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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

Time-dependent Far UV CD data of h-IAPP.The lag time for h-IAPP to form amyloid under these conditions is on the order of 30 hr. The data show a conformational change from random coil to apparent α-helices that occur during the lag phase, followed by conversion to β-sheet structure in the growth phase.DOI:http://dx.doi.org/10.7554/eLife.12977.010
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fig4s1: Time-dependent Far UV CD data of h-IAPP.The lag time for h-IAPP to form amyloid under these conditions is on the order of 30 hr. The data show a conformational change from random coil to apparent α-helices that occur during the lag phase, followed by conversion to β-sheet structure in the growth phase.DOI:http://dx.doi.org/10.7554/eLife.12977.010

Mentions: Our ability to monitor toxicity in a time-resolved fashion allows us to characterize the physio-chemical properties of the toxic intermediates under well-defined conditions. Ultracentrifugation studies demonstrate that h-IAPP toxic species are soluble. Samples of toxic h-IAPP intermediates and amyloid fibrils were pelleted at 20,000 g for 20 min and the soluble peptide remaining in the supernatant was measured. Control experiments confirm that r-IAPP is soluble under these conditions. At least 88% of h-IAPP is pelleted in the sample of fibrils, even at these low g-forces, while 94% of the peptide in the sample of toxic lag phase intermediates remains in the supernatant. TEM and thioflavin-T binding assays confirm the absence of amyloid in the supernatant of ultracentrifuged samples of toxic intermediates, and the presence of amyloid in the pellet obtained from samples of h-IAPP fibrils (Figure 4A–D). The supernatant of intermediates is toxic to β-cells, while the resuspended pellet from samples of amyloid fibrils is not, verifying that cytotoxic entities reside in the soluble phase and are not high molecular weight species (Figure 4E). Characterization of the ensemble of h-IAPP lag phase intermediates by circular dichroism (CD) reveals partial apparent helical structure. Positive signal is observed below 190 nm and a minima at 208 nm (Figure 4F). A second broad minima centered at 220 nm is also detected with a mean residue ellipticity on the order of −6000 (deg-cm2/dmol), consistent with transiently populated partial α-helical structure (Manning and Woody, 1991). However, helical and β-sheet CD signatures overlap in this region of the spectrum. Thus, the broad signal in this region may also include contributions from the presence of some β-sheet structure. Two dimensional infrared (2D IR) studies, described below, indicate that the overall level of β-structure is modest. With further incubation, the CD spectrum of h-IAPP changes and eventually converts into a spectrum indicative of β-structure (Figure 4—figure supplement 1). Aliquots of the supernatant from samples of toxic intermediates were characterized by CD, both before and after ultracentrifugation (Figure 4F). The spectra are superimposable, confirming that the observed CD signal reflects the peptide in the soluble fraction and demonstrates that the overall conformation of the ensemble of oligomeric intermediates in solution remains the same after ultracentrifugation.10.7554/eLife.12977.009Figure 4.Toxic h-IAPP lag phase intermediates are soluble, low order oligomers with partial apparent α-helical 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)

Time-dependent Far UV CD data of h-IAPP.The lag time for h-IAPP to form amyloid under these conditions is on the order of 30 hr. The data show a conformational change from random coil to apparent α-helices that occur during the lag phase, followed by conversion to β-sheet structure in the growth phase.DOI:http://dx.doi.org/10.7554/eLife.12977.010
© Copyright Policy
Related In: Results  -  Collection

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

fig4s1: Time-dependent Far UV CD data of h-IAPP.The lag time for h-IAPP to form amyloid under these conditions is on the order of 30 hr. The data show a conformational change from random coil to apparent α-helices that occur during the lag phase, followed by conversion to β-sheet structure in the growth phase.DOI:http://dx.doi.org/10.7554/eLife.12977.010
Mentions: Our ability to monitor toxicity in a time-resolved fashion allows us to characterize the physio-chemical properties of the toxic intermediates under well-defined conditions. Ultracentrifugation studies demonstrate that h-IAPP toxic species are soluble. Samples of toxic h-IAPP intermediates and amyloid fibrils were pelleted at 20,000 g for 20 min and the soluble peptide remaining in the supernatant was measured. Control experiments confirm that r-IAPP is soluble under these conditions. At least 88% of h-IAPP is pelleted in the sample of fibrils, even at these low g-forces, while 94% of the peptide in the sample of toxic lag phase intermediates remains in the supernatant. TEM and thioflavin-T binding assays confirm the absence of amyloid in the supernatant of ultracentrifuged samples of toxic intermediates, and the presence of amyloid in the pellet obtained from samples of h-IAPP fibrils (Figure 4A–D). The supernatant of intermediates is toxic to β-cells, while the resuspended pellet from samples of amyloid fibrils is not, verifying that cytotoxic entities reside in the soluble phase and are not high molecular weight species (Figure 4E). Characterization of the ensemble of h-IAPP lag phase intermediates by circular dichroism (CD) reveals partial apparent helical structure. Positive signal is observed below 190 nm and a minima at 208 nm (Figure 4F). A second broad minima centered at 220 nm is also detected with a mean residue ellipticity on the order of −6000 (deg-cm2/dmol), consistent with transiently populated partial α-helical structure (Manning and Woody, 1991). However, helical and β-sheet CD signatures overlap in this region of the spectrum. Thus, the broad signal in this region may also include contributions from the presence of some β-sheet structure. Two dimensional infrared (2D IR) studies, described below, indicate that the overall level of β-structure is modest. With further incubation, the CD spectrum of h-IAPP changes and eventually converts into a spectrum indicative of β-structure (Figure 4—figure supplement 1). Aliquots of the supernatant from samples of toxic intermediates were characterized by CD, both before and after ultracentrifugation (Figure 4F). The spectra are superimposable, confirming that the observed CD signal reflects the peptide in the soluble fraction and demonstrates that the overall conformation of the ensemble of oligomeric intermediates in solution remains the same after ultracentrifugation.10.7554/eLife.12977.009Figure 4.Toxic h-IAPP lag phase intermediates are soluble, low order oligomers with partial apparent α-helical 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