asn+-5.77Asp23Asn+-5.77His6Gln+-4.74His13Gln+-4.74His14Gln+-4.74Asp1,7,23Asn+-7.26His6,13,14Gln+-4.08Asp1,7,23 His6,13,14Asn1,7,23 Gln6,13,14+-6.36a The WT Aβ42 sequence is: (H)-DAEFRHDSGYEVHHQKLVFFAEDVSGNKGAIIGLMVGGVVIA-(OH).b The symbols Δ and < signify amino acid deletion and pyroglutamyl N terminus, respectively. The common name for each mutation is listed parenthetically.c The columns indicate on which backbone the primary structure changes were made. The symbols + and - signify whether the specific peptide was or was not studied.d Isoelectric point (pI) values were estimated using MacVector, v. 7.1 (Accelrys, San Diego, CA). Open table in a new tab Isolation of LMW Aβ—LMW fractions of Aβ alloforms were isolated by size exclusion chromatography (SEC), as described previously (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Briefly, 170 μl of a 2 mg/ml peptide solution prepared in Me2SO was fractionated using a 10/30 Superdex 75 HR column eluted at 0.5 ml/min with 10 mm sodium phosphate, pH 7.4. Peaks were detected by UV absorbance at 254 nm. A 10-μl aliquot of each fraction was taken for amino acid analysis a posteriori to determine the peptide concentration. Typical concentrations were 30 ± 10 μm. SEC reproducibly yielded comparable LMW fractions for all of the peptides used. Recent studies (27Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335Crossref PubMed Scopus (1106) Google Scholar) have shown that LMW Aβ prepared using SEC readily forms paranuclei and higher order oligomers, facilitating study of these assembly processes. For this purpose, SEC was superior to base pretreatment protocols (34Fezoui Y. Hartley D.M. Harper J.D. Khurana R. Walsh D.M. Condron M.M. Selkoe D.J. Lansbury P.T. Fink A.L. Teplow D.B. Amyloid: Int. J. Exp. Clin. Invest. 2000; 7: 166-178Crossref PubMed Scopus (232) Google Scholar). Cross-linking and SDS-PAGE Analysis—Freshly isolated LMW peptides were immediately subjected to PICUP, as described (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Briefly, 1 μl of 1 mm Ru(Bpy) and 1 μl of 20 mm ammonium persulfate in 10 mm sodium phosphate, pH 7.4, were added to 18 μl of freshly isolated LMW peptide. The mixture was irradiated for 1 s with visible light, and the reaction was quenched immediately with 10 μl tricine sample buffer (Invitrogen) containing 5% β-mercaptoethanol. Concentration differences caused some variability in the relative abundance of each oligomer but did not alter the overall profile of the oligomer size distribution of each peptide (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). To examine further the question of whether inter-peptide oligomerization differences reflected fundamental differences in peptide assembly and not simply the effects of differing peptide concentrations, a series of cross-linking experiments were performed using Aβ40 and Aβ42 at varying concentrations. At all concentrations, from 1–300 μm, the oligomer size distributions of Aβ40 were distinct from those of Aβ42 (data not shown). This confirmed that the assembly differences we observed in the experiments reported here, and in prior work (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 27Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335Crossref PubMed Scopus (1106) Google Scholar), reflected fundamental differences between peptides and not concentration effects. In principle, radical formation may occur at any site along the polypeptide chain. However, the radical would form at, and further react preferentially with, amino acid side-chains that offer stabilization through aromatic or neighboring-group effects (e.g. Tyr, Phe, or Met (35Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (388) Google Scholar). The primary factors determining how Aβ is cross-linked are the proximity of a susceptible group to the radical and the lifetime of the radical itself. If the lifetime of the radical is long enough to allow intermolecular cross-linking as opposed to quenching by solvent, then the actual chemical nature of the radical is relatively unimportant. Cross-linked Aβ samples were analyzed by SDS-PAGE and silver staining, as described previously (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). The nominal total amount of peptide in each lane of the gels was identical. Gels were dried, scanned, and the intensities of the resulting monomer and oligomer bands quantified by densitometry using the program One-Dscan (Scanalytics, Fairfax, VA). The densitometric data for each of the figures is available as supporting online material. The relative amount of each band, as a percentage of all bands, was determined by calculating the quotient of its intensity and the sum of all band intensities and then multiplying by 100. Effects of Structural Modifications at the C Terminus of Aβ42 on Oligomerization—Our previous studies (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 27Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335Crossref PubMed Scopus (1106) Google Scholar) revealed that LMW Aβ40 and Aβ42 had distinct oligomer size distributions. LMW Aβ40 existed as an equilibrium mixture of monomer, dimer, trimer, and tetramer (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). In contrast, LMW Aβ42 comprised three groups of oligomers: 1) monomer through trimer, displaying decreasing intensity with increasing oligomer order; 2) a Gaussian-like distribution between tetramer and octamer, with a maximum at pentamer and hexamer; and 3) oligomers of M r ∼30–60 kDa, among which two intensity maxima, at dodecamer and octadecamer, were observed (27Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335Crossref PubMed Scopus (1106) Google Scholar). These higher order oligomers appeared to form through self-association of pentamer/hexamer units (paranuclei). The data demonstrated that Ile41 was essential for formation of paranuclei, whereas Ala42 was required for rapid self-association of paranuclei into larger oligomers (27Bitan G. Kirkitadze M.D. Lomakin A. Vollers S.S. Benedek G.B. Teplow D.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335Crossref PubMed Scopus (1106) Google Scholar). To better understand how amino acid side-chain structure in the C-terminal dipeptide of Aβ42 controls oligomerization, seven peptide alloforms (C terminus, Table I) were prepared and their oligomer size distributions were determined by PICUP/SDS-PAGE (Fig. 1). Substitution of Ile41 by Gly, eliminating both the side-chain and the stereocenter of the Cα group, yielded a distribution that was qualitatively similar to that of Aβ40 (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) (cf. Fig. 1, lane 2 and Fig. 2A , lane 1). Monomer through trimer formed in similar amounts, and a rapid decrease in oligomer abundance was observed above trimer, whereas Aβ40 showed abundant tetramer, above which abundances decreased. [Gly41]Aβ42 thus existed in a dynamic equilibrium involving monomer, dimer, and trimer, but not tetramer (the mathematical foundation for this analysis has been published (26Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar)). When Ile41 was substituted by Ala, dimer and trimer were the predominant cross-linking products, a characteristic of Aβ40 distributions. However, in contrast to wild type (WT) Aβ40 distributions, tetramer abundance was low, whereas pentamer abundance was relatively high. This polytonic distribution is characteristic of Aβ42. The oligomer size distribution of [Ala41]Aβ42 thus appeared to be a composite of Aβ40- and Aβ42-like distributions. The majority of the peptide existed as an equilibrium mixture of monomer, dimer, and trimer, as in the case of [Gly41]Aβ42, but the methyl side-chain of Ala41 facilitated limited paranucleus formation. Substitution of Ile41 by Val or Leu led to formation of abundant paranuclei. However, in the oligomer size distribution of [Val41]Aβ42, no oligomers at ∼30–60 kDa were detected. In the distribution of [Leu41]Aβ42 the amount of these high molecular weight oligomers was substantially reduced relative to that of WT Aβ42. Consistent with the low amounts of high molecular weight oligomers, a higher abundance of dimer and trimer were observed for [Val41]Aβ42 and [Leu41]Aβ42 (∼18% dimer and ∼14% trimer for each) relative to WT Aβ42 (∼14% dimer and ∼10% trimer), demonstrating that these oligomers were in equilibrium with tetramer through octamer. Thus, the side-chain in position 41 is involved in the formation and self-association of Aβ42 paranuclei. Examination of the distributions produced by Gly41-, Ala41-, and Val41-substituted Aβ42 reveals a correlation between paranucleus formation and side-chain size. [Val41]Aβ42 and [Leu41]Aβ42, containing iso-propyl and iso-butyl side-chains, respectively, do not facilitate self-association of paranuclei, whereas the sec-butyl side-chain of Ile does. Substitution of Ala42 by Gly or Val had little effect on formation of paranuclei (Fig. 1). However, no high molecular mass oligomers (∼30–60 kDa) were observed for [Gly42]Aβ42, demonstrating a role for the methyl side-chain of Ala42 in the self-association of paranuclei. When the C-terminal carboxyl group was replaced by a carboxamide, a large increase in the abundance of high molecular weight oligomers was seen (Fig. 1). Thus, hydrophobic interactions involving the side-chains in residues 41 and 42 appear to be a driving force in the association of Aβ42 paranuclei into higher oligomers, whereas the C-terminal carboxylate anion moderates this assembly effect. Effects of Structural Modifications in the Central Region of Aβ on Oligomerization—Five naturally occurring, autosomal dominant mutations in the AβPP gene region encoding the CHC of amino acids in Aβ have been reported (25Nilsberth C. Westlind-Danielsson A. Eckman C.B. Condron M.M. Axelman K. Forsell C. Stenh C. Luthman J. 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