Membrane disordering is not sufficient for membrane permeabilization by islet amyloid polypeptide: studies of IAPP(20-29) fragments

Abstract

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and a later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected.

Fig. 1

Effect of human-IAPP_20-29 or rat-IAPP_20-29 on the gel phase to liquid crystalline phase transition of DMPC bilayers as a function of the peptide concentration. Low concentrations of both human-IAPP_20-29 and rat-IAPP_20-29 cause a substantial decrease in the molar heat capacity.

Fig. 2

¹³C-¹H dipolar coupling values of DMPC in magnetically-aligned 3:1 DMPC:DHPC bicelles with and without rat-IAPP_20-29 using 2D PDLF solid-state NMR experiments at 37 °C. (A) Sample ¹H-¹³C dipolar coupling spectral slices extracted from the 2D PDLF spectrum of bicelles containing 2% rat-IAPP_20-29. (B) Dipolar coupling values calculated from the 2D PDLF experiment. Carbons 4-11 are poorly resolved and therefore represented as a single average value. (C) Labeling scheme for DMPC.

Fig. 3

¹⁴N quadrupole coupling spectra of magnetically-aligned 3:1 DMPC:DHPC bicelles containing rat-IAPP_20-29.

Fig 4

Dye leakage assay for human and rat-IAPP_20-29. (A) Normalized fluorescence changes for 1 mole % rat and human-IAPP_20-29 incorporated into the POPC vesicles before the assay. (B) Percent dye leakage in POPG vesicles for rat and human-IAPP_20-29 added externally. Lipid concentration for this experiment is 173 μM.