Induction of negative curvature as a mechanism of cell toxicity by amyloidogenic peptides: the case of islet amyloid polypeptide

Abstract

The death of insulin-producing beta-cells is a key step in the pathogenesis of type 2 diabetes. The amyloidogenic peptide Islet Amyloid Polypeptide (IAPP, also known as amylin) has been shown to disrupt beta-cell membranes leading to beta-cell death. Despite the strong evidence linking IAPP to the destruction of beta-cell membrane integrity and cell death, the mechanism of IAPP toxicity is poorly understood. In particular, the effect of IAPP on the bilayer structure has largely been uncharacterized. In this study, we have determined the effect of the amyloidogenic and toxic hIAPP(1-37) peptide and the nontoxic and nonamyloidogenic rIAPP(1-37) peptide on membranes by a combination of DSC and solid-state NMR spectroscopy. We also characterized the toxic but largely nonamyloidogenic rIAPP(1-19) and hIAPP(1-19) fragments. DSC shows that both amyloidogenic (hIAPP(1-37)) and largely nonamyloidogenic (hIAPP(1-19) and rIAPP(1-19)) toxic versions of the peptide strongly favor the formation of negative curvature in lipid bilayers, while the nontoxic full-length rat IAPP(1-37) peptide does not. This result was confirmed by solid-state NMR spectroscopy which shows that in bicelles composed of regions of high curvature and low curvature, nontoxic rIAPP(1-37) binds to the regions of low curvature while toxic rIAPP(1-19) binds to regions of high curvature. Similarly, solid-state NMR spectroscopy shows that the toxic rIAPP(1-19) peptide significantly disrupts the lipid bilayer structure, whereas the nontoxic rIAPP(1-37) does not have a significant effect. These results indicate IAPP may induce the formation of pores by the induction of excess membrane curvature and can be used to guide the design of compounds that can prevent the cell-toxicity of IAPP. This mechanism may be important to understand the toxicity of other amyloidogenic proteins. Our solid-state NMR results also demonstrate the possibility of using bicelles to measure the affinity of biomolecules for negatively or positively curved regions of the membrane, which we believe will be useful in a variety of biochemical and biophysical investigations related to the cell membrane.

Figure 1. Amino acid sequences of rat and human IAPP sequences used in this study

The differences between the rat and human sequences are shown in red. There are disulfide bonds between residues 2 and 8 and the C-termini of the peptides are amidated like the physiologically expressed peptide.

Figure 2. Differential scanning calorimetry curves of the liquid crystalline (L_α) to inverted hexagonal (H_II) phase transition of DiPOPE multilamellar vesicles containing IAPP

The third DSC heating scan of IAPP peptides and peptide fragments in DiPoPE is shown at the listed molar percentage of peptide. While the L_α to H_II transition temperatures were reproducible, the amount of DiPoPE lipid in the DSC spectrometer cells was sensitive to sample preparation and therefore the intensities of the peaks were not completely reproducible. (A) rIAPP_1-37 in DiPoPE; (B) rIAPP_1-19 in DiPoPE; (C) hIAPP_1-37 in DiPOPE; (D) hIAPP_1-19 in DiPoPE.

Figure 3. Schematic of bicelle structure and ³¹P chemical shift spectra of DMPC:DMPG:DHPC bicelles containing IAPP

(A) A cartoon depiction of magnetically-aligned bicelles in the lamellar phase showing the parallel bicelle lamellae composed of DMPC and DMPG and the perforations composed of DHPC. The large, static magnetic field of the NMR spectrometer is indicated. (B) Zoomed in cartoon depiction of the bicelles, showing the regions of positive and negative curvature. (C) The ³¹P NMR spectrum of the pure bicelle sample. (D) The ³¹P NMR spectrum of the rIAPP_1-19 bicelle sample. (E) The ³¹P NMR spectrum of the rIAPP_1-37 bicelle sample.

Figure 4. The ¹³C chemical shift spectra of the headgroup and glycerol regions of DMPC:DMPG:DHPC bicelles containing IAPP

(A) Structures of the long-chain phospholipids (DMPG and DMPC) indicating the labeling convention. (B) ¹³C spectrum of the headgroup region of the pure bicelle sample, (C) rIAPP_1-19 bicelle sample and (D) rIAPP_1-37 bicelle sample. The glycerol peaks which are shifted relative to the pure bicelle peaks are circled in red.

Figure 5. 2D PDLF spectra of rIAPP and rIAPP_1-19 in DMPC:DMPG:DHPC bicelles

(A) The structures of the long-chain phospholipids of the bicelle samples (DMPG and DMPC) indicating the labeling convention used. Also shown is the 1D ¹³C chemical shift spectrum, indicating the frequency of ¹³C peaks in the horizontal dimension of the PDLF spectra. (B) The 2D ¹H-¹³C PDLF spectrum of bicelles containing rIAPP_1-37 (blue) superimposed upon the pure bicelle spectrum (black). (C) The 2D ¹H-¹³C PDLF spectrum of bicelles containing rIAPP_1-19 (red) superimposed upon the pure bicelle spectrum (black). (D) The ¹³C-¹H dipolar coupling slices corresponding to carbons 12, 13, and 14 of the aliphatic fatty acid chains of DMPG and DMPC. Slices corresponding to the pure bicelle sample are shown in black; bicelles containing rIAPP_1-37, blue; and bicelles containing rIAPP_1-19, red. (E) A zoomed in area of the PDLF spectrum showing the significant change in the dipolar couplings associated with carbons 13 and 14 of DMPG and DMPC induced by rIAPP_1-19.

Figure 6. The order parameter plots associated with DMPC:DMPG:DHPC bicelles containing (A) rIAPP_1-37 and (B) rIAPP_1-19

The order parameter plots are derived from their associated PDLF spectra as described in the methods section. The order parameter plot of pure bicelles is given in blue. Note that one |S_CH| value for carbons 12, 13, and 14 of bicelles containing rIAPP_1-19 is nearly identical to the corresponding value for the pure bicelle sample, indicating some lipids in the sample are unaffected by rIAPP_1-19.

Figure 7. ¹⁴N NMR spectra of DMPC:DMPG:DHPC bicelles containing IAPP

(A) The ¹⁴N quadrupolar NMR spectrum of the pure bicelle sample. The quadrupolar coupling constant (ν_Q) associated with DMPC is 10.2 kHz, and the ν_Q associated with DHPC is 4.2 kHz. DMPG does not have a ¹⁴N nucleus, and therefore there are peaks corresponding to DMPG are absent in the ¹⁴N spectrum. (B) The ¹⁴N spectrum of the rIAPP_1-37 bicelle sample. The ν_Q associated with DMPC is 9.8 kHz and ν_Q associated with DHPC is 3.6 kHz. (C) The ¹⁴N NMR spectrum of the rIAPP_1-19 bicelle sample. The ν_Q associated with DMPC is 10.8 kHz and ν_Q associated with DHPC is 6.4 kHz.