Role of zinc in human islet amyloid polypeptide aggregation

Abstract

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. High-resolution 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.

Figure 1. Zinc inhibits hIAPP amyloid fibril formation at pH 7.5

Amyloid formation by hIAPP as a function of zinc concentration as followed by the amyloid-specific dye Thioflavin T. (A) 5μM and (B) 10 μM hIAPP with indicated concentrations of ZnCl₂. Traces are averages for n=9 (5 μM hIAPP) and n =7 (10 μM hIAPP) runs. The kinetic analysis of these traces and the variation of the final THT fluorescence intensity as a function of zinc concentration (along with the associated error bars) are given in Figures 4 and 2 respectively.

Figure 2. Zinc decreases the equilibrium concentration of hIAPP fibers at pH 7.5

Variation in the fluorescence intensity of the amyloid-bonding dye Thioflavin T as a function of ZnCl₂ concentration after fibrillogenesis has completed as extracted from the experimental results presented in Figure 1. Values for 5 μM and 10 μM were determined after 2 and 1 day(s) respectively. The intensity values were normalized to hIAPP in the absence of zinc. Error bars indicate standard error of measurement (S.E.M) for n=9 (5 μM hIAPP) and n =7 (10 μM hIAPP). Inset shows the effect at lower ZnCl₂ concentrations (0 - 1000 μM).

Figure 3. Zinc reduces the density of hIAPP fibrils while maintaining a similar fibril morphology

Top: Electron micrographs showing the effect of zinc on total amyloid deposition and fiber morphology. Aliquots from a 100 μM hIAPP solution incubated with (A) 0 μM ZnCl₂, (B) 100 μM ZnCl₂ and (C) 1000 μM ZnCl₂ were deposited on copper grids after an incubation period of 2 days and imaged at 6500x magnification. Bottom: Electron micrographs of hIAPP incubated with (D) 0 μM ZnCl₂, (E) 100 μM ZnCl₂ and (F) 1000 μM ZnCl₂ imaged at 15000x magnification.

Figure 4. Zinc has a bimodal effect on hIAPP fibrillogenesis at pH 7.5

Analysis of the kinetics of hIAPP at pH 7.5 according to a sigmoidal growth model. A variation in the lag-time (t_1/2-2/k) before detectable fiber formation as a function of ZnCl₂ at (A) 5 μM and (B) 10 μM hIAPP. Variations in the apparent first-order elongation rate constant (k) at (C) 5 μM and (D) 10 μM hIAPP. Error bars indicate standard error of measurement (S.E.M) for n=9 (5 μM hIAPP) and n =7 (10 μM hIAPP). Insets show the effect from 0–500 μM ZnCl₂.

Figure 5. Inhibition of aggregation is specific for cations that bind imidazole

100 μM NaCl, NH₄Cl,MgCl₂, CaCl₂, or ZnCl₂ was added to 7.5 μM hIAPP in 100 mM Tris, 100 mM NaCl, pH 7.5 buffer and amyloid formation was followed by changes in the fluorescence of the amyloid specific dye Thioflavin T (25 μM). Samples were shaken at 60 Hz. Traces are averages for n= 8 experiments.

Figure 6. Zinc accelerates hIAPP amyloid fibril formation at pH 5.5

Kinetics of hIAPP amyloid formation at pH 5.5. (A) Amyloid formation by 10 μM hIAPP as a function of zinc concentration. (B) Decrease in the lag-time (t_1/2-2/k) before detectable fiber formation as a function of ZnCl2 concentration according to the sigmoidal growth model (Eq. 1). (C) Increase in the elongation rate as a function of ZnCl₂ concentration according to the sigmoidal growth model.

Figure 7

The finger print region of 2D ¹H-¹H NOESY spectra of hIAPP in the absence (A) and presence (B) of 10 mM ZnCl₂ showing the connectivities among Hα nuclei and resonance assignment.

Figure 8. Zinc binding causes a local disruption of the secondary structure in the vicinity of His-18

High-resolution NMR structures of hIAPP in the absence (A) and presence of 10 mM ZnCl₂ (B).

Figure 9

Alpha proton chemical shift indexes (CSI) for hIAPP (A) and hIAPP in the presence of 10 mM zinc (B). The CSI was calculated by subtracting the appropriate random coil shifts reported in the literature. A CSI ≤ 0.1 is considered indicative of helical conformations. Changes in H_α (C) and H_N (D) chemical shifts upon binding to zinc.

Figure 10

Top: Histogram of NOEs versus the amino acid residues for hIAPP (A) without ZnCl₂ and (B) with ZnCl₂, showing the number of intra-residue, sequential (i – j = 1), and medium range (i – j = 2, 3, 4) NOEs. Bottom: Summary of the sequential and medium range NOE connectivities for hIAPP (C) without ZnCl₂ and (D) with ZnCl₂, The intensities of the observed NOEs are represented by the thickness of lines and are classified as strong, medium, and weak, corresponding to upper bound constraints of 2.9, 4.5, and 6 Å, respectively.

Figure 11. Cartoon schematic of how zinc may influence IAPP aggregation and toxicity

(A) In the secretory granule, IAPP is prevented from aggregation by the acidic environment and the inhibitory action of insulin. (B) Exocytosis of the secretory granule simultaneously releases IAPP and zinc into the extracellular space. (C) A high local concentration of IAPP near the plasma membrane is prevented from disrupting the β-cell by transient binding to zinc. (D) Zinc dissociates from IAPP at the low equilibrium concentrations found in the extracellular space, however, IAPP is now diluted past the critical concentration for aggregation.