Human Islet Amyloid Polypeptide (hIAPP) is a highly amyloidogenic protein found in islet cells of patients with type II diabetes. Because hIAPP is highly toxic to beta-cells under certain conditions, it has been proposed that hIAPP is linked to the loss of beta-cells and insulin secretion in type II diabetics. One of the interesting questions surrounding this peptide is how the toxic and aggregation prone hIAPP peptide can be maintained in a safe state at the high concentrations that are found in the secretory granule where it is stored. We show here zinc, which is found at millimolar concentrations in the secretory granule, significantly inhibits hIAPP amyloid fibrillogenesis at concentrations similar to those found in the extracellular environment. Zinc has a dual effect on hIAPP fibrillogenesis: it increases the lag-time for fiber formation and decreases the rate of addition of hIAPP to existing fibers at lower concentrations, while having the opposite effect at higher concentrations. Experiments at an acidic pH which partially neutralizes the change in charge upon zinc binding show inhibition is largely due to an electrostatic effect at His18. Highresolution structures of hIAPP determined from NMR experiments confirm zinc binding to His18 and indicate zinc induces localized disruption of the secondary structure of IAPP in the vicinity of His18 of a putative helical intermediate of IAPP. The inhibition of the formation of aggregated and toxic forms of hIAPP by zinc provides a possible mechanism between the recent discovery of linkage between deleterious mutations in the SLC30A8 zinc transporter, which transports zinc into the secretory granule, and type II diabetes.

Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide secreted with insulin by betacells in the islets of Langerhans. The aggregation of the peptide into either amyloid fibers or small soluble oligomers has been implicated in the death of beta-cells during type 2 diabetes through disruption of the cellular membrane. The actual form of the peptide responsible for beta-cell death has been a subject of controversy. Previous research has indicated that the N-terminal region of the peptide (residues 1-19) is primarily responsible for the membrane disrupting effect of the hIAPP peptide and induces membrane disruption to a similar extent as the full-length peptide without forming amyloid fibers when bound to the membrane. The rat version of the peptide, which is both non-cytotoxic and non-amyloidogenic, differs from the human peptide by only one amino acid residue: Arg18 in the rat version while His18 in the human version. To elucidate the effect of this difference, we have measured in this study the effects of the rat and human versions of IAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] on islet cells and model membranes. Fluorescence microscopy shows a rapid increase in intracellular calcium levels of islet cells after the addition of hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] indicating disruption of the cellular membrane, while the rat version of the IAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] peptide is significantly less effective. Circular dichroism experiments and dye leakage assays on model liposomes show rIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] is deficient in binding to and disrupting lipid membranes at low but not at high peptide to lipid ratios, indicating that the ability of rIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] to form small aggregates necessary for membrane binding and disruption is significantly less than hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . At pH 6.0, where H18 is likely to be protonated, hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] resembles rIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] in its ability to cause membrane disruption. Differential scanning calorimetry suggests a different mode of binding to the membrane for rIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] compared to hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . Human IAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] has a minimal effect on the phase transition of lipid vesicles, suggesting a membrane orientation of the peptide in which the mobility of the acyl chains of the membrane is relatively unaffected. Rat IAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] , however, has a strong effect on the phase transition of lipid vesicles at low concentrations suggesting the pe...

Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide hormone associated with glucose metabolism that is cosecreted with insulin by β-cells in the pancreas. Since human IAPP is a highly amyloidogenic peptide, it has been suggested that the formation of IAPP amyloid fibers is responsible for the death of β-cells during the early stages of type II diabetes. It has been hypothesized that transient membrane-bound α-helical structures of human IAPP are precursors to the formation of these amyloid deposits. On the other hand, rat IAPP forms transient α-helical structures but does not progress further to form amyloid fibrils. To understand the nature of this intermediate state and the difference in toxicity between the rat and human versions of IAPP, we have solved the high-resolution structure of rat IAPP in the membrane-mimicking detergent micelles composed of dodecylphosphocholine. The structure is characterized by a helical region spanning the residues A5 to S23 and a disordered C-terminus. A distortion in the helix is seen at R18 and S19 that may be involved in receptor binding. Paramagnetic quenching NMR experiments indicate that rat IAPP is bound on the surface of the micelle, in agreement with other nontoxic forms of IAPP. A comparison to the detergent-bound structures of other IAPP variants indicates that the N-terminal region may play a crucial role in the self-association and toxicity of IAPP by controlling access to the putative dimerization interface on the hydrophobic face of the amphipathic helix.

SummaryMatrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage podosomes, the podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these podosomes fail to form and instead form focal adhesions. The formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates podosome formation and subsequent RhoA downregulation in the absence of traction force.