Islet amyloid polypeptide‐induced membrane leakage involves uptake of lipids by forming amyloid fibers

Abstract

Fibril formation of islet amyloid polypeptide (IAPP) is associated with cell death of the insulin-producing pancreatic bcells in patients with Type 2 Diabetes Mellitus. A likely cause for the cytotoxicity of human IAPP is that it destroys the barrier properties of the cell membrane. Here, we show by fluorescence confocal microscopy on lipid vesicles that the process of hIAPP amyloid formation is accompanied by a loss of barrier function, whereby lipids are extracted from the membrane and taken up in the forming amyloid deposits. No membrane interaction was observed when preformed fibrils were used. It is proposed that lipid uptake from the cell membrane is responsible for amyloid-induced membrane damage and that this represents a general mechanism underlying the cytotoxicity of amyloid forming proteins.

Amyloid Fiber Formation and Membrane Disruption are Separate Processes Localized in Two Distinct Regions of IAPP, the Type-2-Diabetes-Related Peptide

“…Previous studies have shown that the N-terminal 1-19 region, rather than the amyloidogenic 20-29 region, is primarily responsible for the interaction of the IAPP peptide with membranes. Liposome leakage experiments presented in this study confirm that the pathological membrane disrupting activity of the full-length hIAPP is also shared by hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . The hIAPP 1-19 fragment at a low concentration of peptide induces membrane disruption to a near identical extent as the full-length peptide.…”

“…The hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] and hIAPP [20][21][22][23][24][25][26][27][28][29] peptides were synthesized using standard FMOC (9-fluornylmethoxycarbonyl) methods. A PAL-PEG resin was used to provide an amidated C-terminus.…”

“…The hIAPP 20-29 peptide was synthesized as described above but with both an amidated C-terminus and an acetylated N-terminus. After synthesis, the crude hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] peptide was subjected to air oxidation to form the disulfide bond. The identity of the hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] and hIAPP [20][21][22][23][24][25][26][27][28][29] sequences and formation of the disulfide bond for hIAPP [1][2][3][4]…”

“…Lyophilized hIAPP and hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] were dissolved in HFIP for 1 h to obtain a 4 mg/mL stock solution. Aliquots of the peptide stock solution were lyophilized again and then resolubilized in either 10 mM sodium phosphate buffer, pH 7.3 or 10 mM sodium phosphate buffer with 150 mM NaF, pH 7.3.…”

“…It has previously been shown through fluorescence anisotropy, infrared reflectionabsorption spectroscopy, and lipid monolayer expansion experiments that the N-terminal part of IAPP is the primary membrane binding site of IAPP.11,14,15 Indeed, the 1 -19 fragment of human IAPP (hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] ) inserts into lipid membranes with a higher efficiency than full-length IAPP.11 We report here that the N-terminal region of the peptide (residues 1 -19) can disrupt synthetic lipid vesicles to a similar extent as the full-length IAPP peptide without forming amyloid fibers and may therefore be an important model for the study of membrane disruption by IAPP and other amyloid proteins.…”