Limits...
Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, Zhang J, Tu LH, Middleton CT, Chao B, Sartori DJ, Meng F, Wang H, Wong AG, Zanni MT, Verchere CB, Raleigh DP, Schmidt AM - Elife (2016)

Bottom Line: These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species.They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure.Aromatic interactions modulate, but are not required for toxicity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

No MeSH data available.


Related in: MedlinePlus

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 control (*). (C) TEM image of spherical, toxic, mid-lag phase intermediates produced during amyloid formation by 3xL-IAPP. (D) TEM image of non-toxic amyloid fibrils produced by 3xL-IAPP. (E) Time-resolved Alamar Blue reduction assays of β-cells treated with 3xL-IAPP (●) or buffer (*) at different time points during the course of amyloid formation. (F) Alamar Blue reduction assays measuring β-cell viability in response to increasing doses of h-IAPP or 3xL-IAPP with respect to buffer treated cells: h-IAPP (red), 3xL-IAPP (dark grey) and buffer (gold). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays and TEM studies were carried out using aliquots from the same 40 μM peptide solutions. The peptide concentration in samples assessed in panels B–D was 40 μM. The final peptide concentration in samples assessed in panel E after dilution of the 40 μM peptide solutions into β-cell assays was 28 μM. The final peptide concentrations in dose-response experiments in panel F, after dilution of peptide solutions into β-cell assays was 10.5, 14 and 28 μM. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels B and E are the same size or smaller than the symbols in the graphs (Scale bars: 200 nm; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).DOI:http://dx.doi.org/10.7554/eLife.12977.041
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940161&req=5

fig9: 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 control (*). (C) TEM image of spherical, toxic, mid-lag phase intermediates produced during amyloid formation by 3xL-IAPP. (D) TEM image of non-toxic amyloid fibrils produced by 3xL-IAPP. (E) Time-resolved Alamar Blue reduction assays of β-cells treated with 3xL-IAPP (●) or buffer (*) at different time points during the course of amyloid formation. (F) Alamar Blue reduction assays measuring β-cell viability in response to increasing doses of h-IAPP or 3xL-IAPP with respect to buffer treated cells: h-IAPP (red), 3xL-IAPP (dark grey) and buffer (gold). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays and TEM studies were carried out using aliquots from the same 40 μM peptide solutions. The peptide concentration in samples assessed in panels B–D was 40 μM. The final peptide concentration in samples assessed in panel E after dilution of the 40 μM peptide solutions into β-cell assays was 28 μM. The final peptide concentrations in dose-response experiments in panel F, after dilution of peptide solutions into β-cell assays was 10.5, 14 and 28 μM. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels B and E are the same size or smaller than the symbols in the graphs (Scale bars: 200 nm; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).DOI:http://dx.doi.org/10.7554/eLife.12977.041

Mentions: Aromatic contacts (π-π interactions) have been proposed to play an important role in amyloid formation. Although they are not required for h-IAPP amyloid formation, mutation of the three aromatic residues in h-IAPP to Leu (3xL-IAPP) slows the rate of amyloid formation (Tu and Raleigh, 2013; Marek et al., 2007; Gazit, 2007). Our p-cyanoPhe experiments show that there are no persistent interactions between F15 and Y37 or F23 and Y37 of h-IAPP, but the studies are less sensitive to formation of low levels (<5 to 10%) of conformers with F15/Y37 or F23/Y37 contacts, and do not probe potential interactions between F15 and F23. Consequently, we examined a triple mutant of h-IAPP: F15L, F23L, Y37L-IAPP (3xL-IAPP) that lacks aromatic residues to test whether or not aromatic π-π interactions or aromatic-hydrophobic interactions are required for toxicity (Figure 9A). Thioflavin-T assays and TEM measurements confirm that the triple mutant does form amyloid more slowly than the human peptide (Figure 9B–D). Cell viability studies show, that like h-IAPP, 3XL-IAPP exhibits time dependent toxicity; the amyloid fibrils produced by 3xL-IAPP are not toxic to β-cells, but species populated in the lag phase are, further confirming that toxicity resides with pre-amyloid intermediates (Figure 9B–E). Dose-response studies show that 3xL-IAPP evokes significantly lower levels of toxicity than h-IAPP. At their respective time points of maximum toxicity, 40 μM 3xL-IAPP reduced β-cell viability to 67%, while 20 μM h-IAPP reduced β-cell viability to 35% (Figure 9F). However, 3xL-IAPP is clearly still toxic, indicating that aromatic residues, and hence π-π interactions, are not an absolute requirement for h-IAPP toxicity, but do contribute to it.10.7554/eLife.12977.041Figure 9.Aromatic-aromatic and aromatic-hydrophobic interactions are not required for toxicity.


Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, Zhang J, Tu LH, Middleton CT, Chao B, Sartori DJ, Meng F, Wang H, Wong AG, Zanni MT, Verchere CB, Raleigh DP, Schmidt AM - Elife (2016)

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 control (*). (C) TEM image of spherical, toxic, mid-lag phase intermediates produced during amyloid formation by 3xL-IAPP. (D) TEM image of non-toxic amyloid fibrils produced by 3xL-IAPP. (E) Time-resolved Alamar Blue reduction assays of β-cells treated with 3xL-IAPP (●) or buffer (*) at different time points during the course of amyloid formation. (F) Alamar Blue reduction assays measuring β-cell viability in response to increasing doses of h-IAPP or 3xL-IAPP with respect to buffer treated cells: h-IAPP (red), 3xL-IAPP (dark grey) and buffer (gold). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays and TEM studies were carried out using aliquots from the same 40 μM peptide solutions. The peptide concentration in samples assessed in panels B–D was 40 μM. The final peptide concentration in samples assessed in panel E after dilution of the 40 μM peptide solutions into β-cell assays was 28 μM. The final peptide concentrations in dose-response experiments in panel F, after dilution of peptide solutions into β-cell assays was 10.5, 14 and 28 μM. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels B and E are the same size or smaller than the symbols in the graphs (Scale bars: 200 nm; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).DOI:http://dx.doi.org/10.7554/eLife.12977.041
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940161&req=5

fig9: 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 control (*). (C) TEM image of spherical, toxic, mid-lag phase intermediates produced during amyloid formation by 3xL-IAPP. (D) TEM image of non-toxic amyloid fibrils produced by 3xL-IAPP. (E) Time-resolved Alamar Blue reduction assays of β-cells treated with 3xL-IAPP (●) or buffer (*) at different time points during the course of amyloid formation. (F) Alamar Blue reduction assays measuring β-cell viability in response to increasing doses of h-IAPP or 3xL-IAPP with respect to buffer treated cells: h-IAPP (red), 3xL-IAPP (dark grey) and buffer (gold). Concurrent Alamar Blue reduction assays, thioflavin-T binding assays and TEM studies were carried out using aliquots from the same 40 μM peptide solutions. The peptide concentration in samples assessed in panels B–D was 40 μM. The final peptide concentration in samples assessed in panel E after dilution of the 40 μM peptide solutions into β-cell assays was 28 μM. The final peptide concentrations in dose-response experiments in panel F, after dilution of peptide solutions into β-cell assays was 10.5, 14 and 28 μM. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group. Some of the error bars in panels B and E are the same size or smaller than the symbols in the graphs (Scale bars: 200 nm; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).DOI:http://dx.doi.org/10.7554/eLife.12977.041
Mentions: Aromatic contacts (π-π interactions) have been proposed to play an important role in amyloid formation. Although they are not required for h-IAPP amyloid formation, mutation of the three aromatic residues in h-IAPP to Leu (3xL-IAPP) slows the rate of amyloid formation (Tu and Raleigh, 2013; Marek et al., 2007; Gazit, 2007). Our p-cyanoPhe experiments show that there are no persistent interactions between F15 and Y37 or F23 and Y37 of h-IAPP, but the studies are less sensitive to formation of low levels (<5 to 10%) of conformers with F15/Y37 or F23/Y37 contacts, and do not probe potential interactions between F15 and F23. Consequently, we examined a triple mutant of h-IAPP: F15L, F23L, Y37L-IAPP (3xL-IAPP) that lacks aromatic residues to test whether or not aromatic π-π interactions or aromatic-hydrophobic interactions are required for toxicity (Figure 9A). Thioflavin-T assays and TEM measurements confirm that the triple mutant does form amyloid more slowly than the human peptide (Figure 9B–D). Cell viability studies show, that like h-IAPP, 3XL-IAPP exhibits time dependent toxicity; the amyloid fibrils produced by 3xL-IAPP are not toxic to β-cells, but species populated in the lag phase are, further confirming that toxicity resides with pre-amyloid intermediates (Figure 9B–E). Dose-response studies show that 3xL-IAPP evokes significantly lower levels of toxicity than h-IAPP. At their respective time points of maximum toxicity, 40 μM 3xL-IAPP reduced β-cell viability to 67%, while 20 μM h-IAPP reduced β-cell viability to 35% (Figure 9F). However, 3xL-IAPP is clearly still toxic, indicating that aromatic residues, and hence π-π interactions, are not an absolute requirement for h-IAPP toxicity, but do contribute to it.10.7554/eLife.12977.041Figure 9.Aromatic-aromatic and aromatic-hydrophobic interactions are not required for toxicity.

Bottom Line: These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species.They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure.Aromatic interactions modulate, but are not required for toxicity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

No MeSH data available.


Related in: MedlinePlus