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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

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 supernatant was removed from the pellet containing amyloid fibrils and photochemically cross-linked. The pellet was resuspended in buffer and likewise photochemically cross-linked. (A) Representative SDS-PAGE of the photochemically cross-linked solutions (molecular weight marker: KDaltons). (B) Quantitative analysis of the gels shown in panel A. No peptide was detected in the supernatant.DOI:http://dx.doi.org/10.7554/eLife.12977.014
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fig4s5: 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 supernatant was removed from the pellet containing amyloid fibrils and photochemically cross-linked. The pellet was resuspended in buffer and likewise photochemically cross-linked. (A) Representative SDS-PAGE of the photochemically cross-linked solutions (molecular weight marker: KDaltons). (B) Quantitative analysis of the gels shown in panel A. No peptide was detected in the supernatant.DOI:http://dx.doi.org/10.7554/eLife.12977.014

Mentions: (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 solution of h-IAPP amyloid fibrils produced after 70 h of incubation (Scale bars: 200 nm). (D) Thioflavin-T binding assays confirm absence of amyloid fibrils in solutions of h-IAPP lag phase intermediates before ultracentrifugation and in the ultracentrifuged solutions of h-IAPP lag phase intermediates shown in panel B; they also confirm the presence of amyloid in the resuspended pellets of ultracentrifuged saturation phase solutions shown in panel C. The buffer control sample contains free thioflavin-T and the resulting fluorescence from this control solution is similar to that of free thioflavin-T solution by itself, and is thus the baseline. (E) Alamar Blue reduction assays show that the supernatant of samples of lag phase intermediates are toxic before and after ultracentrifugation, while the resuspended pellet of ultracentrifuged saturation phase samples are not toxic. (F) CD spectra of toxic lag phase intermediates before (red) and after (blue) ultracentrifugation. Data are plotted as mean residue ellipticity. (G) Representative SDS-PAGE of photochemically cross-linked toxic h-IAPP lag phase intermediates: lane-1, markers (molecular weight: KDaltons); lane-2, h-IAPP. (H) Quantitative analysis of the gels shown in panel G show a distribution of low order oligomers at time points of toxicity ranging from monomers to hexamers. Samples assessed in panels B–H were ultracentrifuged at 20,000 g for 20 min. Samples assessed in panels B–F used aliquots from the same peptide solutions. h-IAPP solutions contained 20 µM peptide. The peptide concentration after dilution into β-cell assays was 14 µM. Data represent mean ± SD of three to six replicate wells per condition and a minimum of three to nine replicate experiments per group (***p<0.001). Figure 4—figure supplements 1–5 provide additional biophysical characterization data for h-IAPP and a control peptide.


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)

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 supernatant was removed from the pellet containing amyloid fibrils and photochemically cross-linked. The pellet was resuspended in buffer and likewise photochemically cross-linked. (A) Representative SDS-PAGE of the photochemically cross-linked solutions (molecular weight marker: KDaltons). (B) Quantitative analysis of the gels shown in panel A. No peptide was detected in the supernatant.DOI:http://dx.doi.org/10.7554/eLife.12977.014
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Related In: Results  -  Collection

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fig4s5: 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 supernatant was removed from the pellet containing amyloid fibrils and photochemically cross-linked. The pellet was resuspended in buffer and likewise photochemically cross-linked. (A) Representative SDS-PAGE of the photochemically cross-linked solutions (molecular weight marker: KDaltons). (B) Quantitative analysis of the gels shown in panel A. No peptide was detected in the supernatant.DOI:http://dx.doi.org/10.7554/eLife.12977.014
Mentions: (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 solution of h-IAPP amyloid fibrils produced after 70 h of incubation (Scale bars: 200 nm). (D) Thioflavin-T binding assays confirm absence of amyloid fibrils in solutions of h-IAPP lag phase intermediates before ultracentrifugation and in the ultracentrifuged solutions of h-IAPP lag phase intermediates shown in panel B; they also confirm the presence of amyloid in the resuspended pellets of ultracentrifuged saturation phase solutions shown in panel C. The buffer control sample contains free thioflavin-T and the resulting fluorescence from this control solution is similar to that of free thioflavin-T solution by itself, and is thus the baseline. (E) Alamar Blue reduction assays show that the supernatant of samples of lag phase intermediates are toxic before and after ultracentrifugation, while the resuspended pellet of ultracentrifuged saturation phase samples are not toxic. (F) CD spectra of toxic lag phase intermediates before (red) and after (blue) ultracentrifugation. Data are plotted as mean residue ellipticity. (G) Representative SDS-PAGE of photochemically cross-linked toxic h-IAPP lag phase intermediates: lane-1, markers (molecular weight: KDaltons); lane-2, h-IAPP. (H) Quantitative analysis of the gels shown in panel G show a distribution of low order oligomers at time points of toxicity ranging from monomers to hexamers. Samples assessed in panels B–H were ultracentrifuged at 20,000 g for 20 min. Samples assessed in panels B–F used aliquots from the same peptide solutions. h-IAPP solutions contained 20 µM peptide. The peptide concentration after dilution into β-cell assays was 14 µM. Data represent mean ± SD of three to six replicate wells per condition and a minimum of three to nine replicate experiments per group (***p<0.001). Figure 4—figure supplements 1–5 provide additional biophysical characterization data for h-IAPP and a control peptide.

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