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

The detection of monomers through hexamers is not a consequence of the choice of irradiation time.A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and incubated until the middle of the lag phase was reached. The solution was centrifuged at 20,000 g for 20 min and aliquots of the supernatant were removed and irradiated for 1, 5 and 10 s for photochemical cross-linking. Representative SDS-PAGE of the photochemically cross-linked solutions are shown (molecular weight marker: KDaltons).DOI:http://dx.doi.org/10.7554/eLife.12977.011
© Copyright Policy
Related In: Results  -  Collection

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

fig4s2: The detection of monomers through hexamers is not a consequence of the choice of irradiation time.A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and incubated until the middle of the lag phase was reached. The solution was centrifuged at 20,000 g for 20 min and aliquots of the supernatant were removed and irradiated for 1, 5 and 10 s for photochemical cross-linking. Representative SDS-PAGE of the photochemically cross-linked solutions are shown (molecular weight marker: KDaltons).DOI:http://dx.doi.org/10.7554/eLife.12977.011

Mentions: We next sought to determine the approximate distribution of oligomeric species present in toxic h-IAPP solutions. Aliquots of toxic lag phase intermediates were trapped by in situ photochemical induced cross-linking and examined by SDS-PAGE, allowing differentiation between monomers and different size oligomers in solution. The in situ approach avoids concerns of structural perturbations induced by attaching photoactive groups and has been successfully used to study Aβ (Bitan and Teplow, 2004; Bitan et al., 2001). The data reveal a distribution of oligomers ranging from monomers to hexamers at time points of toxicity, confirming that the ensemble of toxic h-IAPP lag phase intermediates are soluble, low order oligomers (Figure 4G and H). Control studies show that the observed distribution is not an artifact of the irradiation time used for photochemical cross-linking (Figure 4—figure supplement 2). The dead time of the measurement (the time before the first measurement) is on the order of 10 min; a distribution of oligomers ranging from monomers to hexamers is populated within that time frame and the relative populations are similar to those detected later in the lag phase (Figure 4G and H, Figure 4—figure supplement 3). The data are consistent with independent ion mobility mass spectroscopy studies that report that a distribution of h-IAPP monomers to hexamers form within 2 min of initiating amyloid formation, and that the distribution is present later in the lag phase (Young et al., 2014). The rapid formation of oligomers and their persistence through the lag phase is consistent with recently proposed models of h-IAPP amyloid formation that posit that the lag phase could be controlled by a significant structural rearrangement within an oligomeric nucleus that involves crossing a high free energy barrier (Buchanan et al., 2013). Analysis of apparent relative populations of h-IAPP toxic species, deduced from the gel, indicate that dimers, trimers and tetramers are the most populated species. Monomeric proteins can be cross-linked by diffusion and collision of the photochemically modified monomers and it is important to show that the observed distribution differs from that expected for a monomeric protein (Bitan and Teplow, 2004). Thus, we employed a variant of the villin headpiece helical subdomain (HP35*), which is a soluble non-amyloidogenic protein of similar size to IAPP, as a control. The wild-type subdomain contains a single Trp and a Met, but no Tyr. We replaced Trp with Tyr, and Met with nor-leucine to ensure that the control peptide contains the same photochemically active residues as h-IAPP. Quantitative analysis of the silver stained gels reveal that the distribution of cross-linked h-IAPP species is significantly different from that expected for a monomeric protein (Figure 4H and Figure 4—figure supplement 4). We also compared the observed oligomer distributions to those predicted by Teplow and coworkers for diffusing monomers of molecular weight 4 KDaltons (Bitan et al., 2001). That analysis considered the case of spherical monomers which do not interact except by diffusion with random elastic collisions. For low efficiency cross-linking, the model predicts that the most populated species is the monomer, and an approximately exponential decrease in intensity of high order species is predicted. Medium efficiency cross-linking still leads to the monomer being the dominate species, but to a shallower exponential decay in the relative populations. Both cases clearly differ from that observed for h-IAPP. High efficiency cross-linking is predicted to lead to further consumption of monomers and a shift in the maximum to dimers with the predicted monomer and dimer populations being noticeably higher than the predicted trimer, tetramer and pentamer populations. Again, this distribution is fundamentally different from that observed for h-IAPP lag phase species, where trimers are the most highly populated species and the population of pentamers is comparable to the population of monomers. The pattern of cross-linking observed for h-IAPP lag phase species is also very different than observed if pre-formed amyloid fibrils are cross-linked. h-IAPP was allowed to form fibrils and then the samples were centrifuged. No cross-linked h-IAPP oligomers were detected in the supernatant. Re-solubilization of the cross-linked fibrils revealed that the dominant species were monomers with some dimer present (Figure 4—figure supplement 5). The various control experiments together with comparison to independent mass spectrometry studies confirm that the observation of lag phase h-IAPP oligomers is robust.


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 detection of monomers through hexamers is not a consequence of the choice of irradiation time.A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and incubated until the middle of the lag phase was reached. The solution was centrifuged at 20,000 g for 20 min and aliquots of the supernatant were removed and irradiated for 1, 5 and 10 s for photochemical cross-linking. Representative SDS-PAGE of the photochemically cross-linked solutions are shown (molecular weight marker: KDaltons).DOI:http://dx.doi.org/10.7554/eLife.12977.011
© Copyright Policy
Related In: Results  -  Collection

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

fig4s2: The detection of monomers through hexamers is not a consequence of the choice of irradiation time.A 20 µM stock solution of h-IAPP was prepared in Tris HCl buffer (20 mM, 25°C) and incubated until the middle of the lag phase was reached. The solution was centrifuged at 20,000 g for 20 min and aliquots of the supernatant were removed and irradiated for 1, 5 and 10 s for photochemical cross-linking. Representative SDS-PAGE of the photochemically cross-linked solutions are shown (molecular weight marker: KDaltons).DOI:http://dx.doi.org/10.7554/eLife.12977.011
Mentions: We next sought to determine the approximate distribution of oligomeric species present in toxic h-IAPP solutions. Aliquots of toxic lag phase intermediates were trapped by in situ photochemical induced cross-linking and examined by SDS-PAGE, allowing differentiation between monomers and different size oligomers in solution. The in situ approach avoids concerns of structural perturbations induced by attaching photoactive groups and has been successfully used to study Aβ (Bitan and Teplow, 2004; Bitan et al., 2001). The data reveal a distribution of oligomers ranging from monomers to hexamers at time points of toxicity, confirming that the ensemble of toxic h-IAPP lag phase intermediates are soluble, low order oligomers (Figure 4G and H). Control studies show that the observed distribution is not an artifact of the irradiation time used for photochemical cross-linking (Figure 4—figure supplement 2). The dead time of the measurement (the time before the first measurement) is on the order of 10 min; a distribution of oligomers ranging from monomers to hexamers is populated within that time frame and the relative populations are similar to those detected later in the lag phase (Figure 4G and H, Figure 4—figure supplement 3). The data are consistent with independent ion mobility mass spectroscopy studies that report that a distribution of h-IAPP monomers to hexamers form within 2 min of initiating amyloid formation, and that the distribution is present later in the lag phase (Young et al., 2014). The rapid formation of oligomers and their persistence through the lag phase is consistent with recently proposed models of h-IAPP amyloid formation that posit that the lag phase could be controlled by a significant structural rearrangement within an oligomeric nucleus that involves crossing a high free energy barrier (Buchanan et al., 2013). Analysis of apparent relative populations of h-IAPP toxic species, deduced from the gel, indicate that dimers, trimers and tetramers are the most populated species. Monomeric proteins can be cross-linked by diffusion and collision of the photochemically modified monomers and it is important to show that the observed distribution differs from that expected for a monomeric protein (Bitan and Teplow, 2004). Thus, we employed a variant of the villin headpiece helical subdomain (HP35*), which is a soluble non-amyloidogenic protein of similar size to IAPP, as a control. The wild-type subdomain contains a single Trp and a Met, but no Tyr. We replaced Trp with Tyr, and Met with nor-leucine to ensure that the control peptide contains the same photochemically active residues as h-IAPP. Quantitative analysis of the silver stained gels reveal that the distribution of cross-linked h-IAPP species is significantly different from that expected for a monomeric protein (Figure 4H and Figure 4—figure supplement 4). We also compared the observed oligomer distributions to those predicted by Teplow and coworkers for diffusing monomers of molecular weight 4 KDaltons (Bitan et al., 2001). That analysis considered the case of spherical monomers which do not interact except by diffusion with random elastic collisions. For low efficiency cross-linking, the model predicts that the most populated species is the monomer, and an approximately exponential decrease in intensity of high order species is predicted. Medium efficiency cross-linking still leads to the monomer being the dominate species, but to a shallower exponential decay in the relative populations. Both cases clearly differ from that observed for h-IAPP. High efficiency cross-linking is predicted to lead to further consumption of monomers and a shift in the maximum to dimers with the predicted monomer and dimer populations being noticeably higher than the predicted trimer, tetramer and pentamer populations. Again, this distribution is fundamentally different from that observed for h-IAPP lag phase species, where trimers are the most highly populated species and the population of pentamers is comparable to the population of monomers. The pattern of cross-linking observed for h-IAPP lag phase species is also very different than observed if pre-formed amyloid fibrils are cross-linked. h-IAPP was allowed to form fibrils and then the samples were centrifuged. No cross-linked h-IAPP oligomers were detected in the supernatant. Re-solubilization of the cross-linked fibrils revealed that the dominant species were monomers with some dimer present (Figure 4—figure supplement 5). The various control experiments together with comparison to independent mass spectrometry studies confirm that the observation of lag phase h-IAPP oligomers is robust.

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