Analysis of the Minimal Amyloid-forming Fragment of the Islet Amyloid Polypeptide
2001; Elsevier BV; Volume: 276; Issue: 36 Linguagem: Inglês
10.1074/jbc.m102883200
ISSN1083-351X
Autores Tópico(s)Supramolecular Self-Assembly in Materials
ResumoThe development of type II diabetes was shown to be associated with the formation of amyloid fibrils consisted of the islet amyloid polypeptide (IAPP or amylin). Recently, a short functional hexapeptide fragment of IAPP (NH2-NFGAIL-COOH) was found to form fibrils that are very similar to those formed by the full-length polypeptide. To better understand the specific role of the residues that compose the fragment, we performed a systematic alanine scan of the IAPP "basic amyloidogenic units." Turbidity assay experiments demonstrated that the wild-type peptide and the Asn1 → Ala and Gly3 → Ala peptides had the highest rate of aggregate formation, whereas the Phe2 → Ala peptide did not form any detectable aggregates. Dynamic light-scattering experiments demonstrated that all peptides except the Phe2 → Ala form large multimeric structures. Electron microscopy and Congo red staining confirmed that the structures formed by the various peptides are indeed amyloid fibrils. Taken together, the results of our study provide clear experimental evidence for the key role of phenylalanine residue in amyloid formation by IAPP. In contrast, glycine, a residue that was suggested to facilitate amyloid formation in other systems, has only a minor role, if any, in this case. Our results are discussed in the context of the remarkable occurrence of aromatic residues in short functional fragments and potent inhibitors of amyloid-related polypeptides. We hypothesize that π-π interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation. The development of type II diabetes was shown to be associated with the formation of amyloid fibrils consisted of the islet amyloid polypeptide (IAPP or amylin). Recently, a short functional hexapeptide fragment of IAPP (NH2-NFGAIL-COOH) was found to form fibrils that are very similar to those formed by the full-length polypeptide. To better understand the specific role of the residues that compose the fragment, we performed a systematic alanine scan of the IAPP "basic amyloidogenic units." Turbidity assay experiments demonstrated that the wild-type peptide and the Asn1 → Ala and Gly3 → Ala peptides had the highest rate of aggregate formation, whereas the Phe2 → Ala peptide did not form any detectable aggregates. Dynamic light-scattering experiments demonstrated that all peptides except the Phe2 → Ala form large multimeric structures. Electron microscopy and Congo red staining confirmed that the structures formed by the various peptides are indeed amyloid fibrils. Taken together, the results of our study provide clear experimental evidence for the key role of phenylalanine residue in amyloid formation by IAPP. In contrast, glycine, a residue that was suggested to facilitate amyloid formation in other systems, has only a minor role, if any, in this case. Our results are discussed in the context of the remarkable occurrence of aromatic residues in short functional fragments and potent inhibitors of amyloid-related polypeptides. We hypothesize that π-π interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation. islet amyloid polypeptide β-amyloid peptide Congo red Amyloid fibril formation is a central feature in a variety of unrelated pathological situations. A partial list includes Alzheimer's disease, prion diseases, diabetes mellitus (type II diabetes), familial amyloidosis, and light-chain amyloidosis (for review see Refs.1Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1405) Google Scholar, 2Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar, 3Dobson C.M. Trends Biochem. Sci. 1999; 24: 329-332Abstract Full Text Full Text PDF PubMed Scopus (1690) Google Scholar, 4Sipe J.D. Cohen A.S. J. Struct. Biol. 2000; 130: 88-98Crossref PubMed Scopus (834) Google Scholar). Islet amyloids are found in more than 95% of the patients with type II diabetes mellitus and are most likely an important factor in the development of β-cells failure (5Westermark P. Wernstedt C. O'Brien T.D. Hayden D.W. Johnson K.H. Am. J. Pathol. 1987; 127: 414-417PubMed Google Scholar, 6Luskey K.L. Diabetes Care. 1992; 15: 297-299Crossref PubMed Scopus (13) Google Scholar, 7Kahn S.E. Andrikopoulos S. Verchere C.B. Diabetes. 1999; 48: 241-253Crossref PubMed Scopus (424) Google Scholar, 8Höppener J.W.M. Ahrén B. Lips C.J.M. N. Engl. J. Med. 2000; 343: 411-419Crossref PubMed Scopus (441) Google Scholar). The islet amyloid fibrils consist predominantly of the islet amyloid polypeptide (IAPP1 or amylin), a 37-amino acid polypeptide hormone that is produced by pancreatic β-cells (5Westermark P. Wernstedt C. O'Brien T.D. Hayden D.W. Johnson K.H. Am. J. Pathol. 1987; 127: 414-417PubMed Google Scholar, 9Westermark P. Wernstedt C. Wilander E. Sletten K. Biochem. Biophys. Res. Commun. 1986; 140: 827-831Crossref PubMed Scopus (412) Google Scholar, 10Clark A. Cooper G.J.S. Lewis C.E. Morris J.F. Willis A.C. Reid K.B. Turner R.C. Lancet. 1987; 2: 231-234Abstract PubMed Scopus (320) Google Scholar, 11Cooper G.J.S. Willis A.C. Clark A. Turner R.C. Sim R.B. Reid K.B.M. Proc. Natl. Acad. Sci., U. S. A. 1987; 84: 8628-8632Crossref PubMed Scopus (1162) Google Scholar, 12Westermark P. Wernstedt C. Wilander E. Hayden D.W. O'Brien T.D. Johnson K.H. Proc. Natl. Acad. Sci., U. S. A. 1987; 84: 3881-3885Crossref PubMed Scopus (871) Google Scholar, 13Westermark P. Engström U. Johnson K.H. Westermark G.T. Betsholtz C. Proc. Natl. Acad. Sci., U. S. A. 1990; 87: 5036-5040Crossref PubMed Scopus (696) Google Scholar, 14Sipe J.D. Crit. Rev. Clin. Lab. Sci. 1994; 31: 325Crossref PubMed Scopus (189) Google Scholar, 15Moriarty D.F. Raleigh D.P. Biochemistry. 1999; 38: 1811-1818Crossref PubMed Scopus (174) Google Scholar). IAPP plays a central role in glucose homeostasis in its soluble form (16Rink T.J. Beaumont K. Koda J. Young A. Trends Pharmacol. Sci. 1993; 14: 113-118Abstract Full Text PDF PubMed Scopus (99) Google Scholar). Although the molecular mechanism of IAPP amyloidogenesis in vivo is not fully understood, thein vitro mechanism has been studied extensively. The 37-amino acid IAPP was shown to form amyloid fibrils in vitro (5Westermark P. Wernstedt C. O'Brien T.D. Hayden D.W. Johnson K.H. Am. J. Pathol. 1987; 127: 414-417PubMed Google Scholar, 10Clark A. Cooper G.J.S. Lewis C.E. Morris J.F. Willis A.C. Reid K.B. Turner R.C. Lancet. 1987; 2: 231-234Abstract PubMed Scopus (320) Google Scholar, 11Cooper G.J.S. Willis A.C. Clark A. Turner R.C. Sim R.B. Reid K.B.M. Proc. Natl. Acad. Sci., U. S. A. 1987; 84: 8628-8632Crossref PubMed Scopus (1162) Google Scholar, 16Rink T.J. Beaumont K. Koda J. Young A. Trends Pharmacol. Sci. 1993; 14: 113-118Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 17Goldsbury C. Goldie K. Pellaud J. Seelig J. Frey P. Müller S.A. Kistler J. Cooper G.J.S. Aebi U. J. Struct. Biol. 2000; 130: 352-362Crossref PubMed Scopus (288) Google Scholar). These fibrils were shown to be cytotoxic to pancreatic β-cell culture and thus are assumed to play a major role in the diabetes mechanism (18Lorenzo A. Razzaboni B. Weir G.C. Yankner B.A. Nature. 1994; 368: 756-760Crossref PubMed Scopus (723) Google Scholar, 19Lorenzo A. Yankner B.A. Proc. Natl. Acad. Sci., U. S. A. 1994; 91: 12243-12247Crossref PubMed Scopus (1291) Google Scholar). The kinetics of amyloid formation by IAPP as determined by turbidity assay is consistent with a nucleation-dependent mechanism of polymerization (20Kapurniotu A. Bernhagen J. Greenfield N. Al-Abed Y. Teichberg S. Frank R.W. Voelter W. Bucala R. Eur. J. Biochem. 1998; 251: 208-216Crossref PubMed Scopus (89) Google Scholar, 21Kayed R. Bernhagen J. Greenfield N. Sweimeh K. Brunner H. Voelter W. Kapurniotu A. J. Mol. Biol. 1999; 287: 781-796Crossref PubMed Scopus (314) Google Scholar, 22Larson J.L. Ko E. Miranker A.D. Protein Sci. 2000; 9: 427-431Crossref PubMed Scopus (48) Google Scholar, 23Ashburn T.T. Lansbury Jr., P.T. J. Am. Chem. Soc. 1993; 115: 11012-11013Crossref Scopus (65) Google Scholar).Recently, a six-residue peptide fragment of the human IAPP (with the amino acid sequence NFGAIL using the 1-letter code) was shown to form amyloid fibrils that are very similar to those formed by the full-length polypeptide (24Tenidis K. Waldner M. Bernhagen J. Fischle W. Bergmann M. Weber M. Merkle M.L. Voelter W. Brunner H. Kapurniotu A. J. Mol. Biol. 2000; 295: 1055-1071Crossref PubMed Scopus (356) Google Scholar). Furthermore, rodent IAPP, which does not form amyloid in vitro, is almost identical to human IAPP apart from a seven-amino acid block that includes most of this hexapeptide motif (Fig. 1 A). Therefore, this six-amino acid motif seems to serve as the "basic amyloidogenic unit" of the human IAPP polypeptide. A shorter five-residue fragment (FGAIL) also forms ordered amyloid fibrils. However, those fibrils are somewhat different in their morphology as compared with the full-length peptide (24Tenidis K. Waldner M. Bernhagen J. Fischle W. Bergmann M. Weber M. Merkle M.L. Voelter W. Brunner H. Kapurniotu A. J. Mol. Biol. 2000; 295: 1055-1071Crossref PubMed Scopus (356) Google Scholar). A shorter peptide corresponding to the GAIL sequence did not form any fibrils at all (24Tenidis K. Waldner M. Bernhagen J. Fischle W. Bergmann M. Weber M. Merkle M.L. Voelter W. Brunner H. Kapurniotu A. J. Mol. Biol. 2000; 295: 1055-1071Crossref PubMed Scopus (356) Google Scholar).Although different amyloid-related sequences do not reveal any sequence homology, they all share similar ultrastructural and physicochemical properties (reviewed in Refs. 1Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1405) Google Scholar, 4Sipe J.D. Cohen A.S. J. Struct. Biol. 2000; 130: 88-98Crossref PubMed Scopus (834) Google Scholar, and 25Cohen A.S. Shirhama T. Skinner M. Heris J.R. Electron Microscopy of Proteins 3. Academic Press, New York1982: 165-205Google Scholar). Amyloid deposits are characteristic of fibrils rich with β-pleated sheet structures, which are on average 7–10 nm in diameter and of varying length. Another well known characteristic of amyloid fibrils is the observation of a green birefringence after staining of the amyloid with Congo red (CR) dye (26Taylor D.L. Allen R.D. Benditt E.P. J. Histochem. Cytochem. 1974; 22: 1105-1112Crossref PubMed Scopus (43) Google Scholar, 27Cooper J.H. Lab. Investig. 1974; 31: 232-238PubMed Google Scholar). Amyloid formation is different from a simple process of nonspecific aggregation because amyloid fibrils show ordered structures that also have a characteristic x-ray diffraction pattern (1Harper J.D. Lansbury Jr., P.T. Annu. Rev. Biochem. 1997; 66: 385-407Crossref PubMed Scopus (1405) Google Scholar, 4Sipe J.D. Cohen A.S. J. Struct. Biol. 2000; 130: 88-98Crossref PubMed Scopus (834) Google Scholar). All of the above suggest that a specific pattern of molecular interactions, rather than nonspecific hydrophobic interactions, would lead to such an ordered process. Nevertheless, common structural elements that mediate the interactions that lead to these organized structures were not identified yet. The determination of such interactions, which underlay molecular recognition and self-assembly, is crucial for profound understanding of the amyloid formation process. Furthermore, such knowledge is valuable for the future design of drugs that can block these interactions and thus have a key clinical importance.Here we determine the role of each residue of the six amino acids that comprise the basic amyloidogenic unit of the IAPP by performing a systematic alanine scan of the motif followed by a structural and functional analysis of the various peptides. We studied the aggregation kinetics of the various peptides using a turbidity assay followed by dynamic light-scattering assays to estimate the size of aggregates that were formed by the different peptides. Finally, we determined the fibrillogenic nature of the different structures formed by the peptides using ultrastructural analysis by electron microscopy and CR staining.RESULTSThe minimal amyloid-forming fragment of IAPP provides a unique case of an extremely short peptide fragment that contains all OF the structural information needed to mediate the molecular recognition and self-assembly processes that lead to amyloid formation. To pinpoint the role of each amino acid in the formation of amyloid fibrils by this short fragment, a systematic substitution of the amino acid residues of the basic amyloidogenic unit with alanine was performed. We decided to substitute the various amino acids with alanine in order to specifically change the molecular interface of the peptides without dramatically changing their hydrophobicity or tendency to form β-sheet structures. The alanine scan was performed in the context of the block that is unique to human IAPP (Fig.1 A). This block includes two serine residues that follow the NFGAIL motif in the full-length polypeptide. These eight amino acid peptides were used to increase the solubility of the short peptides studied that are rather hydrophobic. Fig. 1 B shows a schematic representation of the chemical structure of the wild-type peptide, and Fig. 1 C indicates the amino acid substitutions in the different mutant peptides that were used in the study.Kinetic AggregationTo gain initial insights regarding the aggregation potential of the various peptides studied, a turbidity assay was performed. Freshly made stocks of the wild-type peptide and the various peptide mutants were made in Me2SO. The peptides were than diluted to a buffer solution, and the turbidity was monitored by following the absorbance at 405 nm as a function of time (Fig. 2). The wild-type peptide fragment showed an aggregation kinetic profile very similar to those previously reported for the nonseeded six-amino acid IAPP peptide (24Tenidis K. Waldner M. Bernhagen J. Fischle W. Bergmann M. Weber M. Merkle M.L. Voelter W. Brunner H. Kapurniotu A. J. Mol. Biol. 2000; 295: 1055-1071Crossref PubMed Scopus (356) Google Scholar). Such a profile is strongly indicative of a nucleation-dependent polymerization mechanism (28Jarrett J.T. Lansbury Jr., P.T. Biochemistry. 1992; 31: 12345-12352Crossref PubMed Scopus (277) Google Scholar). Peptide G3A showed a profile that was very similar to that of the wild-type peptide. The N1A peptide showed higher kinetics of aggregate formation, albeit with a different kinetic profile than the wild-type peptide. The aggregation of N1A seemed to be less nucleation-dependent. Substitution of the isoleucine or leucine to alanine (peptides I5A and L6A, respectively) reduced the kinetics of aggregation but did not abolish it completely. The most dramatic effect was that of the substitution of the phenylalanine residue to alanine (peptide F2A). This substitution led to a total loss of the peptide ability to form any aggregates as far as could be detected by the assay.Figure 2Turbidity assay. The kinetics of aggregate formation of the IAPP peptide fragment and its derivatives as followed by turbidity at 405 nm is shown. A, the turbidity of the peptide solution in 10 mm Tris buffer, pH 7.2, and 4% Me2SO prepared as described under "Experimental Procedures" was followed for 60 min. B, scale-up of the initial phase of aggregation. Data points are taken from one representative experiment of at least five performed for each of the peptides.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mean Particle Size MeasurementAlthough the turbidity assay had provided us with an important estimate regarding the aggregation potential and kinetics of the various peptides, it did not give information about the size of the actual aggregates formed. Therefore, we studied the average size of the aggregates formed by the various peptides, using dynamic light scattering. The autocorrelation data were fit to derive average apparent hydrodynamic diameters. The average apparent hydrodynamic diameters of the structures that were formed by the various peptides after a 1-h incubation are presented in Fig.3. Although the physical size of the various structures may be somewhat different that the apparent hydrodynamic diameter because of irregularity of the amyloid structure, it certainly gave a clear indication of the order of magnitude of the structure formed and provided us with a quantitative criterion to compare the average sizes of the structures formed by the various peptides. The apparent hydrodynamic diameter of the structures formed by the various peptides seemed to be generally consistent with the results obtained by the turbidity assay with some subtle differences. As with the turbidity assay, the wild-type peptide and G3A peptide behaved in a very similar way (Fig. 3). Both peptides formed particles of very similar hydrodynamic diameters. Smaller structures were detected within the derivative peptides: N1A, I5A, and L6A (Fig. 3). As with the turbidity assay, the dynamic light-scattering experiments clearly suggested that no large particles are formed by the F2A peptide under the experimental conditions (Fig. 3).Figure 3Light-scattering experiments.Calculated mean particle size of the IAPP peptide fragment and its derivatives determined by light scattering measurements. Measurements were performed for 100 μm peptides in 10 mm Tris buffer, pH 7.2, 1% Me2SO after 60 min of incubation. The results are presented as the mean of 3–5 independent experiments. The error bars represent the standard error.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Congo Red Staining and BirefringenceNext we studied whether the structures formed by the various peptides are indeed amyloid fibrils. One of the best known characteristics of amyloid fibrils is their ability to show typical birefringence upon binding of CR dye. The CR staining method combined with detection using cross-polarizers was applied to test the amyloidogenicity of the IAPP peptide fragment and its derivatives. Amyloid fibrils are known to bind CR and exhibit a gold-green birefringence under polarized light (26Taylor D.L. Allen R.D. Benditt E.P. J. Histochem. Cytochem. 1974; 22: 1105-1112Crossref PubMed Scopus (43) Google Scholar, 27Cooper J.H. Lab. Investig. 1974; 31: 232-238PubMed Google Scholar). The wild-type peptide, as well as peptides N1A (AFGAILSS) and G3A (NFAAILSS), bound CR and exhibited the characteristic birefringence (Fig.4, G, A, and E for normal field and H, B, and F for polarized light, respectively). Peptides I5A (NFGAALSS) and L6A (NFGAIASS) bound CR and exhibited rare but characteristic birefringence (Fig. 4, Iand K for normal field and J and L for polarized light, respectively). Peptide F2A (NAGAIL) showed no capability of binding CR (Fig. 4 C for normal field and 4D for polarized light). As a negative control we dried a buffer solution on a slide and then applied a CR solution (Fig. 4,M and N for normal and polarized light, respectively). There was no detectable difference between this negative control and the staining of F2A peptide deposits.Figure 4Congo red binding assay. Normal field microscopic examination of aged solutions of N1A peptide (A), F2A peptide (C), G3A peptide (E), wild-type IAPP peptide fragment (NFGAIL) (G), I5A peptide (I) , and L6A peptide (K) following staining with CR is shown. Microscopic examination under polarized light of N1A peptide (B), F2A peptide (D), G3A peptide (F), wild-type amylin peptide (NFGAIL) (H), I5A peptide (J) , and L6A peptide (L) is also shown. Buffer with CR is shown as a negative control with (N) and without (M) polarized light, respectively. Supersaturated solutions were prepared and aged for 4 days as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)To study whether the F2A peptide cannot form amyloid fibrils whatsoever or whether the undetectable CR binding is a result of extremely slow kinetics, a solution of the peptide under the same experimental conditions was incubated for 2 weeks. Although some degree of aggregation was observed after 2 weeks of incubation of the F2A peptide, CR staining showed no amyloid structure (results not shown). As a positive control, we incubated the wild-type peptide under the same conditions, and a typical CR birefringence was observed.Electron MicroscopyWe went further to carry out ultrastructural visualization of the structures formed by the various peptides. The occurrence and characteristics of the amyloid fibrils formed by the various peptides were studied by electron microscopy using negative staining. For this aim, we used peptides solutions (with the concentration of 2 mm) that were incubated overnight in 10 mm Tris buffer, pH 7.2, at room temperature. Filamentous structures were observed for all the peptides except F2A (Fig.5). Appearance of fibrils formed by the I5A and L6A peptides (Fig. 5, E and F, respectively) was at a lower frequency as compared with the wild-type (Fig. 5 D), N1A, and G3A peptides (Fig. 5, A andC, respectively). The solution containing peptide F2A consisted of amorphous aggregates only (Fig. 5 B). In the case of peptides F2A, I5A, and L6A, the electron microscopy pictures do not exhibit a representative field but rather rare ones. Although those results support the quantitative results presented in the previous sections, it provides a qualitative evidence for the morphology of the fibrils.Figure 5Electron microscopy. Electron microscopic examination of aged solutions (concentration of 2 mm) of the amylin peptide and its derivatives are shown in this order: A, N1A peptide; B, F2A peptide;C, G3A peptide; D, wild-type amylin peptide;E, I5A peptide; and F, L6A peptide. Thescale bar represents 100 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The tangled net-like structures that are observed with the wild-type peptide, N1A, and G3A are reasonable results of the fast kinetics of formation of these fibrils in the conditions described under "Experimental Procedures." More separated structures and longer fibrils, albeit less frequent, were observed with peptides I5A and L6A. These longer fibrils may be a result of a slower kinetics that allows a more ordered organization. At any rate, the qualitative information that was obtained by the electron microscope is clearly consistent with the findings using the other assays.DISCUSSIONDespite the key clinical importance of the process of amyloid formation by diverse peptides and proteins, there is a profound lack of understanding regarding the structural elements that specifically mediate this process of self-assembly and molecular recognition. The short minimal active fragments of IAPP (Fig. 1) provide an excellent model system to address this question in the context of a very short amyloidogenic motif. The results presented in this article clearly indicate that a specific pattern of molecular recognition, rather than nonspecific hydrophobic interactions, directs this process of self-assembly in the case of IAPP fibrils formation. A major finding presented here is the key role of the phenylalanine residue in the process of amyloid formation by the short active fragment of IAPP. The turbidity assay (Fig. 2), dynamic light scattering (Fig. 3), CR binding experiments (Fig. 4), and electron microscopy examination (Fig. 5) all indicate the essential role of the phenylalanine residue in the ability of the short peptide to form amyloid fibrils. These results are consistent with the observation of dramatic reduction in the rate of aggregation of a longer IAPP fragment when the phenylalanine residue was changed to a proline or leucine (15Moriarty D.F. Raleigh D.P. Biochemistry. 1999; 38: 1811-1818Crossref PubMed Scopus (174) Google Scholar,23Ashburn T.T. Lansbury Jr., P.T. J. Am. Chem. Soc. 1993; 115: 11012-11013Crossref Scopus (65) Google Scholar).The isoleucine and leucine residues in the C terminus of the IAPP recognition element have an important role in the kinetics of amyloid formation (Fig. 2). However, unlike the F2A peptide, the I5A and L6A peptides will eventually form typical amyloid structures (Figs. 4 and5), albeit with significantly slower kinetics (Fig. 2). Nevertheless, it is possible that the slow kinetics have a pathological significance, as in vivo amyloid formation of wild-type proteins is a very slow process (and thus amyloid diseases are generally associated with old age). It may be that under normal physiological conditions no fibrilization will occur upon the substitution of isoleucine or leucine to alanine in the context of a full-length IAPP.The role of the asparagine residue as determined in this study is consistent with the study of the hexa- and pentapeptides (NFGAIL and FGAIL, respectively (24Tenidis K. Waldner M. Bernhagen J. Fischle W. Bergmann M. Weber M. Merkle M.L. Voelter W. Brunner H. Kapurniotu A. J. Mol. Biol. 2000; 295: 1055-1071Crossref PubMed Scopus (356) Google Scholar)). We found that the presence of an alanine residue instead of an asparagine at the N terminus of the peptide actually accelerates the kinetics of the aggregation process (Fig. 2), but the hydrodynamic diameter (Fig. 3) and the morphology (Fig. 5) of the fibrils are also somewhat different from the wild-type fragment. This is consistent with the findings that the FGAIL pentapeptide forms fibrils but with different morphology as compared with the NFGAIL hexapeptide and the full-length IAPP (24Tenidis K. Waldner M. Bernhagen J. Fischle W. Bergmann M. Weber M. Merkle M.L. Voelter W. Brunner H. Kapurniotu A. J. Mol. Biol. 2000; 295: 1055-1071Crossref PubMed Scopus (356) Google Scholar). Taken together, it seems that the asparagine residue is not essential for amyloid formation but has a role in the kinetics of aggregation and modulation of the fine structure of the fibrils.Another interesting finding presented here is the fact that the G3A peptide behaved in a manner very similar to the wild-type peptide. This observation suggests that the glycine residue does not play a significant role in the formation of amyloids by the IAPP peptides. This observation is not trivial, as a priori glycine seems to be a residue that may have importance in amyloid formation because of its structural flexibility (27Cooper J.H. Lab. Investig. 1974; 31: 232-238PubMed Google Scholar, 29Lansbury Jr., P.T. Biochemistry. 1992; 31: 6865-6870Crossref PubMed Scopus (149) Google Scholar, 30Richardson J.S. Richardson D.C. Trends Biochem. Sci. 1989; 14: 304-309Abstract Full Text PDF PubMed Scopus (189) Google Scholar). Nonetheless, our results here are consistent with the study of synthetic peptides corresponding to a wild-type Alzheimer's β-amyloid (Aβ) peptide fragment and a corresponding peptide with an alanine to glycine mutation (the "Flemish mutation"). The study of the aggregation of the peptidesin vitro showed no significant change in the rate amyloid aggregation of the two peptides (31Wisniewski T. Ghiso J. Frangione B. Biochem. Biophys. Res. Commun. 1991; 179: 1247-1254Crossref PubMed Scopus (197) Google Scholar, 32Clements A. Walsh D.M. Williams C.H. Allsop D. Neurosci. Lett. 1993; 161: 17-20Crossref PubMed Scopus (80) Google Scholar).The major role of the phenylalanine residue in the formation of amyloid by the IAPP short fragment is consistent with the key role that was found for phenylalanine residues in the amyloid formation by the Aβ polypeptide. A short fragment of Aβ that contains two phenylalanine residues (QKLVFF) was shown to bind specifically to the full-length peptide (33Tjernberg L.O. Näslund J. Lindqvist F. Johansson J. Karlström A.R. Thyberg J. Terenius L. Nordstedt C. J. Biol. Chem. 1996; 271: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar). Furthermore, this short peptide could inhibit amyloid formation by the full-length Aβ (33Tjernberg L.O. Näslund J. Lindqvist F. Johansson J. Karlström A.R. Thyberg J. Terenius L. Nordstedt C. J. Biol. Chem. 1996; 271: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar). Follow-up studies have shown that not only the QKLVFF peptide (33Tjernberg L.O. Näslund J. Lindqvist F. Johansson J. Karlström A.R. Thyberg J. Terenius L. Nordstedt C. J. Biol. Chem. 1996; 271: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar) but also a LVFFA peptide and its derivatives (34Findeis M.A. Musso G.M. Arico-Muendel C.C. Benjamin H.W. Hundal A.M. Lee J.-J. Chin J. Kelley M. Wakefield J. Hayward N.J. Molineaux S.M. Biochemistry. 1999; 38: 6791-6800Crossref PubMed Scopus (228) Google Scholar, 35Pallitto M.M. Ghanta J. Heinzelman P. Kiessling L.L. Murphy R.M. Biochemistry. 1999; 38: 3570-3578Crossref PubMed Scopus (196) Google Scholar) and a LPFFD peptide (36Soto C. Sigurdsson E.M. Morelli L. Kumar R.A. Castano E.M. Frangione B. Nat. Med. 1998; 4: 822-826Crossref PubMed Scopus (786) Google Scholar) are all potent inhibitors of amyloid formation by the Aβ polypeptide. Comparing the sequences of the various peptides clearly indicate that the pair of phenylalanine residues ("FF motif") is th
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