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Seeding Specificity in Amyloid Growth Induced by Heterologous Fibrils

2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês

10.1074/jbc.m311300200

1083-351X

Brian O’Nuallain, Angela D. Williams, Per Westermark, Ronald Wetzel,

Aluminum toxicity and tolerance in plants and animals

Over residues 15–36, which comprise the H-bonded core of the amyloid fibrils it forms, the Alzheimer's disease plaque peptide amyloid β (Aβ) possesses a very similar sequence to that of another short, amyloidogenic peptide, islet amyloid polypeptide (IAPP). Using elongation rates to quantify seeding efficiency, we inquired into the relationship between primary sequence similarity and seeding efficiency between Aβ-(1–40) and amyloid fibrils produced from IAPP as well as other proteins. In both a solution phase and a microtiter plate elongation assay, IAPP fibrils are poor seeds for Aβ-(1–40) elongation, exhibiting weight-normalized efficiencies of only 1–2% compared with Aβ-(1–40) fibrils. Amyloid fibrils of peptides with sequences completely unrelated to Aβ also exhibit poor to negligible seeding ability for Aβ elongation. Fibrils from a number of point mutants of Aβ-(1–40) exhibit intermediate seeding abilities for wild-type Aβ elongation, with differing efficiencies depending on whether or not the mutation is in the amyloid core region. The results suggest that amyloid fibrils from different proteins exhibit structural differences that control seeding efficiencies. Preliminary results also suggest that identical sequences can grow into different conformations of amyloid fibrils as detected by seeding efficiencies. The results have a number of implications for amyloid structure and biology. Over residues 15–36, which comprise the H-bonded core of the amyloid fibrils it forms, the Alzheimer's disease plaque peptide amyloid β (Aβ) possesses a very similar sequence to that of another short, amyloidogenic peptide, islet amyloid polypeptide (IAPP). Using elongation rates to quantify seeding efficiency, we inquired into the relationship between primary sequence similarity and seeding efficiency between Aβ-(1–40) and amyloid fibrils produced from IAPP as well as other proteins. In both a solution phase and a microtiter plate elongation assay, IAPP fibrils are poor seeds for Aβ-(1–40) elongation, exhibiting weight-normalized efficiencies of only 1–2% compared with Aβ-(1–40) fibrils. Amyloid fibrils of peptides with sequences completely unrelated to Aβ also exhibit poor to negligible seeding ability for Aβ elongation. Fibrils from a number of point mutants of Aβ-(1–40) exhibit intermediate seeding abilities for wild-type Aβ elongation, with differing efficiencies depending on whether or not the mutation is in the amyloid core region. The results suggest that amyloid fibrils from different proteins exhibit structural differences that control seeding efficiencies. Preliminary results also suggest that identical sequences can grow into different conformations of amyloid fibrils as detected by seeding efficiencies. The results have a number of implications for amyloid structure and biology. One of the major unanswered questions of amyloid fibril formation is the specificity with which primary sequence determines both amyloidogenicity and the details of amyloid structure. The observation that many globular proteins can be induced to generate amyloid 1Amyloid fibrils are classically defined as originating in tissue-associated deposits that yield bright green birefringence under the polarizing light microscope after Congo red staining. We refer here to in vitro generated fibrils as amyloid, cognizant that they may not resemble tissue-derived amyloid fibrils in all structural and functional respects (see “Discussion”). fibrils, albeit in some cases only under non-native conditions, has led to the notion that the amyloid folding motif is a kind of default structure in the protein folding pathway that many or all polypeptides can engage when their normal folding pathways are compromised (1Fandrich M. Fletcher M.A. Dobson C.M. Nature. 2001; 410: 165-166Crossref PubMed Scopus (735) Google Scholar, 2Dobson C.M. Biochem. Soc. Symp. 2001; 68: 1-26Crossref PubMed Scopus (123) Google Scholar). This seems a reasonable hypothesis, especially if the dominant driving force for amyloid formation is the formation of the backbone H-bonding network of these β-sheet-rich structures (3Fandrich M. Dobson C.M. EMBO J. 2002; 21: 5682-5690Crossref PubMed Scopus (464) Google Scholar), since all polypeptides regardless of sequence share the polypeptide backbone. Amyloid stability, however, appears to be derived from a combination of forces, including but not limited to H-bonding, much in the manner of how globular proteins are stabilized (4Williams A. Portelius E. Kheterpal I. Guo J. Cook K. Xu Y. Wetzel R. J. Mol. Biol. 2004; 335: 833-842Crossref PubMed Scopus (346) Google Scholar). In addition, mutations associated with modest changes in side chain hydrophobicity, charge, etc., have been observed to have significant impact on fibril growth kinetics and stability (5Hilbich C. Kisters-Woike B. Reed J. Masters C.L. Beyreuther K. J. Mol. Biol. 1992; 228: 460-473Crossref PubMed Scopus (380) Google Scholar, 6Wood S.J. Wetzel R. Martin J.D. Hurle M.R. Biochem. 1995; 34: 724-730Crossref PubMed Scopus (319) Google Scholar, 7Esler W.P. Stimson E.R. Ghilardi J.R. Lu Y.A. Felix A.M. Vinters H.V. Mantyh P.W. Lee J.P. Maggio J.E. Biochemistry. 1996; 35: 13914-13921Crossref PubMed Scopus (176) Google Scholar, 8Morimoto A. Irie K. Murakami K. Ohigashi H. Shindo M. Nagao M. Shimizu T. Shirasawa T. Biochem. Biophys. Res. Commun. 2002; 295: 306-311Crossref PubMed Scopus (50) Google Scholar, 9Murakami K. Irie K. Morimoto A. Ohigashi H. Shindo M. Nagao M. Shimizu T. Shirasawa T. Biochem. Biophys. Res. Commun. 2002; 294: 5-10Crossref PubMed Scopus (123) Google Scholar, 10Chiti F. Stefani M. Taddei N. Ramponi G. Dobson C.M. Nature. 2003; 424: 805-808Crossref PubMed Scopus (931) Google Scholar). These considerations raise an interesting conundrum; if side chain packing contributes significantly to amyloid stability, how is it possible that so many proteins whose sequences evolved to fold into stable, globular structures (including many that contain little or no β-sheet structure in the native state) can be equally well accommodated into the β-sheet-rich amyloid folding motif? Does amyloid fibril formation follow the same or different folding rules compared with other collapsed states of polypeptide sequences? One measure of packing specificity in the amyloid fibril is the efficiency with which fibrils composed of one protein sequence can act as seeds for the elongation of another amyloidogenic polypeptide. The ability of the folded structure at the growth point of the amyloid fibril to serve as a template for the recruitment of an incoming monomer can be likened to the abilities of domains and subdomains of globular proteins to pack together with high complementarity and specificity. Although such “cross-seeding” between two different amyloid proteins has been described (11Jarrett J.T. Lansbury Jr., P.T. Biochemistry. 1992; 31: 12345-12352Crossref PubMed Scopus (280) Google Scholar, 12Han H. Weinreb P.H. Lansbury Jr., P.T. Chem. Biol. 1995; 2: 163-169Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 13Hasegawa K. Yamaguchi I. Omata S. Gejyo F. Naiki H. Biochemistry. 1999; 38: 15514-15521Crossref PubMed Scopus (193) Google Scholar, 14Morozova-Roche L.A. Zurdo J. Spencer A. Noppe W. Receveur V. Archer D.B. Joniau M. Dobson C.M. J. Struct. Biol. 2000; 130: 339-351Crossref PubMed Scopus (289) Google Scholar), the data are often qualitative and/or difficult to assess quantitatively due to limitations in our understanding of the amyloid assembly mechanism. For example, it is well known that the addition of a fibril seed can eliminate or reduce the lag time in the spontaneous, nucleation-dependent generation of Aβ 2The abbreviations used are: Aβ, amyloid β protein; IAPP, islet amyloid polypeptide; ThT, thioflavin T; PBS, phosphate-buffered saline; PBSA, PBS plus sodium azide; WT, wild type; AD, Alzheimer's disease; HPLC, high performance liquid chromatography. fibrils from rigorously disaggregated monomers (15Evans K.C. Berger E.P. Cho C.-G. Weisgraber K.H. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 763-767Crossref PubMed Scopus (344) Google Scholar, 16Wood S.J. Chan W. Wetzel R. Biochemistry. 1996; 35: 12623-12628Crossref PubMed Scopus (106) Google Scholar). However, it is not clear how to quantitatively interpret reductions in lag time or to compare one fibril to another with respect to their relative abilities as seeds to reduce lag times. Furthermore, some previous experimental explorations of seeding and cross-seeding may also have been compromised by the use of monomeric protein preparations that contained small but significant amounts of aggregate seeds. Cross-seeding ability is not only of interest in terms of the structural aspects of amyloid fibril formation. Although the mechanisms by which amyloid fibril formation is initiated in vivo are still being worked out, it is very likely that, once these initial seeds are formed, fibrils grow in vivo through elongation by monomer additions (17Esler W.P. Stimson E.R. Ghilardi J.R. Vinters H.V. Lee J.P. Mantyh P.W. Maggio J.E. Biochemistry. 1996; 35: 749-757Crossref PubMed Scopus (124) Google Scholar). The ability of amyloid fibrils to be efficiently elongated in vivo has impact on a number of biological aspects of amyloid function. These include the questions of species and strain specificity in yeast (18Chien P. Weissman J.S. Nature. 2001; 410: 223-227Crossref PubMed Scopus (133) Google Scholar, 19DePace A.H. Weissman J.S. Nat. Struct. Biol. 2002; 9: 389-396PubMed Google Scholar) and mammalian (20Horiuchi M. Priola S.A. Chabry J. Caughey B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5836-5841Crossref PubMed Scopus (137) Google Scholar) prions, the feasibility of the recruitment-sequestration mechanism (21McCampbell A. Fischbeck K.H. Nat. Med. 2001; 7: 528-530Crossref PubMed Scopus (78) Google Scholar, 22Chen S. Berthelier V. Yang W. Wetzel R. J. Mol. Biol. 2001; 311: 173-182Crossref PubMed Scopus (282) Google Scholar), and the ability of short poly-Gln sequences to influence the nucleation of long poly-Gln repeats 3A. M. Bhattacharyya and R. Wetzel, submitted for publication. in diseases such as Huntington's disease, the specificity of intracellular protein aggregation (24Rajan R.S. Illing M.E. Bence N.F. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13060-13065Crossref PubMed Scopus (187) Google Scholar), and the better understanding of the mechanism of amyloid enhancement factor in the classic mouse amyloidosis model (25Lundmark K. Westermark G.T. Nystrom S. Murphy C.L. Solomon A. Westermark P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6979-6984Crossref PubMed Scopus (235) Google Scholar). Promiscuous cross-seeding among amyloid fibrils would suggest the possibility that one amyloid disease might influence another. For example, because the vast majority of type II diabetes patients have pancreatic amyloid deposits composed of islet amyloid polypeptide (26Westermark P. Wilander E. Diabetologia. 1978; 15: 417-421Crossref PubMed Scopus (217) Google Scholar) and because IAPP and Aβ share striking sequence similarity (see “Results”), it might be possible that cross-seeding of Aβ amyloid formation by IAPP fibrils is the mechanism by which diabetics appear to exhibit an elevated risk for developing AD (27Ott A. Stolk R.P. van Harskamp F. Pols H.A. Hofman A. Breteler M.M. Neurology. 1999; 53: 1937-1942Crossref PubMed Google Scholar). To test this latter hypothesis, we studied the abilities of Aβ and IAPP fibrils to act as seeds for the elongation reactions of Aβ and IAPP monomeric peptides. We used the pseudo-first order elongation kinetics in two separate assay modes as measures of relative seeding ability. To put these results into context we also surveyed a broad spectrum of amyloid fibrils as seeds for Aβ elongation. The results show that in general seeding of fibril elongation is highly specific, being exquisitely sensitive to point mutations at certain positions in the amyloidogenic peptide. IAPP fibrils prove to be very poor seeds for Aβ-(1–40) elongation, suggesting that heterologous seeding of amyloid formation probably does not influence AD risk in diabetics. Materials—Synthetic wild-type Aβ-(1–40) and Aβ-(1–42), the “Arctic” mutant (E22G), some proline mutants, and a Cys-1 analog of Aβ-(1–40) were all obtained via custom syntheses from the Keck Biotechnology Center at Yale University. The 1–30 fragment of the immunoglobulin light chain Len and oxidized, full-length, C-terminal-amidated IAPP were also obtained from the Keck Center. Aβ-(1–40) proline mutants F4P, R6P, and L17P were obtained from the Stanford University Protein and Nucleic Acids Biotechnology Facility. Recombinant human immunoglobulin κ6 light chain variable domain (JTO5) was a gift of Dr. Jonathon Wall (University of Tennessee Graduate School of Medicine), and Aβ-(25–35) was a gift of Charles Murphy (University of Tennessee Graduate School of Medicine). Amyloid-like aggregates of synthetic Gln28 and Gln47 polyglutamine peptides (28Chen S. Berthelier V. Hamilton J.B. O'Nuallain B. Wetzel R. Biochemistry. 2002; 41: 7391-7399Crossref PubMed Scopus (282) Google Scholar) were obtained from Tina Richey (University of Tennessee Graduate School of Medicine). Amyloid fibrils of β2-microglobulin were gifts of Susan Jones and Sheena Radford (University of Leeds). Fibrils of the yeast prion protein Ure2p (29Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1343Crossref PubMed Scopus (264) Google Scholar) were gifts of Kimberley Taylor and Reed Wickner (National Institutes of Health). Bovine collagen and bovine serum albumin (essentially fatty acid free) were purchased from Sigma. N-terminal-biotinylated Aβ-(1–40) was prepared by alkylating a Cys-1 analog of Aβ-(1–40) (Keck Biotechnology Center custom synthesis) with PEO (polyethylene oxide)-iodoacetyl biotin (Pierce). All other chemicals were of analytical grade. General Methods—The concentrations of disaggregated peptides were estimated from peak areas of the A215 absorbance trace in analytical HPLC of aliquots of peptide solutions using standard curves generated from peptide standards calibrated by amino acid composition analysis, as described previously (30Kheterpal I. Williams A. Murphy C. Bledsoe B. Wetzel R. Biochemistry. 2001; 40: 11757-11767Crossref PubMed Scopus (198) Google Scholar). Reverse phase HPLC was carried out on a ZORBAX SB-C3 column (Agilent Technologies) with a 1–51% (v/v) acetonitrile gradient in 0.05% aqueous trifluoroacetic acid (Pierce). ThT fluorescence measurements (31LeVine H. Methods Enzymol. 1999; 309: 274-284Crossref PubMed Scopus (1213) Google Scholar) of reaction samples were carried out by diluting aliquots of the reaction into PBS containing 10 μm ThT. Thioflavin T fluorescence was then monitored by excitation at 450 nm and fluorescence emission at 482 nm. Unless otherwise indicated, all quantitative experimental results shown are from measurements done in triplicate. Error bars in the figures represent S.D. Preparation of Soluble, Disaggregated Peptides, and Sonicated Fibril Seeds—Wild-type and mutant Aβ peptides were treated to remove aggregates as described (30Kheterpal I. Williams A. Murphy C. Bledsoe B. Wetzel R. Biochemistry. 2001; 40: 11757-11767Crossref PubMed Scopus (198) Google Scholar, 32Kheterpal I. Zhou S. Cook K.D. Wetzel R.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13597-13601Crossref PubMed Scopus (166) Google Scholar). Thus, synthetic lyophilized powder was exposed to sequential applications of trifluoroacetic acid and hexafluoroisopropanol and evaporated under an argon stream, trace volatile solvents were removed under high vacuum, and the peptide residue was dissolved in 2 mm NaOH followed immediately with the addition of a 10× PBS buffer to generate 1× PBS plus 0.05% sodium azide (PBSA). To remove any residual aggregates these solutions were centrifuged (51,500 × g, 17 h, 4 °C), and the upper [¾] of the supernatant was carefully removed and analyzed for Aβ concentration by HPLC as described above (generally about 60 μm). Solutions were used immediately to conduct kinetics experiments (described below) or to make fibrils. Fibrils were made by spontaneous fibril assembly from disaggregated monomeric peptide as described previously (33O'Nuallain B. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1485-1490Crossref PubMed Scopus (301) Google Scholar). For some less stable proline mutants of Aβ-(1–40), fibril formation reactions were conducted at higher concentrations of monomeric peptide (4Williams A. Portelius E. Kheterpal I. Guo J. Cook K. Xu Y. Wetzel R. J. Mol. Biol. 2004; 335: 833-842Crossref PubMed Scopus (346) Google Scholar). Fibril formation reactions were monitored by ThT fluorescence and by analysis of centrifugation supernatants by HPLC. Human IAPP was solubilized and disaggregated using a 1:1 mixture of trifluoroacetic acid/hexafluoroisopropanol (34Chen S. Wetzel R. Protein Sci. 2001; 10: 887-891Crossref PubMed Scopus (156) Google Scholar) as previously described (33O'Nuallain B. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1485-1490Crossref PubMed Scopus (301) Google Scholar). Disaggregated IAPP (about 30 μm) was used immediately in elongation kinetics assays (described below) or to make fibrils as described previously (33O'Nuallain B. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1485-1490Crossref PubMed Scopus (301) Google Scholar). JTO5 fibrils were prepared as previously described (35Wall J. Schell M. Murphy C. Hrncic R. Stevens F.J. Solomon A. Biochemistry. 1999; 38: 14101-14108Crossref PubMed Scopus (160) Google Scholar). Preparation of reduced and alkylated ovalbumin (ovalbumin-RA) was described previously (33O'Nuallain B. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1485-1490Crossref PubMed Scopus (301) Google Scholar). To improve seeding potency (11Jarrett J.T. Lansbury Jr., P.T. Biochemistry. 1992; 31: 12345-12352Crossref PubMed Scopus (280) Google Scholar) and potentially normalize fibril size all aggregates were sonicated with a probe sonicator on ice for 2.5 min in 30-s bursts in PBSA containing 1 mm dithiothreitol (a precaution against oxidation; however, we have no evidence that oxidation occurs during sonication in the absence of dithiothreitol). The fact that the series of mutated Aβ fibrils yields results consistent with what is known about Aβ fibril structure (see “Results” and “Discussion”) suggests that the sonication step does produce fibrils with very similar average molecular weights, which can therefore be assumed to have approximately the same number of growth sites per weight of fibril. Recent experimental titration of fibril growth sites further supports this assumption. 4S. Shivaprasad, B. O'Nuallain, and R. Wetzel, unpublished data. Initial IAPP aggregates from spontaneous aggregation experiments consist mainly of spheroidal aggregates and smaller amounts of amyloid fibrils and amorphous material (see “Results”). Except for collagen and reduced and alkylated ovalbumin, other aggregates prepared for use as seeds exhibited typical amyloid fibril structure in electron micrographs as well as typical ThT responses (data not shown). Solution Phase Aggregation Assays—Aggregation kinetics studies were conducted using relatively high concentrations of peptide using modifications of the procedure described by Naiki and Nakakuki (36Naiki H. Nakakuki K. Lab. Invest. 1996; 74: 374-383PubMed Google Scholar) and Naiki and Gejyo (37Naiki H. Gejyo F. Methods Enzymol. 1999; 309: 305-318Crossref PubMed Scopus (267) Google Scholar). A typical solution phase assay was carried out by incubating (unshaken) a solution of ∼30 μm concentrations of freshly disaggregated Aβ or IAPP in PBSA in a water bath at 37 °C. For seeded reactions, solutions were preincubated to reach temperature, then seeded with 10–70 μg/ml aggregate at time 0. Reactions were followed by removing aliquots of gently mixed suspensions and measuring ThT fluorescence. For each time point, an aliquot was removed from each of three independent samples and diluted into PBSA containing ThT. Because the ThT signal varies linearly with fibril mass for any given amyloid fibril, the signal can be used as a measure of the completeness of the fibril formation reaction and, when appropriately plotted, will yield values for the pseudo-first order rate constant (37Naiki H. Gejyo F. Methods Enzymol. 1999; 309: 305-318Crossref PubMed Scopus (267) Google Scholar). Solid Phase, Microtiter Plate-based Aggregation Assays—Progress curves for polypeptide aggregation were also determined using a solid phase microtiter plate assay modified from previously described methods (38Esler W.P. Stimson E.R. Ghilardi J.R. Felix A.M. Lu Y.A. Vinters H.V. Mantyh P.W. Maggio J.E. Nat. Biotechnol. 1997; 15: 258-263Crossref PubMed Scopus (80) Google Scholar, 39Berthelier V. Wetzel R. Potter N.T. Methods in Molecular Biology. Humana Press Inc., Totowa, NJ2003: 295-303Google Scholar). N-terminal-biotinylated Aβ-(1–40) (see “General Methods” under “Experimental Procedures”) was disaggregated, dissolved at a low micromolar concentration in PBS buffer, separated into aliquots, snap-frozen in liquid nitrogen, and stored at -80 °C. Sonicated fibrils or other aggregates were applied to the microplate wells by incubating 100 μl/well of a 1 μg/ml suspension of aggregate (i.e. 100 ng of aggregate/well) in PBSA for 18 h at 37 °C in activated high binding microtiter plates (EIA/RIA Plates, Costar, Atlanta, GA). The wells were then washed twice with PBSA containing 0.05% Tween 20 (Fisher) (assay buffer). This is the standard washing procedure carried out throughout this experiment. To ensure that aggregation rates are not significantly affected by variable efficiencies of aggregate adhesion to the plastic, we confirmed good immobilization by recovering unbound protein from the wells as part of the wash protocol and estimating the amount of non-absorbed protein using a protein assay (33O'Nuallain B. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1485-1490Crossref PubMed Scopus (301) Google Scholar). In all cases more than 95% of the aggregate remained on the plastic. Wells were blocked with 1% BSA in PBSA (blocking buffer) for 1 h at 37 °C, then 100 μl of assay buffer was added into each well, and the plate was sealed with an adhesive overlay. For the assay, the microtiter plate was pre-equilibrated at 37 °C. The kinetics data were collected by determining individual time points in reverse temporal order. For each replicate series of each analysis on the plate, a 100-μl aliquot of a 25 nm solution of biotinyl-Aβ in assay buffer was added to a well by first removing the assay buffer in which the well was stored. This constitutes the longest time point of the kinetics run. The plate was resealed and returned to 37 °C incubation. At the appropriate time, the plate was again removed, and the storage buffer was removed from the wells designated for this time point and replaced with fresh aliquots of biotinyl-Aβ solution. This process was repeated until the last incubation, constituting the earliest time point, was completed. After the last incremental incubation for the kinetics the plate was removed from 37 °C incubation and washed with assay buffer, 100 μl was added per well of a 1:1000 dilution of a europium-streptavidin conjugate (PerkinElmer Life Sciences) in blocking buffer containing 0.05% Tween 20, and the plate was incubated at room temperature for 1 h. The plate was then washed 3 times, and europium was released from the microplate well surface using 100 μl/well of chelation buffer (Enhancement Solution, PerkinElmer Life Sciences). After 10 min europium was measured by time-resolved fluorometry (40Diamandis E.P. Clin. Biochem. 1988; 21: 139-150Crossref PubMed Google Scholar) in a Victor2 1420 Multilabel Counter (PerkinElmer Life Sciences) using the programmed parameters for europium. fmol of europium were determined from a standard curve and converted to fmol of biotin using the stated europium content of the commercial streptavidin reagent. Congo Red Staining and Birefringence of IAPP Aggregates—Congo red staining of aggregates was carried out using a modified version (Sigma: procedure HT60) of the protocol of Puchtler et al. (41Puchtler H. Sweat F. Levine M. J. Histochem. Cytochem. 1962; 10: 355-364Crossref Google Scholar). For each experiment 20 μl of freshly prepared IAPP aggregates (about 0.2 mg/ml) was dried to a glass microscope slide overnight at 37 °C and then Congo red-stained. Birefringence was determined with an Olympus microscope (20×) equipped with a polarizing stage. Electron Micrographs—Aggregates (0.1–0.4 mg/ml) were adsorbed onto carbon and Formvar-coated copper grids and then negatively stained with 0.5% uranyl acetate. Stained samples were examined and photographed in a Hitachi H-800. Sequence Similarity between Aβ and IAPP—The Aβ peptide is derived from the proteolytic processing of the cross-membrane region of the receptor-like, type I cross-membrane protein amyloid-β protein precursor (42Selkoe D.J. Annu. Rev. Neurosci. 1994; 17: 489-517Crossref PubMed Scopus (829) Google Scholar). IAPP, or amylin, is derived from the proteolytic processing of a propeptide and appears to play a role along with insulin in the regulation of glucose metabolism (43Gebre-Medhin S. Mulder H. Pekny M. Westermark G. Tornell J. Westermark P. Sundler F. Ahren B. Betsholtz C. Biochem. Biophys. Res. Commun. 1998; 250: 271-277Crossref PubMed Scopus (138) Google Scholar). Despite the absence of a larger sequence homology between their precursor proteins and no obvious relationship in known functions, the mature peptides Aβ and IAPP have very similar sequences, as shown in Fig. 1. After introduction of a 1-residue gap in Aβ to maximize the alignment, the 2 peptides overall share 25% sequence identity and 50% sequence similarity (Fig. 1). When the overlap region is confined to that portion of the Aβ sequence, residues 15–37, known to be involved in the H-bonded core region of the amyloid fibril (4Williams A. Portelius E. Kheterpal I. Guo J. Cook K. Xu Y. Wetzel R. J. Mol. Biol. 2004; 335: 833-842Crossref PubMed Scopus (346) Google Scholar, 44Torok M. Milton S. Kayed R. Wu P. McIntire T. Glabe C.G. Langen R. J. Biol. 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Amyloid. 2001; 8: 242-249Crossref PubMed Scopus (52) Google Scholar), the extents of identity and similarity climb further to 43 and 70%, respectively. This striking similarity introduces the questions of whether these two highly amyloidogenic peptides use their shared sequences in similar ways to engage the amyloid folding motif and the extent to which they might thereby exhibit specificity in amyloid formation as measured by their sensitivities to heterologous seeding. The hypothesis that the shared sequence element is involved in the core structure of amyloid fibrils from both peptides is supported by the sharp reduction in amyloid growth observed when proline residues are substituted within this region in both Aβ (4Williams A. Portelius E. Kheterpal I. Guo J. Cook K. Xu Y. Wetzel R. J. Mol. Biol. 2004; 335: 833-842Crossref PubMed Scopus (346) Google Scholar) and IAPP (48Westermark P. Engstrom U. Johnson K.H. Westermark G.T. Betsholtz C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5036-5040Crossref PubMed Scopus (712) Google Scholar, 49Moriarty D.F. Raleigh D.P. Biochemistry. 1999; 38: 1811-1818Crossref PubMed Scopus (175) Google Scholar). Given these facts it is not unreasonable to suppose that a growing Aβ amyloid fibril might be able to relatively easily accommodate IAPP molecules into the fibril structure. It should be stressed that, although the sequence similarities discussed above are in a structural sense undeniable, we infer no significance to this similarity outside of the context of amyloid formation. That is, it is highly unlikely that these sequences share a common precursor in evolution, and there is no reason to believe that their common sequence elements are associated with any similarity in the normal function of these polypeptides. Growth of IAPP and Aβ Amyloid by Spontaneous, Nucleation-dependent Polymerization and by Homologous Seeding—The spontaneous, nucleation-dependent amyloid formation reaction by IAPP is complicated by the existence of two amyloid-related products. The first-formed product in the unseeded reaction consists of clusters of spheroidal bodies (Fig. 2A) that, despite their non-fibrillar morphologies, exhibit in our hands a number of other amyloid-like features. These include the ability to be labeled with the amyloid-selective (41Puchtler H. Sweat F. Levine M. J. Histochem. Cytochem. 1962; 10: 355-364Crossref Google Scholar, 50LeVine III, H. Amyloid. 1995; 2: 1-6Crossref Scopus (264) Google Scholar) dyes Congo red (Fig. 2, G and H) and thioflavin-T (see below). Their structural relationship to the classic amyloid fibril is also suggested by their response to sonication, which generates from these globular aggregates clus