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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

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.DOI:http://dx.doi.org/10.7554/eLife.12977.024
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fig6s1: 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.DOI:http://dx.doi.org/10.7554/eLife.12977.024

Mentions: We probed the conformational properties of toxic h-IAPP oligomers in more detail to determine how they compared with those reported for toxic species formed by other amyloidogenic proteins. Of particular interest is a comparison with the Aβ peptide of AD, given the similarity between the two polypeptides and recent studies that suggest a link between AD and T2D (Yang and Song, 2013). h-IAPP and Aβ40 have 25% amino acid identity and 50% similarity with segments believed to be important for the self-assembly of each peptide, h-IAPP (Park et al., 2012; Zhang et al., 2003; Zraika et al., 2009; Cooper et al., 2010; Bolognesi et al., 2010; Chen et al., 2013; Chimon et al., 2007; Glabe, 2008; Kim et al., 2009; Laganowsky et al., 2012) and Aβ40 (Chimon et al., 2007; Glabe, 2008; Kim et al., 2009; Laganowsky et al., 2012; Mannini et al., 2014; Bucciantini et al., 2002; Lendel et al., 2014) having high similarity (Figure 6—figure supplement 1). Aβ fibrils can seed amyloid formation by h-IAPP in vitro and in an animal model, and the two polypeptides interact in vitro (Andreetto et al., 2010; O'Nuallain et al., 2004; Oskarsson et al., 2015). Recent work has revealed significant levels of β-sheet structure in toxic oligomers from several proteins, including Aβ (Chimon et al., 2007; Laganowsky et al., 2012; Lendel et al., 2014; Sandberg et al., 2010; Do et al., 2016). We detected apparent partial helical structure in the ensemble of toxic h-IAPP lag phase oligomers by CD (Figure 4E, 5H and Figure 4—figure supplement 1). Observation of partial helical structure is consistent with studies of truncated h-IAPP analogs fused to maltose binding protein; as well as studies of h-IAPP aromatic residue mutants, and NMR studies of soluble IAPP variants (Wiltzius et al., 2009; Williamson et al., 2009; Tu and Raleigh, 2013). It is also known that helical structure can be stabilized in h-IAPP by binding to negatively charged surfaces such as sulfated glycosaminoglycans or to vesicles containing significant amounts of anionic lipids (Wiltzius et al., 2009; Williamson et al., 2009; Tu and Raleigh, 2013; Brender et al., 2012; Knight et al., 2006; Meng et al., 2007). CD is well suited to probe helical structure, but is less sensitive to the details of β-sheet structure; individual β-sheets can exhibit significant differences in their CD signal. Thus, we applied newly developed 2D IR methods. 2D IR is a sensitive probe of β-sheet structure in aggregating systems (Buchanan et al., 2013; Wang et al., 2011; Strasfeld et al., 2009). The spectrum of h-IAPP amyloid fibrils has significant intensity along the diagonal in the β-sheet region between 1615 and 1625 cm-1 (Figure 6A) and is in good agreement with published spectra of h-IAPP amyloid fibrils. A flexible, partially structured intermediate will yield a significantly less intense 2D IR spectrum than a well-ordered β-structure, since the parallel β-sheet structure in amyloid fibrils leads to a large transition dipole. The spectrum of the intermediates is much less intense than the spectrum of the amyloid fibrils, indicating only modest levels of β-sheet structure (Figure 6B). Based on the relative areas in Figure 6C, the upper limit for the β-sheet content in the ensemble of lag phase intermediates populated under these conditions is estimated to be on the order of 15%. This does not preclude a high level of β-structure in a short segment of the protein. Recent isotope edited 2D IR studies using 10- to 20-fold higher h-IAPP concentrations than used herein (the isotope edited studies cannot currently be conducted at 40 µM peptide for technical reasons) suggest that h-IAPP forms an intermediate with well developed, parallel, in–register β-structure in the FGAIL region during amyloid formation under those conditions (Buchanan et al., 2013). A β-sheet of this size is fully consistent with the 2D IR data presented here.10.7554/eLife.12977.023Figure 6.The ensemble of toxic h-IAPP oligomers contain only modest amounts of overall β-sheet structure.


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)

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.DOI:http://dx.doi.org/10.7554/eLife.12977.024
© Copyright Policy
Related In: Results  -  Collection

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

fig6s1: 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.DOI:http://dx.doi.org/10.7554/eLife.12977.024
Mentions: We probed the conformational properties of toxic h-IAPP oligomers in more detail to determine how they compared with those reported for toxic species formed by other amyloidogenic proteins. Of particular interest is a comparison with the Aβ peptide of AD, given the similarity between the two polypeptides and recent studies that suggest a link between AD and T2D (Yang and Song, 2013). h-IAPP and Aβ40 have 25% amino acid identity and 50% similarity with segments believed to be important for the self-assembly of each peptide, h-IAPP (Park et al., 2012; Zhang et al., 2003; Zraika et al., 2009; Cooper et al., 2010; Bolognesi et al., 2010; Chen et al., 2013; Chimon et al., 2007; Glabe, 2008; Kim et al., 2009; Laganowsky et al., 2012) and Aβ40 (Chimon et al., 2007; Glabe, 2008; Kim et al., 2009; Laganowsky et al., 2012; Mannini et al., 2014; Bucciantini et al., 2002; Lendel et al., 2014) having high similarity (Figure 6—figure supplement 1). Aβ fibrils can seed amyloid formation by h-IAPP in vitro and in an animal model, and the two polypeptides interact in vitro (Andreetto et al., 2010; O'Nuallain et al., 2004; Oskarsson et al., 2015). Recent work has revealed significant levels of β-sheet structure in toxic oligomers from several proteins, including Aβ (Chimon et al., 2007; Laganowsky et al., 2012; Lendel et al., 2014; Sandberg et al., 2010; Do et al., 2016). We detected apparent partial helical structure in the ensemble of toxic h-IAPP lag phase oligomers by CD (Figure 4E, 5H and Figure 4—figure supplement 1). Observation of partial helical structure is consistent with studies of truncated h-IAPP analogs fused to maltose binding protein; as well as studies of h-IAPP aromatic residue mutants, and NMR studies of soluble IAPP variants (Wiltzius et al., 2009; Williamson et al., 2009; Tu and Raleigh, 2013). It is also known that helical structure can be stabilized in h-IAPP by binding to negatively charged surfaces such as sulfated glycosaminoglycans or to vesicles containing significant amounts of anionic lipids (Wiltzius et al., 2009; Williamson et al., 2009; Tu and Raleigh, 2013; Brender et al., 2012; Knight et al., 2006; Meng et al., 2007). CD is well suited to probe helical structure, but is less sensitive to the details of β-sheet structure; individual β-sheets can exhibit significant differences in their CD signal. Thus, we applied newly developed 2D IR methods. 2D IR is a sensitive probe of β-sheet structure in aggregating systems (Buchanan et al., 2013; Wang et al., 2011; Strasfeld et al., 2009). The spectrum of h-IAPP amyloid fibrils has significant intensity along the diagonal in the β-sheet region between 1615 and 1625 cm-1 (Figure 6A) and is in good agreement with published spectra of h-IAPP amyloid fibrils. A flexible, partially structured intermediate will yield a significantly less intense 2D IR spectrum than a well-ordered β-structure, since the parallel β-sheet structure in amyloid fibrils leads to a large transition dipole. The spectrum of the intermediates is much less intense than the spectrum of the amyloid fibrils, indicating only modest levels of β-sheet structure (Figure 6B). Based on the relative areas in Figure 6C, the upper limit for the β-sheet content in the ensemble of lag phase intermediates populated under these conditions is estimated to be on the order of 15%. This does not preclude a high level of β-structure in a short segment of the protein. Recent isotope edited 2D IR studies using 10- to 20-fold higher h-IAPP concentrations than used herein (the isotope edited studies cannot currently be conducted at 40 µM peptide for technical reasons) suggest that h-IAPP forms an intermediate with well developed, parallel, in–register β-structure in the FGAIL region during amyloid formation under those conditions (Buchanan et al., 2013). A β-sheet of this size is fully consistent with the 2D IR data presented here.10.7554/eLife.12977.023Figure 6.The ensemble of toxic h-IAPP oligomers contain only modest amounts of overall β-sheet structure.

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