Calcitonin (CT) is an amyloid fibril forming peptide. Since salmon calcitonin (sCT), having Leu residues (Leu12, Leu16 or Leu19) instead of Tyr12, Phe16 or Phe19 for human calcitonin (hCT), is known to form the fibrils much slower than hCT, hCTs mutated to Leu residues at the position of 16 (F16L-hCT), 19 (F19L-hCT), and 12, 16 and 19 (TL-hCT) were examined to reveal the role of aromatic side-chains on amyloid fibrillation using solid-state (13)C NMR. The detailed kinetics were analyzed using a two-step reaction mechanism such as nucleation and fibril elongation with the rate constants of k1 and k2, respectively. The k2 values of hCT mutants were significantly slower than that of hCT at a neutral pH, although they were almost the same at an acidic pH. The (13)C chemical shifts of the labeled sites showed that the conformations of monomeric hCT mutants take α-helices as viewed from the Gly10 moiety. The hCT mutants formed fibrils and during the fibril formation, the α-helix around Gly10-Phe22 changed to the β-sheet, and the major structures around Ala26-Ala31 were random coil in the fibrils. Molecular dynamics simulation was performed for the β-sheet system of hCT9-23 and its mutants F16L-hCT9-23, F19L-hCT9-23 and TL-hCT9-23. In one of the stable fibril structures, Phe16 of hCT interacts with Phe19 of the next strand alternatively. In the hCT mutants, lack of Phe16 and Phe19 interaction causes significant instability as compared with the hCT fibril, leading to the reduction of k2 values, as observed experimentally in the hCT mutants at a neutral pH.

The abnormal aggregation of amyloid β-protein (Aβ) is considered central in the pathogenesis of Alzheimer’s disease. We focused on membrane-mediated amyloidogenesis and found that amyloid fibrils formed on monosialoganglioside GM1 clusters were more toxic than those formed in aqueous solution. In this study, we investigated the structure of the toxic fibrils by Aβ-(1–40) in detail in comparison with less-toxic fibrils formed in aqueous solution. The less-toxic fibrils contain in-resister parallel β-sheets, whereas the structure of the toxic fibrils is unknown. Atomic force microscopy revealed that the toxic fibrils had a flat, tape-like morphology composed of a single β-sheet layer. Isotope-edited infrared spectroscopy indicated that almost the entire sequence of Aβ is included in the β-sheet. Chemical cross-linking experiments using Cys-substituted Aβs suggested that the fibrils mainly contained both in-resister parallel and two-residue-shifted antiparallel β-sheet structures. Solid-state NMR experiments also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure.

Human calcitonin (hCT) is a 32-amino acid peptide hormone that contains an intrachain disulfide bridge between Cys1 and Cys7 and a proline amide at the C-terminus. hCT tends to associate to form a fibril precipitate of the same type as amyloid fibrils, and hence has been studied as a model of amyloid fibril formation. The fibrillation process in N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) solution was examined using transmission electron microscopy. The rate of hCT fibrillation in HEPES solution was much lower than in phosphate buffer and acetic acid solution. Spherical intermediate aggregates (nuclei) were observed during the early stage of fibril formation. Short proto-fibrils appeared on the surface of the spherical intermediates. Subsequently, the spherical intermediates transformed directly into long proto-fibrils, which then elongated into mature hCT fibrils. The fibrillation process was also examined using solid-state (13)C-NMR spectroscopy, which indicated that the fibril structure was a β-sheet in the central region and a mixture of random coils and β-sheets at the C-terminus. The kinetics of fibril formation was examined in terms of a two-step autocatalytic reaction mechanism. The first-step nucleation rate (k1) was lower in HEPES solution than in phosphate buffer and acetic acid solution because the half-life of the intermediates is significantly longer in HEPES solution. In contrast, the second-step fibril elongation rate (k2) was similar in HEPES solution and acidic solutions. Specific interaction of HEPES molecules with hCT may stabilize the spherical intermediates and consequently inhibit the fibril elongation process of hCT.

Human calcitonin (hCT) known as peptide thyroid hormone, plays a central role in calcium-phosphorus metabolism. HCT forms amyloid fibril in concentrated aqueous solution. We have shown that the fibrillation mechanism of hCT can be analyzed using a two-step autocatalytic reaction mechanism. In this study, we particularly investigated the early process in the fibril formation in HEPES solution. The morphology of the early process was examined by means of TEM, and turned out that the fibril formation in HEPES is much slower than that in acetic acid solution. It was nated that intermediate was seen in the early process. This observation clearly supports that hCT fibril takes a two-step autocatalytic reaction mechanism. 3P068β 2 ミクログロブリンのアミロイド幹形成領域のスキャン 探索 Amyloid formation of β 2 -microglobulin is responsible for dialysis-related amyloidosis. The most relevant region to amyloidogenicity is considered to be (20-41). Structural models of the amyloid have been reported using isotope-labeling IR and solid-state NMR for K3-peptide that corresponds to . Herein, we developed 2-dimensional scanning method in order to identify a pair of β-strands that form the amyloid-stem, a cross-β structure in fibrils. The survey was carried out with the peptides consisting of systematically selected two 6-residue sequences and a designed turninducing linker, Asp-( D -Ala). The specific residue-residue interactions between thus-identified two β-strands in K3-peptide region were found to have an important role for amyloidogenicity. Human calcitonin (hCT) is a 32-residue peptide hormone, involves in bone calcium metabolism and known as amyloid peptide. However, the detailed fibrillization mechanism is not well understood. In this study, the structure and fibrillation kinetics of hCT in solution containing neutral and acidic phospholipids (bicelles and micelles) were observed using NMR and UVVis spectroscopies. The fibrillation kinetics was revealed using a two-step autocatalytic reaction model composed of fibril nucleation (k 1 ) and fibril elongation reaction (k 2 ). In the presence of lipids, the first reaction was accelerated but the second reaction was decelerated. The surface condensation effect and the electrostatic interaction between hCT and lipid may play a role in fibril formation. 3P069 ヒトカルシトニンの酸性膜存在下でのアミロイド線維形成機 構の解明 3P070 インスリン B 鎖に見られる多様なアミロイド線維前駆中間 体の観察 Observation of various types of amyloid prefibrillar intermediates of insulin B chainShoko Tsuhara, Eri Chatani (Grad.Sch.of Sci.,Kobe.Univ)Amyloid fibrils are protein aggregates associated with many diseases. To elucidate detailed mechanism of amyloid fibril formation, we have investigated the fibrillation process of insulin B chain. Immediately after dissolving this peptide in buffer solution, significant ThT fluorescence intensity was observed, and the needle-like mature fibrils were formed subsequently without any lag phase, suggesting the formation of a prefibrillar intermediate at the initial stage. Interestingly, different types of prefibrillar intermediates in terms o...

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