Fig 1.
(A) Alignment of the primary sequence of h-amylin and Aβ1–40. The sequence alignment was performed using the program ALIGN (http://www.ch.embnet.org/software/LALIGN_form.html). Red and blue represent sequence identity and sequence similarity respectively. h-Amylin contains a conserved disulfide between Cys-2 and Cys-7 and an amidated C-terminus. (B) Structure of brilliant blue G (BBG).
Fig 2.
Brilliant blue G (BBG) moderately inhibits h-amylin amyloid formation but interferes with thioflavin-T based assays.
Thioflavin-T monitored kinetics assays are shown: (A) h-amylin alone (black) and h-amylin with an equimolar amount of BBG added at the beginning of the experiment (red); (B) h-amylin alone (black); h-amylin with a 5-fold excess of BBG added at the beginning of the experiment (blue) and (C) h-amylin alone (black) and h-amylin with a 10-fold excess amount of BBG added at the beginning of the experiment (green). (D) TEM images were recorded at 20 h (yellow star) and after 93 h (blue star) for all samples: h-amylin alone (black), h-amylin with an equimolar concentration of BBG (red), h-amylin with a 5-fold excess of BBG (blue) and h-amylin with a 10-fold excess amount of BBG (green). Experiments were conducted using 16 μM h-amylin, 32 μM thioflavin-T in 20 mM Tris-HCl with 140 mM KCl at pH 7.4 and 25°C. Scale bars represent 100 nm.
Fig 3.
Brilliant blue G remodels h-amylin amyloid fibers, but interferes with amyloid disaggregation assays.
Thioflavin-T monitored kinetic curve, h-amylin samples were allowed to form amyloid fibers and (A) an equimolar concentration (based on monomer concentration) and (B) a 10-fold excess concentration (based on monomer concentration) of BBG were added at 20 h. (C) TEM image of a sample collected just prior to the addition of BBG (black star) and TEM images collected at 1 h after addition of BBG (blue star) and at 72 h after addition of BBG (red star). Experiments were conducted with 16 μM h-amylin, 32 μM thioflavin-T in 20 mM Tris-HCl with 140 mM KCl at pH 7.4 and 25°C. Scale bars represent 100 nm.
Fig 4.
BBG does not efficiently protect cultured β-cells from h-amylin induced toxicity.
(A), dose-dependent h-amylin toxicity effects on INS-1 cells as measured by resazurin reduction (Alamar Blue) assays. A concertation of h-amylin corresponding to the estimated concentration of h-amylin required to reduce cell viability by 90%, EC90, (40 μM) was used for the BBG cell protection experiments. (B), viability of h-amylin treated (filled circles) and untreated (open circles) INS-1 cells exposed to different concentrations of BBG, as measured by Alamar Blue assays and given as percentage of untreated control (no added amylin). (C), viability of h-amylin treated INS-1 cells exposed to different concentrations of BBG, as measured by the CellTiter-Glo assay and given as percentage of untreated control (no added amylin). (D), plasma membrane integrity of h-amylin treated INS-1 cells exposed to different concentrations of BBG, as measured by the CellTox Green assay and given as percentage of maximal cell permeability. The data from the experiments are plotted as the mean ± SD, n = 3.
Fig 5.
The h-Amylin fibril structure contains patches of exposed aromatic and basic residues.
(A) A cross sectional view of the h-amylin fibril with R11, H18 and the three aromatic residues F15, F23, and Y37 shon in space filling presentation. (B) A top down view of the h-amylin fibril model. Five h-amylin molecules are shown per stack. Arg, His and aromatic residues are color coded: Arg-11, Red; His-18, Purple; Phe-15, Green; Phe-23, Green; Tyr-37, Yellow. The structure is based on the solid state NMR model of the amylin amyloid fiber [44].