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Ion mobility spectrometry-mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors.

Young LM, Cao P, Raleigh DP, Ashcroft AE, Radford SE - J. Am. Chem. Soc. (2013)

Bottom Line: We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly.Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition.The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds , Leeds LS2 9JT, U.K.

ABSTRACT
The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

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Oligomers formed fromrIAPP resemble those of hIAPP. (a) ESI-IMS-MSdriftscope plot of rIAPP oligomers present at 2 min after dilutioninto 20 mM ammonium acetate buffer, pH 6.8 to a final peptide concentrationof 50 μM. The number adjacent to each peak denotes oligomerorder with charge state of the oligomer in superscript. (b) Aggregationof rIAPP (diamonds) and hIAPP (circles) monitored using turbidityat 635 nm. In both cases, 50 μM peptide was incubated in 20mM ammonium acetate buffer, pH 6.8 (37 °C, 600 rpm). (c) Negativestain TEM image of rIAPP aggregates after 5 days of incubation (37°C, 600 rpm); scale bar = 100 nm. (d) ThT fluorescence intensityof rIAPP (50 μM peptide, 37 °C, pH 6.8, 600 rpm). The dataare normalized to the signal intensity of a hIAPP fibril formationend point at the same peptide concentration.
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fig3: Oligomers formed fromrIAPP resemble those of hIAPP. (a) ESI-IMS-MSdriftscope plot of rIAPP oligomers present at 2 min after dilutioninto 20 mM ammonium acetate buffer, pH 6.8 to a final peptide concentrationof 50 μM. The number adjacent to each peak denotes oligomerorder with charge state of the oligomer in superscript. (b) Aggregationof rIAPP (diamonds) and hIAPP (circles) monitored using turbidityat 635 nm. In both cases, 50 μM peptide was incubated in 20mM ammonium acetate buffer, pH 6.8 (37 °C, 600 rpm). (c) Negativestain TEM image of rIAPP aggregates after 5 days of incubation (37°C, 600 rpm); scale bar = 100 nm. (d) ThT fluorescence intensityof rIAPP (50 μM peptide, 37 °C, pH 6.8, 600 rpm). The dataare normalized to the signal intensity of a hIAPP fibril formationend point at the same peptide concentration.

Mentions: For rIAPP, which does notform ordered amyloid fibrils when incubated at near neutral pH,5 a surprisingly similar array of oligomers wasobserved using ESI-IMS-MS compared with that detected for hIAPP (Figure 3a, Supporting Information Figures S1a and S2). Akin to the results observed for hIAPP, multipleconformers are observed for rIAPP monomer and oligomers, albeit atdifferent relative intensities compared with those observed for hIAPP.Accordingly, hIAPP consistently occupies a greater proportion of moreexpanded conformers than rIAPP (Supporting Information Figure S1b). To investigate the relative stabilities of the differentmonomeric conformers of hIAPP and rIAPP, the dependence of the ionarrival time distribution versus increasing the trap energy (usedto effect CID) was examined (Supporting Information, Figure S1c). The results of these experiments showed that hIAPPmonomer 3+ ions unfold at lower trap collision energies than thoserequired for the rIAPP monomer, with hIAPP more readily convertingto expanded conformers at lower trap voltages (Supporting Information Figure S1c).


Ion mobility spectrometry-mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors.

Young LM, Cao P, Raleigh DP, Ashcroft AE, Radford SE - J. Am. Chem. Soc. (2013)

Oligomers formed fromrIAPP resemble those of hIAPP. (a) ESI-IMS-MSdriftscope plot of rIAPP oligomers present at 2 min after dilutioninto 20 mM ammonium acetate buffer, pH 6.8 to a final peptide concentrationof 50 μM. The number adjacent to each peak denotes oligomerorder with charge state of the oligomer in superscript. (b) Aggregationof rIAPP (diamonds) and hIAPP (circles) monitored using turbidityat 635 nm. In both cases, 50 μM peptide was incubated in 20mM ammonium acetate buffer, pH 6.8 (37 °C, 600 rpm). (c) Negativestain TEM image of rIAPP aggregates after 5 days of incubation (37°C, 600 rpm); scale bar = 100 nm. (d) ThT fluorescence intensityof rIAPP (50 μM peptide, 37 °C, pH 6.8, 600 rpm). The dataare normalized to the signal intensity of a hIAPP fibril formationend point at the same peptide concentration.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928500&req=5

fig3: Oligomers formed fromrIAPP resemble those of hIAPP. (a) ESI-IMS-MSdriftscope plot of rIAPP oligomers present at 2 min after dilutioninto 20 mM ammonium acetate buffer, pH 6.8 to a final peptide concentrationof 50 μM. The number adjacent to each peak denotes oligomerorder with charge state of the oligomer in superscript. (b) Aggregationof rIAPP (diamonds) and hIAPP (circles) monitored using turbidityat 635 nm. In both cases, 50 μM peptide was incubated in 20mM ammonium acetate buffer, pH 6.8 (37 °C, 600 rpm). (c) Negativestain TEM image of rIAPP aggregates after 5 days of incubation (37°C, 600 rpm); scale bar = 100 nm. (d) ThT fluorescence intensityof rIAPP (50 μM peptide, 37 °C, pH 6.8, 600 rpm). The dataare normalized to the signal intensity of a hIAPP fibril formationend point at the same peptide concentration.
Mentions: For rIAPP, which does notform ordered amyloid fibrils when incubated at near neutral pH,5 a surprisingly similar array of oligomers wasobserved using ESI-IMS-MS compared with that detected for hIAPP (Figure 3a, Supporting Information Figures S1a and S2). Akin to the results observed for hIAPP, multipleconformers are observed for rIAPP monomer and oligomers, albeit atdifferent relative intensities compared with those observed for hIAPP.Accordingly, hIAPP consistently occupies a greater proportion of moreexpanded conformers than rIAPP (Supporting Information Figure S1b). To investigate the relative stabilities of the differentmonomeric conformers of hIAPP and rIAPP, the dependence of the ionarrival time distribution versus increasing the trap energy (usedto effect CID) was examined (Supporting Information, Figure S1c). The results of these experiments showed that hIAPPmonomer 3+ ions unfold at lower trap collision energies than thoserequired for the rIAPP monomer, with hIAPP more readily convertingto expanded conformers at lower trap voltages (Supporting Information Figure S1c).

Bottom Line: We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly.Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition.The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds , Leeds LS2 9JT, U.K.

ABSTRACT
The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

Show MeSH
Related in: MedlinePlus