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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

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 fibrils (red, - - - -). (C) Kinetic assays monitored by thioflavin-T fluorescence (●) and bis-ANS fluorescence (▲) confirm that bis-ANS binds to amyloid fibrils and not lag phase intermediates. (D) Structure of Nile Red. (E) Nile Red 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 fibrils (red, - - - -). The fluorescence intensity of Nile Red in buffer is comparable to the fluorescence intensity of Nile Red in the presence of h-IAPP. (F) Kinetic assays monitored by thioflavin-T fluorescence (●) and Nile Red fluorescence (▲) confirm that Nile Red does not bind to h-IAPP at any time during amyloid fibril formation.DOI:http://dx.doi.org/10.7554/eLife.12977.026
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fig7s1: 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 fibrils (red, - - - -). (C) Kinetic assays monitored by thioflavin-T fluorescence (●) and bis-ANS fluorescence (▲) confirm that bis-ANS binds to amyloid fibrils and not lag phase intermediates. (D) Structure of Nile Red. (E) Nile Red 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 fibrils (red, - - - -). The fluorescence intensity of Nile Red in buffer is comparable to the fluorescence intensity of Nile Red in the presence of h-IAPP. (F) Kinetic assays monitored by thioflavin-T fluorescence (●) and Nile Red fluorescence (▲) confirm that Nile Red does not bind to h-IAPP at any time during amyloid fibril formation.DOI:http://dx.doi.org/10.7554/eLife.12977.026

Mentions: ANS, a dye that is widely employed in protein folding studies to detect exposed hydrophobic patches and molten globule states (Figure 7A), binds to toxic pre-amyloid oligomers formed by a range of other amyloidogenic proteins, including various oligomers formed by the Aβ peptide, lysozyme, the α-synuclein protein of Parkinson’s disease, SH3 domains, HypF-N, bovine serum albumin, concanavalin and others (Bolognesi et al., 2010; Mannini et al., 2014; Frare et al., 2009; Lorenzen et al., 2014; Bhattacharya et al., 2011; Fu et al., 2015; Ghosh et al., 2015; Paslawski et al., 2014; Vetri et al., 2013). We tested if toxic h-IAPP lag phase intermediates bind ANS. No ANS binding is observed in the lag phase, but is observed during the growth phase of amyloid formation and in the saturation phase containing fibrils (Figure 7B and C). Thus, the properties of h-IAPP toxic oligomers are distinct from those recently described for certain other amyloidogenic proteins (Bolognesi et al., 2010; Kim et al., 2009; Laganowsky et al., 2012; Mannini et al., 2014; Lendel et al., 2014; Sandberg et al., 2010; Frare et al., 2009; Lorenzen et al., 2014; Stroud et al., 2012). 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) has also been used to probe molten globule states, the formation of exposed hydrophobic patches, and, in limited applications, amyloid formation (Younan and Viles, 2015; Hawe et al., 2008). Binding of bis-ANS to partially folded states often leads to a larger fluorescence change than ANS binding. We also tested the ability of h-IAPP toxic intermediates to bind bis-ANS. No binding to lag phase species is observed, but the dye, like ANS, binds to h-IAPP amyloid fibrils (Figure 7—figure supplement 1). Nile Red, like ANS, is used as a fluorescent probe of hydrophobic protein surfaces and has been shown to bind to pre-amyloid oligomers formed by some amyloidogenic proteins, but recent studies, conducted under different conditions than employed in our work, show that it does not bind to h-IAPP lag phase intermediates (Hawe et al., 2008; Jha et al., 2014; Krishnan et al., 2012; Sackett and Wolff, 1987). We independently confirmed that it does not bind to the lag phase species populated in our experiments (Figure 7—figure supplement 1).10.7554/eLife.12977.025Figure 7.The ensemble of toxic h-IAPP oligomers do not bind ANS.


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)

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 fibrils (red, - - - -). (C) Kinetic assays monitored by thioflavin-T fluorescence (●) and bis-ANS fluorescence (▲) confirm that bis-ANS binds to amyloid fibrils and not lag phase intermediates. (D) Structure of Nile Red. (E) Nile Red 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 fibrils (red, - - - -). The fluorescence intensity of Nile Red in buffer is comparable to the fluorescence intensity of Nile Red in the presence of h-IAPP. (F) Kinetic assays monitored by thioflavin-T fluorescence (●) and Nile Red fluorescence (▲) confirm that Nile Red does not bind to h-IAPP at any time during amyloid fibril formation.DOI:http://dx.doi.org/10.7554/eLife.12977.026
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Related In: Results  -  Collection

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fig7s1: 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 fibrils (red, - - - -). (C) Kinetic assays monitored by thioflavin-T fluorescence (●) and bis-ANS fluorescence (▲) confirm that bis-ANS binds to amyloid fibrils and not lag phase intermediates. (D) Structure of Nile Red. (E) Nile Red 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 fibrils (red, - - - -). The fluorescence intensity of Nile Red in buffer is comparable to the fluorescence intensity of Nile Red in the presence of h-IAPP. (F) Kinetic assays monitored by thioflavin-T fluorescence (●) and Nile Red fluorescence (▲) confirm that Nile Red does not bind to h-IAPP at any time during amyloid fibril formation.DOI:http://dx.doi.org/10.7554/eLife.12977.026
Mentions: ANS, a dye that is widely employed in protein folding studies to detect exposed hydrophobic patches and molten globule states (Figure 7A), binds to toxic pre-amyloid oligomers formed by a range of other amyloidogenic proteins, including various oligomers formed by the Aβ peptide, lysozyme, the α-synuclein protein of Parkinson’s disease, SH3 domains, HypF-N, bovine serum albumin, concanavalin and others (Bolognesi et al., 2010; Mannini et al., 2014; Frare et al., 2009; Lorenzen et al., 2014; Bhattacharya et al., 2011; Fu et al., 2015; Ghosh et al., 2015; Paslawski et al., 2014; Vetri et al., 2013). We tested if toxic h-IAPP lag phase intermediates bind ANS. No ANS binding is observed in the lag phase, but is observed during the growth phase of amyloid formation and in the saturation phase containing fibrils (Figure 7B and C). Thus, the properties of h-IAPP toxic oligomers are distinct from those recently described for certain other amyloidogenic proteins (Bolognesi et al., 2010; Kim et al., 2009; Laganowsky et al., 2012; Mannini et al., 2014; Lendel et al., 2014; Sandberg et al., 2010; Frare et al., 2009; Lorenzen et al., 2014; Stroud et al., 2012). 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) has also been used to probe molten globule states, the formation of exposed hydrophobic patches, and, in limited applications, amyloid formation (Younan and Viles, 2015; Hawe et al., 2008). Binding of bis-ANS to partially folded states often leads to a larger fluorescence change than ANS binding. We also tested the ability of h-IAPP toxic intermediates to bind bis-ANS. No binding to lag phase species is observed, but the dye, like ANS, binds to h-IAPP amyloid fibrils (Figure 7—figure supplement 1). Nile Red, like ANS, is used as a fluorescent probe of hydrophobic protein surfaces and has been shown to bind to pre-amyloid oligomers formed by some amyloidogenic proteins, but recent studies, conducted under different conditions than employed in our work, show that it does not bind to h-IAPP lag phase intermediates (Hawe et al., 2008; Jha et al., 2014; Krishnan et al., 2012; Sackett and Wolff, 1987). We independently confirmed that it does not bind to the lag phase species populated in our experiments (Figure 7—figure supplement 1).10.7554/eLife.12977.025Figure 7.The ensemble of toxic h-IAPP oligomers do not bind ANS.

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