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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

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 derived from species that do not form islet amyloid in vivo. Rat and mouse IAPP have identical sequences. The residues in rat/mouse IAPP (r-IAPP) that differ from the corresponding residues in human IAPP (h-IAPP) are indicated in black.DOI:http://dx.doi.org/10.7554/eLife.12977.016
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fig5s1: 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 derived from species that do not form islet amyloid in vivo. Rat and mouse IAPP have identical sequences. The residues in rat/mouse IAPP (r-IAPP) that differ from the corresponding residues in human IAPP (h-IAPP) are indicated in black.DOI:http://dx.doi.org/10.7554/eLife.12977.016

Mentions: IAPP is expressed by all mammals examined to date; the amino acid sequences are ~80% conserved between species, however not all IAPP sequences are toxic or form amyloid in vivo (Figure 5—figure supplement 1) (Betsholtz et al., 1989; Cao et al., 2013b; Westermark et al., 1990). We wondered if non-amyloidogenic, non-toxic variants of IAPP oligomerize, and if so, whether the size distribution and/or the structure of the oligomers produced were significantly different. r-IAPP is non-toxic and non-amyloidogenic in vivo and is widely used as a negative control in biological and biophysical/biochemical studies of h-IAPP (Westermark et al., 2011). However, it aggregates and forms oligomers. To further validate the use of r-IAPP as a negative control, we carried out dose-response experiments to test the effect of incubating INS-1 β-cells with up to 6-fold higher concentrations (84 µM) of r-IAPP for up to 19-fold longer incubation times on cells (96 h) than used in the assays employed herein to assess h-IAPP toxicity. No detectable toxicity was observed for r-IAPP, even at these significantly higher concentrations (Figure 5—figure supplement 2). The r-IAPP sequence differs from the h-IAPP sequence at six positions and contains three Pro residues and a His-18 to Arg replacement (Figure 5A). Pro is a well-known breaker of secondary structure and substitution of His with Arg will increase the net charge of the peptide. Cross-linking studies (Figure 5B) reveal a broadly similar distribution of oligomers produced in solution by r-IAPP and h-IAPP, with detected species ranging from monomers to hexamers. There are some differences in the relative intensities of the different oligomeric states, but these are relatively modest and it is difficult to unambiguously deduce their significance. The important feature is that the rat polypeptide clearly oligomerizes and forms dimers to hexamers similar to that of the human peptide; yet, it is not toxic (Figure 5C). Characterization by thioflavin-T binding assays and TEM confirm that r-IAPP does not form amyloid fibrils during these experiments (Figure 5D and E). Independent ion mobility mass spectroscopy (IM-MS) studies have also shown that h-IAPP and r-IAPP form similar distributions of oligomers (Young et al., 2014). Cell viability assays carried out simultaneously with biophysical measurements using aliquots from the same stock solutions show that r-IAPP is not toxic at any time point under these conditions, even though it oligomerizes (Figure 5F and G). Additional studies show that r-IAPP is not toxic over a 28 day time course (data not shown). CD studies reveal that r-IAPP oligomers appear less structured than their h-IAPP counterparts, as indicated by a positive signal below 190 nm and less intense signal between 218 to 222 nm (Figure 5H) in the spectrum of lag phase oligomers. While the differences in the CD spectra of r-IAPP and h-IAPP are moderate, they are significant and reproducible using different preparations of both peptides and in independent experiments conducted by different investigators. In contrast to h-IAPP, the CD spectrum of non-amyloidogenic r-IAPP does not change with time and is independent of concentration over the range tested (Figure 5—figure supplement 3). To further test if toxicity is decoupled from general aggregation, we examined an I26P point mutant of h-IAPP (I26P-IAPP) (Figure 5A). We have previously shown that I26P-IAPP inhibits amyloid formation by h-IAPP and does not form amyloid by itself under the conditions of our studies (Abedini et al., 2007; Meng et al., 2010). I26P-IAPP is similar to h-IAPP in hydrophobicity and has an identical net charge. Like r-IAPP, this mutant forms low order oligomers with an apparent size distribution that is similar to h-IAPP oligomers, as judged by photochemical induced cross-linking studies, but is non-amyloidogenic under these conditions over the 35+ h duration of these studies, as judged by thioflavin-T binding assays and TEM (Figure 5D and I). The CD spectrum of I26P-IAPP is similar to that of r-IAPP (Figure 5H and Figure 5—figure supplement 4). Cell viability assays carried out in parallel with biophysical studies show that I26P-IAPP is not toxic at any time point in our studies (Figure 5F and J). As an additional control, we analyzed a recently described non-toxic variant of h-IAPP (Wang et al., 2014a) (Figure 5—figure supplement 5A). The H18R, G24P, I26P triple mutant of h-IAPP (TM-IAPP) has been shown to be non-amyloidogenic and non-toxic (Wang et al., 2014b). Photochemical induced cross-linking and CD studies show that this variant also oligomerizes, even though it remains as random coil as judged by CD (Figure 5—figure supplement 5B–D). Thus, all three of the different non-toxic variants, which have similar hydrophobicity to h-IAPP, oligomerize (Figure 5—figure supplements 6 and 7). It is not possible to resolve the structural differences between the transiently populated ensemble of h-IAPP oligomers and the ensembles populated by r-IAPP, I26P-IAPP and TM-IAPP. However, the key point is that properties of the polypeptides beyond their ability to oligomerize are clearly important determinants of cellular toxicity. It is interesting to note that there are differences in the distribution of h-IAPP oligomers and the non-toxic r-IAPP and I26P-IAPP variants. Relatively more dimer is detected for h-IAPP compared to these two non-toxic variants and a reduction in the relative population of pentamers and hexamers is also detected. This may reflect actual differences in the distribution of oligomers in solution or it may include contributions from changes in cross-linking efficiency. The data cannot differentiate between the two potential explanations. The key feature is that these results decouple general aggregation and oligomer formation from toxic species formation, and suggest that the conformational properties of oligomers, and not their size, are important determinants of cellular toxicity.10.7554/eLife.12977.015Figure 5.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.


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)

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 derived from species that do not form islet amyloid in vivo. Rat and mouse IAPP have identical sequences. The residues in rat/mouse IAPP (r-IAPP) that differ from the corresponding residues in human IAPP (h-IAPP) are indicated in black.DOI:http://dx.doi.org/10.7554/eLife.12977.016
© Copyright Policy
Related In: Results  -  Collection

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

fig5s1: 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 derived from species that do not form islet amyloid in vivo. Rat and mouse IAPP have identical sequences. The residues in rat/mouse IAPP (r-IAPP) that differ from the corresponding residues in human IAPP (h-IAPP) are indicated in black.DOI:http://dx.doi.org/10.7554/eLife.12977.016
Mentions: IAPP is expressed by all mammals examined to date; the amino acid sequences are ~80% conserved between species, however not all IAPP sequences are toxic or form amyloid in vivo (Figure 5—figure supplement 1) (Betsholtz et al., 1989; Cao et al., 2013b; Westermark et al., 1990). We wondered if non-amyloidogenic, non-toxic variants of IAPP oligomerize, and if so, whether the size distribution and/or the structure of the oligomers produced were significantly different. r-IAPP is non-toxic and non-amyloidogenic in vivo and is widely used as a negative control in biological and biophysical/biochemical studies of h-IAPP (Westermark et al., 2011). However, it aggregates and forms oligomers. To further validate the use of r-IAPP as a negative control, we carried out dose-response experiments to test the effect of incubating INS-1 β-cells with up to 6-fold higher concentrations (84 µM) of r-IAPP for up to 19-fold longer incubation times on cells (96 h) than used in the assays employed herein to assess h-IAPP toxicity. No detectable toxicity was observed for r-IAPP, even at these significantly higher concentrations (Figure 5—figure supplement 2). The r-IAPP sequence differs from the h-IAPP sequence at six positions and contains three Pro residues and a His-18 to Arg replacement (Figure 5A). Pro is a well-known breaker of secondary structure and substitution of His with Arg will increase the net charge of the peptide. Cross-linking studies (Figure 5B) reveal a broadly similar distribution of oligomers produced in solution by r-IAPP and h-IAPP, with detected species ranging from monomers to hexamers. There are some differences in the relative intensities of the different oligomeric states, but these are relatively modest and it is difficult to unambiguously deduce their significance. The important feature is that the rat polypeptide clearly oligomerizes and forms dimers to hexamers similar to that of the human peptide; yet, it is not toxic (Figure 5C). Characterization by thioflavin-T binding assays and TEM confirm that r-IAPP does not form amyloid fibrils during these experiments (Figure 5D and E). Independent ion mobility mass spectroscopy (IM-MS) studies have also shown that h-IAPP and r-IAPP form similar distributions of oligomers (Young et al., 2014). Cell viability assays carried out simultaneously with biophysical measurements using aliquots from the same stock solutions show that r-IAPP is not toxic at any time point under these conditions, even though it oligomerizes (Figure 5F and G). Additional studies show that r-IAPP is not toxic over a 28 day time course (data not shown). CD studies reveal that r-IAPP oligomers appear less structured than their h-IAPP counterparts, as indicated by a positive signal below 190 nm and less intense signal between 218 to 222 nm (Figure 5H) in the spectrum of lag phase oligomers. While the differences in the CD spectra of r-IAPP and h-IAPP are moderate, they are significant and reproducible using different preparations of both peptides and in independent experiments conducted by different investigators. In contrast to h-IAPP, the CD spectrum of non-amyloidogenic r-IAPP does not change with time and is independent of concentration over the range tested (Figure 5—figure supplement 3). To further test if toxicity is decoupled from general aggregation, we examined an I26P point mutant of h-IAPP (I26P-IAPP) (Figure 5A). We have previously shown that I26P-IAPP inhibits amyloid formation by h-IAPP and does not form amyloid by itself under the conditions of our studies (Abedini et al., 2007; Meng et al., 2010). I26P-IAPP is similar to h-IAPP in hydrophobicity and has an identical net charge. Like r-IAPP, this mutant forms low order oligomers with an apparent size distribution that is similar to h-IAPP oligomers, as judged by photochemical induced cross-linking studies, but is non-amyloidogenic under these conditions over the 35+ h duration of these studies, as judged by thioflavin-T binding assays and TEM (Figure 5D and I). The CD spectrum of I26P-IAPP is similar to that of r-IAPP (Figure 5H and Figure 5—figure supplement 4). Cell viability assays carried out in parallel with biophysical studies show that I26P-IAPP is not toxic at any time point in our studies (Figure 5F and J). As an additional control, we analyzed a recently described non-toxic variant of h-IAPP (Wang et al., 2014a) (Figure 5—figure supplement 5A). The H18R, G24P, I26P triple mutant of h-IAPP (TM-IAPP) has been shown to be non-amyloidogenic and non-toxic (Wang et al., 2014b). Photochemical induced cross-linking and CD studies show that this variant also oligomerizes, even though it remains as random coil as judged by CD (Figure 5—figure supplement 5B–D). Thus, all three of the different non-toxic variants, which have similar hydrophobicity to h-IAPP, oligomerize (Figure 5—figure supplements 6 and 7). It is not possible to resolve the structural differences between the transiently populated ensemble of h-IAPP oligomers and the ensembles populated by r-IAPP, I26P-IAPP and TM-IAPP. However, the key point is that properties of the polypeptides beyond their ability to oligomerize are clearly important determinants of cellular toxicity. It is interesting to note that there are differences in the distribution of h-IAPP oligomers and the non-toxic r-IAPP and I26P-IAPP variants. Relatively more dimer is detected for h-IAPP compared to these two non-toxic variants and a reduction in the relative population of pentamers and hexamers is also detected. This may reflect actual differences in the distribution of oligomers in solution or it may include contributions from changes in cross-linking efficiency. The data cannot differentiate between the two potential explanations. The key feature is that these results decouple general aggregation and oligomer formation from toxic species formation, and suggest that the conformational properties of oligomers, and not their size, are important determinants of cellular toxicity.10.7554/eLife.12977.015Figure 5.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.

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