Probing the sources of the apparent irreproducibility of amyloid formation: drastic changes in kinetics and a switch in mechanism due to micellelike oligomer formation at critical concentrations of IAPP

Abstract

The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions sometimes leading to large changes in aggregation rates. Using the amyloidogenic protein IAPP (islet amyloid polypeptide protein, also known as amylin) as an example, we show that a part of this phenomenon may be related to the formation of micellelike oligomers at specific critical concentrations and temperatures. We show that pyrene fluorescence can sensitively detect micellelike oligomer formation by IAPP and discriminate between micellelike oligomers from fibers and monomers, making pyrene one of the few chemical probes specific to a prefibrillar oligomer. We further show that oligomers of this type reversibly form at critical concentrations in the low micromolar range and at specific critical temperatures. Micellelike oligomer formation has several consequences for amyloid formation by IAPP. First, the kinetics of fiber formation increase substantially as the critical concentration is approached but are nearly independent of concentration below it, suggesting a direct role for the oligomers in fiber formation. Second, the critical concentration is strongly correlated with the propensity to form amyloid: higher critical concentrations are observed for both IAPP variants with lower amyloidogenicity and for native IAPP at acidic pH in which aggregation is greatly slowed. Furthermore, using the DEST NMR technique, we show that the pathway of amyloid formation switches as the critical point is approached, with self-interactions primarily near the N-terminus below the critical temperature and near the central region above the critical temperature, reconciling two apparently conflicting views of the initiation of IAPP aggregation.

Figure 1.. The rate of amyloid formation of hIAPP increases sharply near a threshold concentration.

Time to one half completion of amyloid formation as a function of hIAPP concentration for the native amidated hIAPP sequence (left) and the free acid version (right) as measured by the amyloid specific dye ThT at 25°C with orbital shaking (pH 7.3 10 mM sodium phosphate buffer, 100 mM NaCl). The dashed lines indicate the approximate threshold concentrations from the aggregation experiment. Error bars represent S.E.M. for six (hIAPP) or three (hIAPP free acid) experiments.

Figure 2.. Pyrene specifically detects concentration dependent oligomer formation over amyloid fibers.

(A) Fluorescence emission spectra of 1 μM pyrene with the indicated concentrations of hIAPP upon excitation at 334 nm. (B) Plots of the pyrene I_I/I_III ratio measured during a titration with freshly dissolved hIAPP at pH 7.3 (black circles) or preformed fibers of hIAPP (allowed to aggregate beforehand for 24 hours at 37 °C with shaking, red circles). (C) Corresponding plots of the pyrene I_I/I_III ratio measured with hIAPP free acid. Note the difference in the x-axis scale from (B). All measurements were performed in phosphate buffer 20 mM, 50 mM NaCl, 1 μM pyrene at 25 °C. Errors bars indicate S.E.M. (measurement preformed in triplicate).

Figure 3.. Concentration dependent oligomer formation of hIAPP is reversible.

Plots of the pyrene I_I/I_III ratio measured either by titration with freshly dissolved hIAPP at pH 7.3 (black circles) or by dilution from 4 μM hIAPP (red circles). All measures were performed in 20 mM phosphate buffer, 50 mM NaCl, 25 °C. The pyrene concentration is kept constant at 1 μM for all experiments.

Figure 4.. Oligomer formation of hIAPP only occurs above an apparent critical solution temperature.

Plot of the pyrene I_I/I_III ratio in the presence of 4 μM hIAPP at pH 7.3 at increasing temperature values (black circles). As control the pyrene I_I/I_III ratio in absence of hIAPP was collected (red circles). All measures were performed at pH 7.3 in 20 mM phosphate buffer, 50 mM NaCl, 1 μM pyrene.

Figure 5.. Peak intensity in the NMR spectra of hIAPP free acid sharply decreases at a critical temperature at pH 7.3 but not pH 5.

Temperature dependence of the ¹⁵N HSQC spectra of 78 μM hIAPP free acid in 20 mM sodium phosphate buffer, 50 mM NaCl at pH 5 (A) and pH7.3 (B). (C) Average of the peak intensity relative to the value at 4°C as a function of temperature. Error bars represent the S.E.M. considering N to be the number of residues.

Figure 6.. Non- or weakly amyloidogenic IAPP variants do not form pyrene detectable oligomers.

Plots of the pyrene II/IIII ratio measured during a titration with freshly dissolved hIAPP at pH 5 (cyan circles), non-amyloidogenic amidated rat IAPP (maroon circles), or weakly amyloidogenic hIAPP_1–19 (grey circles). All measures were performed in 20 mM phosphate buffer, 50 mM NaCl, 1 μM pyrene at 25 °C.

Figure 7.. Dilution changes the ¹H NMR spectra of hIAPP free acid.

¹H NMR spectra of 10 and 40 μM hIAPP free acid at 37 °C in 20 mM sodium phosphate buffer, 50 mM NaCl at pH 7.3 normalized to the number of scans only (A) and normalized to both the concentration and number of scans (B). Inset Strongly shielded peaks previously correlated with oligomer formation.

Figure 8.. hIAPP forms a different type of oligomer following brief incubation at 4°C or 25°C.

Top Different aliquots of the same solution of 10 μM hIAPP (initially at 4°C, 10 mM phosphate buffer, 100 mM NaCl, pH 7.4) were either left at 4°C (A) or heated to 25°C (B) and then quickly deposited on SiO₂, frozen with liquid N₂, and then lyophilized to preserve the morphology of the original aggregates and the by tapping mode AFM in air (40% humidity). Bottom: Section analysis of samples initially prepared at 4°C (C) and 25°C (D) showing the height distribution along the green line in (A) and (B).

Figure 9.. Changes in self-interaction profile with pH and temperature through DEST NMR experiments.

(A) DEST NMR spectra of 78 μM hIAPP free acid at pH 5 and 4°C, pH 7.3 and 4°C (C) pH 7.3 and 10°C in 20 mM phosphate buffer, 50 mM NaCl. Pink contours represent saturation at 30 kHz off-resonance, blue contours from 5 kHz off-resonance saturation. Right: Changes in relative intensity upon saturation expressed as the ratio between the intensity at 5 kHz and 30 kHz off-resonance saturation.