Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics
- New York University School of Medicine, United States
- University of British Columbia, Canada
- Stony Brook University, United States
- University of Wisconsin-Madison, United States
Figures
Figure 1
A schematic diagram of the process of amyloid formation by h-IAPP.
(A) Amino acid sequence of wild-type h-IAPP. The mature, bioactive form of the polypeptide has an amidated C-terminus and a disulfide bridge indicated by the bracket between Cys-2 and Cys-7. (B) …
Figure 2 with 2 supplements
Toxic h-IAPP species are transiently populated lag phase intermediates.
(A) A schematic diagram of the experimental design for the kinetic assays. Protein aggregation was initiated by dissolving amyloidogenic IAPP, non-amyloidogenic IAPP variants or r-IAPP in 20 mM Tris …
Figure 2—figure supplement 1
Dilution of h-IAPP by 30% does not change the distribution of the toxic oligomers.
A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and incubated until the middle of the lag phase was reached. The solution was centrifuged at 20,000 g for 20 min and an …
Figure 2—figure supplement 2
Dilution of h-IAPP by 30% into cell culture medium does not change the kinetics of amyloid formation.
A stock solution of h-IAPP (20 µM) was prepared in Tris HCl buffer (20 mM, 25°C) and the reaction was monitored by thioflavin-T fluorescence. Aliquots of the stock solution were removed after 10 h …
Figure 3 with 2 supplements
h-IAPP lag phase intermediates upregulate pro-inflammatory cytokines and oxidative stress.
(A and B) qPCR studies of INS-1 β-cells treated with h-IAPP: Lag phase intermediates (blue) upregulate (A) Ccl2 and (B) Il1b, but time-zero species (black), amyloid fibrils (red) and r-IAPP at the …
Figure 3—figure supplement 1
h-IAPP lag phase intermediates induce ROS production in INS-1 β-cells.
(A) Alamar Blue reduction assays and (B) DHE fluorescence assays of β-cells treated for 1 h with h-IAPP lag phase intermediates (blue), amyloid fibrils (red) or buffer (gold) show significant …
Figure 3—figure supplement 2
Toxic h-IAPP lag phase intermediates induce β-cell apoptosis, but freshly dissolved h-IAPP (time-zero) and amyloid fibrils do not.
Cleaved caspase-3 colorimetric assays of β-cells treated with exogenous h-IAPP lag phase intermediates (blue) show an increase in cleaved caspase-3, but time-zero h-IAPP species (black), h-IAPP …
Figure 4 with 5 supplements
Toxic h-IAPP lag phase intermediates are soluble, low order oligomers with partial apparent α-helical structure.
(A–C) TEM images: (A) r-IAPP, (B) supernatant of ultracentrifuged solution of h-IAPP lag phase intermediates produced after 10 h of incubation, and (C) resuspended pellet of ultracentrifuged …
Figure 4—figure supplement 1
Time-dependent Far UV CD data of h-IAPP.
The lag time for h-IAPP to form amyloid under these conditions is on the order of 30 hr. The data show a conformational change from random coil to apparent α-helices that occur during the lag phase, …
Figure 4—figure supplement 2
The detection of monomers through hexamers is not a consequence of the choice of irradiation time.
A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and incubated until the middle of the lag phase was reached. The solution was centrifuged at 20,000 g for 20 min and …
Figure 4—figure supplement 3
The distribution of photochemically cross-linked oligomers detected for h-IAPP at 'time-zero' is similar to those detected for toxic h-IAPP lag phase intermediates.
A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and aliquots were removed after several minutes and irradiated for 10 s for photochemical cross-linking. (A) …
Figure 4—figure supplement 4
The distribution of photochemically cross-linked oligomers detected for h-IAPP is different from that observed for a monomeric protein of similar size.
(A) Sequence of h-IAPP and a variant of the villin headpiece helical subdomain (HP35*) in which the single Met residue was replaced by Nor-Leu and the single Trp by Tyr. These substitutions were …
Figure 4—figure supplement 5
The distribution of photochemically cross-linked oligomers detected for solutions of non-toxic h-IAPP fibrils is significantly different than for toxic h-IAPP lag phase intermediates.
A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and incubated until the saturation phase was reached. The solution was centrifuged at 20,000 g for 20 min and the …
Figure 5 with 7 supplements
r-IAPP and I26P-IAPP form oligomers that are similar in size to those formed by h-IAPP, but do not form amyloid under the conditions of these studies, and are not toxic.
(A) Primary sequence of h-IAPP, r-IAPP and I26P-IAPP. Mature polypeptides contain a disulfide between Cys2 and Cys7, indicated by brackets, and an amidated C-terminus. Amino acid positions that …
Figure 5—figure supplement 1
Primary sequences of IAPP from different species.
Sequences depicted in red have been shown to form amyloid in vitro and/or are from species that are known to form islet amyloid in vivo. Sequences in blue are non-amyloidogenic in vitro and/or are …
Figure 5—figure supplement 2
Dose-response studies show r-IAPP is not toxic.
Alamar Blue reduction assays of rat INS-1 β-cells treated with increasing concentrations of r-IAPP. Peptide solutions were prepared directly in cell culture medium (28, 56 and 84 µM peptide) and …
Figure 5—figure supplement 3
The CD spectrum of r-IAPP reveals random coil conformation and is independent of concentration and time.
CD data show a random coil conformation for r-IAPP at every dose examined at (A) time-zero and (B) after 5 h of incubation: 28 µM (blue), 56 µM (red) and 84 µM (green). Spectra are plotted in mdeg, …
Figure 5—figure supplement 4
Time-dependent far UV CD data of I26P-IAPP.
CD data of I26P-IAPP collected at different time points over 75 h of aggregation confirm that this peptide does not form amyloid under these experimental conditions.
Figure 5—figure supplement 5
A designed, non-toxic H18R, G24P, I26P triple mutant of h-IAPP (TM-IAPP) oligomerizes.
(A) Sequence alignment of h-IAPP with TM-IAPP. Both peptides contain a disulfide bond and have amidated C-termini. (B) Representative SDS-PAGE gel of a photochemically cross-linked sample of TM-IAPP …
Figure 5—figure supplement 6
Hydropathy plots for IAPP peptides.
Plots of hydrophobicity versus residue number for h-IAPP (red), r-IAPP (green), TM-IAPP (purple) and I26P-IAPP (blue) constructed with a three residue sliding window. (A and B) Calculations …
Figure 5—figure supplement 7
Average per residue hydrophobicity for different IAPP peptides.
The hydrophobicity values were taken from the octanol to water scale of Wimley and White (Wimley et al., 1996). Larger positive numbers indicate an increased hydrophobicity.
Figure 6 with 1 supplement
The ensemble of toxic h-IAPP oligomers contain only modest amounts of overall β-sheet structure.
2D IR spectra of h-IAPP: (A) Amyloid fibrils are rich in β-sheet structure, but (B) lag phase intermediates show no significant (<15%) β-sheet structure. The spectra in panels A and B are plotted on …
Figure 6—figure supplement 1
Sequence alignment of h-IAPP with Aβ40.
The disulfide bond in h-IAPP and the amidated C-terminus are shown. Solid vertical lines indicate amino acid identity; dashed lines indicate amino acid similarity.
Figure 7 with 14 supplements
The ensemble of toxic h-IAPP oligomers do not bind ANS.
(A) Structural model of an ANS molecule. (B) ANS fluorescence emission spectra of h-IAPP at time-zero (black, ····), lag phase intermediates (blue, —) and amyloid fibrils (red, - - - -). (C) Kinetic …
Figure 7—figure supplement 1
Bis-ANS and Nile Red do not bind to h-IAPP lag phase intermediates.
(A) Structure of bis-ANS. (B) Bis-ANS fluorescence emission spectra in the presence of h-IAPP at time-zero (black, ····), with h-IAPP lag phase intermediates (blue, —), and with h-IAPP amyloid …
Figure 7—figure supplement 2
Fluorescence detected thioflavin-T binding assay (black) showing the kinetics of amyloid formation by a solution of h-IAPP used in the proteolytic digestion studies presented in Figure 7—figure supplements 3–14.
Arrows indicate time points at which aliquots were removed and subjected to Proteinase K treatment. The time points are denoted as: (red, S1) time-zero, (blue, S2) early lag phase intermediates, …
Figure 7—figure supplement 3
Characterization of h-IAPP time-zero species by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken at time-zero directly after initiation of aggregation, in the absence of Proteinase K. Data show the expected molecular weight for h-IAPP (3903 …
Figure 7—figure supplement 4
Characterization of h-IAPP early lag phase species by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken early in the lag phase of amyloid formation, in the absence of Proteinase K. Data show the expected molecular weight for h-IAPP (3903 Daltons). The …
Figure 7—figure supplement 5
Characterization of h-IAPP mid-lag phase species by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken in the middle of the lag phase of amyloid formation, in the absence of Proteinase K. Data show the expected molecular weight for h-IAPP (3903 …
Figure 7—figure supplement 6
Characterization of h-IAPP amyloid fibrils by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken in the saturation phase of amyloid formation, in the absence of Proteinase K. Data show the expected molecular weight for h-IAPP (3903 Daltons). The …
Figure 7—figure supplement 7
Characterization of h-IAPP time-zero species by five minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken at time-zero directly after initiation of aggregation, after 5 min treatment with Proteinase K. Data show that freshly dissolved h-IAPP are rapidly …
Figure 7—figure supplement 8
Characterization of h-IAPP early lag phase species by five minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken early in the lag phase of amyloid formation, after 5 min treatment with Proteinase K. Data show that early h-IAPP lag phase intermediates are rapidly …
Figure 7—figure supplement 9
Characterization of h-IAPP mid-lag phase species by five minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken in the middle of the lag phase of amyloid formation, after 5 min treatment with Proteinase K. Data show that h-IAPP lag phase intermediates are …
Figure 7—figure supplement 10
Characterization of h-IAPP amyloid fibrils by five minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken in the saturation phase of amyloid formation, after 5 min treatment with Proteinase K. Data show the expected molecular weight of h-IAPP (3903 …
Figure 7—figure supplement 11
Characterization of h-IAPP time-zero species by forty minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken at time-zero directly after initiation of aggregation, after 40 min treatment with Proteinase K. Data show that freshly dissolved h-IAPP are …
Figure 7—figure supplement 12
Characterization of h-IAPP early lag phase species by forty minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken early in the lag phase of amyloid formation, after 40 min treatment with Proteinase K. Data show that early h-IAPP lag phase intermediates are …
Figure 7—figure supplement 13
Characterization of h-IAPP mid-lag phase species by forty minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken in the middle of the lag phase of amyloid formation, after 40 min treatment with Proteinase K. Data show that h-IAPP lag phase intermediates are …
Figure 7—figure supplement 14
Characterization of h-IAPP amyloid fibrils by forty minute Proteinase K digestion as monitored by MALDI-TOF MS.
MALDI-TOF mass spectra of h-IAPP aliquots taken in the saturation phase of amyloid formation, after 40 min treatment with Proteinase K. Data show the expected molecular weight for h-IAPP (3903 …
Figure 8
Aromatic residues in the ensemble of toxic h-IAPP oligomers are solvent exposed.
(A) Primary sequences of h-IAPP and p-cyano-phenylalanine variants; red X=cyanophenylalanine. (B) A structural model of the h-IAPP amyloid fibril. (C) Location of aromatic residues in h-IAPP which …
Figure 9
Aromatic-aromatic and aromatic-hydrophobic interactions are not required for toxicity.
(A) Primary sequences of h-IAPP and 3xL-IAPP. Amino acid positions differing from h-IAPP are indicated in red. (B) Thioflavin-T monitored kinetics of amyloid formation by 3xL-IAPP (●) and buffer …
Figure 10
I26P-IAPP inhibits h-IAPP amyloid formation, but prolongs cytotoxicity.
(A) Time-resolved Alamar Blue reduction assays of β-cells treated with: I26P-IAPP (♦), h-IAPP (●) and 1:1 I26P-IAPP/h-IAPP (■). Light microscopy: (†) Viable β-cells treated with h-IAPP amyloid …
