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. 2008 May 21;130(20):6424-9.
doi: 10.1021/ja710484d. Epub 2008 Apr 30.

Amyloid fiber formation and membrane disruption are separate processes localized in two distinct regions of IAPP, the type-2-diabetes-related peptide

Jeffrey R Brender  1 , Edgar L Lee, Marchello A Cavitt, Ari Gafni, Duncan G Steel, Ayyalusamy Ramamoorthy
Affiliations

Amyloid fiber formation and membrane disruption are separate processes localized in two distinct regions of IAPP, the type-2-diabetes-related peptide

Jeffrey R Brender et al. J Am Chem Soc. .

Abstract

Aggregation of Islet Amyloid Polypeptide (IAPP) has been implicated in the development of type II diabetes. Because IAPP is a highly amyloidogenic peptide, it has been suggested that the formation of IAPP amyloid fibers causes disruption of the cellular membrane and is responsible for the death of beta-cells during type II diabetes. 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 1-19. The hIAPP 1-19 fragment at a low concentration of peptide induces membrane disruption to a near identical extent as the full-length peptide. At higher peptide concentrations, the hIAPP 1-19 fragment induces a greater extent of membrane disruption than the full-length peptide. Similar to the full-length peptide, hIAPP 1-19 exhibits a random coil conformation in solution and adopts an alpha-helical conformation upon binding to lipid membranes. However, unlike the full-length peptide, the hIAPP 1-19 fragment did not form amyloid fibers when incubated with POPG vesicles. These results indicate that membrane disruption can occur independently from amyloid formation in IAPP, and the sequences responsible for amyloid formation and membrane disruption are located in different regions of the peptide.

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Figures

Figure 1
Figure 1
Dye leakage from POPG vesicles induced by the full-length hIAPP and the hIAPP1–19 fragment. Carboxyfluorescein containing POPG liposomes (1.5 mM) were added to 250 nM (A) or 1 mM (B) solutions of either full-length hIAPP or the hIAPP1–19 fragment along with the listed concentrations of dye-free POPG liposomes. The fraction of dye leaked was recorded at 100 s. Each experiment was repeated three times; error bars indicate 1 standard deviation from the average. All experiments were performed in 50 mM sodium phosphate buffer at pH 7.5.
Figure 2
Figure 2
CD spectra of hIAPP1–19 (black solid line) and the full-length hIAPP (red line) peptides in (A) a low ionic strength (25 µM, sodium phosphate buffer pH 7.3) solution and (B) a high ionic strength (25 µM, sodium phosphate buffer pH 7.3 with 150 mM NaF) solution. The lower intensity of the signal from the full-length peptide may be indicative of a lower solubility at a higher ionic strength.
Figure 3
Figure 3
CD spectra of (A) hIAPP1–19 and (B) full length IAPP in a low ionic strength solution (25 µM, sodium phosphate buffer pH 7.3) measured as a function of time after the addition of 400 µM POPG large unilamellar vesicles.
Figure 4
Figure 4
CD spectra of (A) hIAPP1–19 and (B) full-length hIAPP in a high ionic strength solution (25 µM, sodium phosphate buffer and 150 mM NaF at pH 7.3) measured as a function of time after the addition of 400 µM POPG large unilamellar vesicles.
Figure 5
Figure 5
Binding of the amyloid-specific dye Thioflavin T to hIAPP1–19 and full-length hIAPP measured by fluorescence. Thioflavin T (25 µM) was incubated with 25 µM of either the full-length hIAPP peptide or the 1–19 fragment and 400 µM of POPG large unilamellar vesicles.
Figure 6
Figure 6
(A) Electron micrograph images of POPG vesicles incubated with full-length hIAPP for 7 days prior to measurement. Amyloid fibers of hIAPP can be seen branching out from the POPG vesicles. (B) Electron micrograph images of POPG vesicles incubated with hIAPP1–19 for 15 days prior to the measurement. Amyloid fibers were not detected; the circular structures are indicative of POPG vesicles. (C) Electron micrograph images of POPG vesicles incubated without a peptide for 15 days prior to measurement. The scale bars represent 500 nm.
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