Macromolecular Crowding as a Suppressor of Human IAPP Fibril Formation and Cytotoxicity

<div><p>The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most <i>in vitro</i> studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.</p></div

Influence of the crowder type on IAPP restriction and binding.

<p><b>A</b> Crowder concentration-dependent lateral diffusion constant, <i>D</i>, of 10 µM fluorescent labeled rIAPP (rIAPP-K-Bodipy FL) as monitored by FCS. The polymer-like network forming molecules Ficoll and dextran as well as the proteins BSA and lysozyme were used as crowding reagents at concentrations ranging from 10 µM to 40% (w/v). <b>B</b> Content of rIAPP bound to the different crowding agents as analyzed from the FCS results of the equimolar rIAPP-K-Bodipy FL to crowder mixture (10 µM each). Data points are mean values (errors bars indicate standard deviations) of ≥3 individual measurements.</p

Effect of crowding on hIAPP cytotoxicity and on cell membrane enhanced hIAPP aggregation.

<p><b>A</b> Cell viability of pancreatic β-cells after exposure to hIAPP (10 µM) in crowded environments of different type (Ficoll, dextran, BSA) and concentration (10 and 20% (w/v)). As controls, viability of β-cells incubated without hIAPP and crowder reagents (grey bar) as well as with hIAPP (10 µM) without crowding reagents (black bar) are shown. <b>B</b> Time dependent ThT fluorescence spectroscopy assay of 10 µM hIAPP in a solution containing 0.5 mg/mL cell membrane vesicles (lipids extracted from the pancreatic β-cell line INS-1E) and 10 to 40% Ficoll as crowding reagent. The upper panel shows absolute ThT fluorescence intensities; whereas the corresponding normalized data are presented in the lower panel. Both data sets show mean values (errors bars indicate standard deviations) of ≥6 experiments.</p

Competing reaction pathways of hIAPP aggregation under crowding conditions.

<p>The horizontal path displays the usual hIAPP aggregation mechanism from a natural monomeric disordered structure via formation of nuclei and oligomers to fibril accumulation. Vertically, the competing crowder type dependent stabilization of globular non-toxic off-pathway species is depicted.</p

Schematic illustration of the crowder systems' molecular characteristics.

<p><b>A</b> High concentrations of Ficoll and dextran exhibit a network-like structure. <b>B</b>, <b>C</b> The proteins BSA and lysozyme can be described as hard sphere systems with BSA (<b>B</b>) having a larger hydrodynamic radius than lysozyme (<b>C</b>), leading to differences in the dimensions of void volume.</p

Monitoring crowder binding effects on hIAPP aggregation.

<p>Time dependent ThT fluorescence spectroscopic measurements of equimolar solutions of hIAPP and crowding reagents (10 µM each). The left panel shows absolute ThT fluorescence intensities, whereas the corresponding intensity normalized data are presented in the right panel. Data points are mean values (errors bars indicate standard deviations) of ≥6 experiments.</p

Structural differences of hIAPP aggregates upon incubation in crowded environments as detected by AFM.

<p>AFM images showing the structures of 50 µM hIAPP after ∼15 h of incubation in solutions with and without crowding reagents. <b>A</b> hIAPP only. <b>B</b>–<b>E</b> Incubation of hIAPP in 20% or 40% (w/v) of Ficoll, dextran, BSA and lysozyme. For an easier comparison of the hIAPP fibril sizes, insets in the left images of <b>D</b> and <b>E</b> show the same magnification as the other images displaying fibrils. All scale bars correspond to 1 µm. The color scale represents a height of 30 nm for images <b>A</b>–<b>C</b> and 5 nm for images <b>D</b> and <b>E</b>.</p

The hIAPP aggregation behavior under highly crowded conditions monitored by ThT fluorescence spectroscopy.

<p>Aggregation time courses of hIAPP (10 µM) in solutions using (<b>A</b>) Ficoll, (<b>B</b>) dextran, (<b>C</b>) BSA and (<b>D</b>) lysozyme at concentrations of 10–40% (w/v) as crowding reagents. Left panels show absolute ThT fluorescence intensities, whereas the corresponding intensity normalized data are presented in the right panels. Data points are mean values (errors bars indicate standard deviations) of ≥6 experiments.</p