NMR Spectroscopic Investigation of Early Events in IAPP Amyloid Fibril Formation

Abstract

The islet amyloid polypeptide (IAPP) is a hormone, secreted by pancreatic b-cells along with glucagon and insulin. A balanced ratio of insulin and IAPP keeps the glucose metabolism of humans under control.[1] Disturbance of this balance results in type II diabetes mellitus. Most patients diagnosed with type II diabetes have a deposition of extracellular amyloid in the pancreas, and biochemical analysis has revealed that the main component of the amyloid deposit is IAPP. IAPP comprises 37 amino acids and is C-terminally amidated. In addition, cysteines 2 and 7 form a disulfide bond. [2,3] Under physiological conditions, IAPP remains soluble and a random coil structure has been reported by circular dichroism (CD) spectroscopy. [4] However, in the pathological condition of type II diabetes, IAPP adopts a fibrillar structure rich in b-sheets. [5] It is still unclear what triggers the conversion of soluble monomeric IAPP into insoluble amyloid fibrils. Mostly biophysical and mutagenesis approaches have been used to understand IAPP fibril formation in vitro. [6,7] A wealth of information about the role of the amino acid sequence in IAPP fibril formation has been obtained from rat IAPP (rIAPP), which differs in only six amino acid residues but does not form amyloid fibrils.[8] The non-aggregating nature of rIAPP was attributed to three proline residues in region 20-29 in the peptide. One other important difference in IAPP residues in the two species is the presence of arginine at position 18 in rat and histidine in humans. Although the preventive properties of the proline residues in IAPP fibrillation have been well studied, [8] biophysical studies that pinpoint the roles of specific residues in the initiation of IAPP amyloid formation are missing. Various real-time spectroscopic methods have been used to monitor amyloid fibril formation in bulk solution, [9,10] as well as in the presence of membranes, [11,12] but residue-specific information on the native peptide, which can best be obtained by NMR spectroscopy, is so far lacking. In a recent NMR spectroscopic study it was shown that rIAPP forms transient stretches of helical structure in its monomeric form.[13] In another study on human IAPP free acid, it has been indicated that the N terminus has a propensity for a-helical structure formation, but owing to the fast aggregation rate these studies were carried out only on the charged IAPP free acid. [14] In an attempt to study the early stages of glucagon amyloid fibril formation, one-dimensional, correlation and diffusion experiments were carried out by NMR spectroscopy. [15] Other approaches such as the application of high pressure to dissociate the fibril or to stabilize oligomeric structures during amyloid formation have also been shown to provide insight into the fibrillation reaction. [16,17] In this study we elucidated the monomeric structure of fulllength human IAPP (hIAPP, Figure 1) and the role of specific amino acid residues in the initiation of IAPP fibril formation by NMR spectroscopy. IAPP is known to be o...

Structure and Thermodynamics of Amylin Dimer Studied by Hamiltonian-Temperature Replica Exchange Molecular Dynamics Simulations

“…10 Our simulations suggest that some helix-helix interactions may exist in hIAPP in the presence of a disulfide bond as the WT-hIAPP dimer shows strong interchain hydrophobic interactions in the N-terminal region between the side chains of L12-F15 and L12-L16 which have 80% propensity to form an R-helix structure. This disulfide bond is essential for the formation of the N-terminal R-helix which, in turns, provides a support for the C-terminal β-sheet.…”

“…[3][4][5][6][7][8][9][10][11][12][13][14][15] In spite of these efforts, however, a detailed understanding of the first steps of aggregation is still missing. In particular, little is known about the structure of low molecular weight oligomers formed in the prefibrillar stage.…”

“…Brender et al, 7 for example, showed that membrane disruption and amyloid formation are two separate processes that happen in different regions of hIAPP. Liposome leakage experiments on full-length hIAPP and hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] fragments at low concentration confirmed that the pathological membrane-disrupting activity comes from the IAPP N-terminal region as both sequences induce almost identical ABSTRACT: The loss of the insulin-producing β-cells in the pancreatic islets of Langerhans, responsible for type-II diabetes, is associated with islet amyloid deposits. The main component of these deposits is the amyloid fibrils formed by the 37-residue human islet amyloid polypeptide (hIAPP also known as amylin).…”

“…However, the peptide is exposed to the water, whereas hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] is buried within the micelle. Interestingly, protonation of His18 of hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] drives the peptide to the surface of the membrane and decreases its toxicity. 29,30 To better understand the role of the disulfide bridge and the amino acid sequence on the early stage of aggregation in solution, we study the full-length hIAPP dimer, with and without the disulfide bridge, and compare its conformational properties with that of the monomer, already studied by us, 20 as well as with rIAPP in both monomeric and dimeric forms.…”

Hot‐Spot‐Regionen der Aβ‐IAPP‐Wechselwirkungsdomänen als hochaffine Bindungsstellen bei Kreuz‐ und Selbstassoziation

“…[6,18,[20][21][22]29] Daher untersuchten wir als Nächstes die Bindung der IAPP-Peptidarraymembran an Biotin-IAPP-GI. Dabei ergab sich, dass alle IAPP-Dekamere des IAPP(1-20)-Bereichs stark an Biotin-IAPP-GI binden und dass zusätzlich für die Sequenzen des C-terminalen IAPP-Bereichs eine schwache Wechselwirkung nachgewiesen werden konnte (Abbildung 3).…”