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Pyroglutamylated amyloid-β peptide reverses cross β-sheets by a prion-like mechanism.

Matos JO, Goldblatt G, Jeon J, Chen B, Tatulian SA - J Phys Chem B (2014)

Bottom Line: The amyloid hypothesis causatively relates the fibrillar deposits of amyloid β peptide (Aβ) to Alzheimer's disease (AD).Pyroglutamylated Aβ (pE-Aβ) is present in AD brains and exerts augmented neurotoxicity, which is believed to result from its higher β-sheet propensity and faster fibrillization.Circular dichroism and FTIR data indicate that while the unmodified Aβ readily forms β-sheet fibrils in aqueous media, pE-Aβ displays increased α-helical and decreased β-sheet propensity.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Graduate Program, University of Central Florida , 4000 Central Florida Boulevard, Orlando, Florida 32816, United States.

ABSTRACT
The amyloid hypothesis causatively relates the fibrillar deposits of amyloid β peptide (Aβ) to Alzheimer's disease (AD). More recent data, however, identify the soluble oligomers as the major cytotoxic entities. Pyroglutamylated Aβ (pE-Aβ) is present in AD brains and exerts augmented neurotoxicity, which is believed to result from its higher β-sheet propensity and faster fibrillization. While this concept is based on a set of experimental results, others have reported similar β-sheet contents in unmodified and pyroglutamylated Aβ, and slower aggregation of pE-Aβ as compared to unmodified Aβ, leaving the issue unresolved. Here, we assess the structural differences between Aβ and pE-Aβ peptides that may underlie their distinct cytotoxicities. Transmission electron microscopy identifies a larger number of prefibrillar aggregates of pE-Aβ at early stages of aggregation and suggests that pE-Aβ affects the fibrillogenesis even at low molar fractions. Circular dichroism and FTIR data indicate that while the unmodified Aβ readily forms β-sheet fibrils in aqueous media, pE-Aβ displays increased α-helical and decreased β-sheet propensity. Moreover, isotope-edited FTIR spectroscopy shows that pE-Aβ reverses β-sheet formation and hence fibrillogenesis of the unmodified Aβ peptide via a prion-like mechanism. These data provide a novel structural mechanism for pE-Aβ hypertoxicity; pE-Aβ undergoes faster formation of prefibrillar aggregates due to its increased hydrophobicity, thus shifting the initial stages of fibrillogenesis toward smaller, hypertoxic oligomers of partial α-helical structure.

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CD spectra of dry and water-suspendedpeptides. (a) Aβ1-42 (green), AβpE3-42 (turquoise),AβpE3-42/Aβ1–42 =1:9 (blue), and AβpE3-42/Aβ1–42 = 1:1 (red) were dissolved in HFIP, followed by 15 min of desiccationin a 4 mm × 4 mm quartz cuvette and collection of the spectra.(b–e) Aqueous buffer of 50 mM NaCl + 50 mM Na,K-phosphate (pH7.2) was added to a 50 μM final concentration of Aβ1–42 (b), AβpE3-42 (c), AβpE3-42/Aβ1–42 = 1:9 (d), andAβpE3-42/Aβ1–42 =1:1 (e), and spectra were acquired after 1 h (blue) and 24 h (red)of incubation at 37 °C with constant stirring.
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fig2: CD spectra of dry and water-suspendedpeptides. (a) Aβ1-42 (green), AβpE3-42 (turquoise),AβpE3-42/Aβ1–42 =1:9 (blue), and AβpE3-42/Aβ1–42 = 1:1 (red) were dissolved in HFIP, followed by 15 min of desiccationin a 4 mm × 4 mm quartz cuvette and collection of the spectra.(b–e) Aqueous buffer of 50 mM NaCl + 50 mM Na,K-phosphate (pH7.2) was added to a 50 μM final concentration of Aβ1–42 (b), AβpE3-42 (c), AβpE3-42/Aβ1–42 = 1:9 (d), andAβpE3-42/Aβ1–42 =1:1 (e), and spectra were acquired after 1 h (blue) and 24 h (red)of incubation at 37 °C with constant stirring.

Mentions: It has been recognized that the fibrillarmorphology is determinedby the molecular structure of the peptides,23,30,38,39 but CD studiesprovided conflicting data on the relative secondary structural changesin Aβ and pE-Aβ during fibrillogenesis (see above). Tomonitor the structural transitions in the peptides during fibrillogenesis,peptide samples dried from HFIP solution were used as a starting point,before the onset of aggregation. CD spectra of Aβ1–42, AβpE3-42, and their combinations in dryform shown in Figure 2a indicate mostly α-helicalstructure with two minima around 222 and 208 nm.40,41 The spectrum of AβpE3-42 has a significantlyreduced ratio of ellipticities θ208/θ222, indicative of a more flexible or disordered α-helix.42 These results concur with solution NMR datashowing α-helical conformation for both Aβ1–42 and AβpE3–40 in organic solvents.9,29 Upon addition of an aqueous buffer and incubation at 37 °Cwith constant stirring, the peptides undergo significant structuralchanges. Aβ1–42 promptly adopts and maintainsβ-sheet structure, as evidenced by a deep minimum at 215–216nm of spectra measured at 1 and 24 h of incubation (Figure 2b). The spectra of AβpE3-42, on the other hand, show a wide well between 208 and 222 nm, mostlikely indicating a combination of α-helical and β-sheetstructures (Figure 2c). These data suggestsubstantially different structures of Aβ1–42 and AβpE3-42, while the former readily adoptsβ-sheets, the latter shows increased α-helical propensity.The CD spectra of the 1:9 AβpE3-42/Aβ1–42 combination display β-sheet features, i.e.a prominent minimum at 216 nm at 1 h and at 219 nm at 24 h of incubation(Figure 2d). The higher intensity and the redshift of the signal at 24 h may reflect gradual suspension of thepeptide into the aqueous medium and decreased solvent accessibilityupon aggregation.40 It should be notedthat the spectra of Figure 2d are dominatedby the structural features of Aβ1–42 whichare present at a large molar excess (90%). At 1:1 molar ratio, the1-h spectrum shows a minimum at 209 and a shoulder at 223 nm (Figure 2e), implying α-helix structure, possibly includinga β-sheet component, as in the case of pure AβpE3-42 (cf. blue spectra in Figure 2c and e). At24 h, the spectrum has a β-sheet minimum at 216 nm and a shoulderat 227 nm, likely generated by a turn structure. It is remarkablethat AβpE3-42 exerts a dominant structuraleffect, especially at the early stages of fibrillogenesis. Thus, consistentwith the TEM data, CD results indicate that (a) Aβ1–42 and AβpE3-42 evidently follow distinct structuralpathways of fibrillogenesis and (b) AβpE3-42 is able to divert the overall path toward less β-sheet andmore α-helical intermediates.


Pyroglutamylated amyloid-β peptide reverses cross β-sheets by a prion-like mechanism.

Matos JO, Goldblatt G, Jeon J, Chen B, Tatulian SA - J Phys Chem B (2014)

CD spectra of dry and water-suspendedpeptides. (a) Aβ1-42 (green), AβpE3-42 (turquoise),AβpE3-42/Aβ1–42 =1:9 (blue), and AβpE3-42/Aβ1–42 = 1:1 (red) were dissolved in HFIP, followed by 15 min of desiccationin a 4 mm × 4 mm quartz cuvette and collection of the spectra.(b–e) Aqueous buffer of 50 mM NaCl + 50 mM Na,K-phosphate (pH7.2) was added to a 50 μM final concentration of Aβ1–42 (b), AβpE3-42 (c), AβpE3-42/Aβ1–42 = 1:9 (d), andAβpE3-42/Aβ1–42 =1:1 (e), and spectra were acquired after 1 h (blue) and 24 h (red)of incubation at 37 °C with constant stirring.
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fig2: CD spectra of dry and water-suspendedpeptides. (a) Aβ1-42 (green), AβpE3-42 (turquoise),AβpE3-42/Aβ1–42 =1:9 (blue), and AβpE3-42/Aβ1–42 = 1:1 (red) were dissolved in HFIP, followed by 15 min of desiccationin a 4 mm × 4 mm quartz cuvette and collection of the spectra.(b–e) Aqueous buffer of 50 mM NaCl + 50 mM Na,K-phosphate (pH7.2) was added to a 50 μM final concentration of Aβ1–42 (b), AβpE3-42 (c), AβpE3-42/Aβ1–42 = 1:9 (d), andAβpE3-42/Aβ1–42 =1:1 (e), and spectra were acquired after 1 h (blue) and 24 h (red)of incubation at 37 °C with constant stirring.
Mentions: It has been recognized that the fibrillarmorphology is determinedby the molecular structure of the peptides,23,30,38,39 but CD studiesprovided conflicting data on the relative secondary structural changesin Aβ and pE-Aβ during fibrillogenesis (see above). Tomonitor the structural transitions in the peptides during fibrillogenesis,peptide samples dried from HFIP solution were used as a starting point,before the onset of aggregation. CD spectra of Aβ1–42, AβpE3-42, and their combinations in dryform shown in Figure 2a indicate mostly α-helicalstructure with two minima around 222 and 208 nm.40,41 The spectrum of AβpE3-42 has a significantlyreduced ratio of ellipticities θ208/θ222, indicative of a more flexible or disordered α-helix.42 These results concur with solution NMR datashowing α-helical conformation for both Aβ1–42 and AβpE3–40 in organic solvents.9,29 Upon addition of an aqueous buffer and incubation at 37 °Cwith constant stirring, the peptides undergo significant structuralchanges. Aβ1–42 promptly adopts and maintainsβ-sheet structure, as evidenced by a deep minimum at 215–216nm of spectra measured at 1 and 24 h of incubation (Figure 2b). The spectra of AβpE3-42, on the other hand, show a wide well between 208 and 222 nm, mostlikely indicating a combination of α-helical and β-sheetstructures (Figure 2c). These data suggestsubstantially different structures of Aβ1–42 and AβpE3-42, while the former readily adoptsβ-sheets, the latter shows increased α-helical propensity.The CD spectra of the 1:9 AβpE3-42/Aβ1–42 combination display β-sheet features, i.e.a prominent minimum at 216 nm at 1 h and at 219 nm at 24 h of incubation(Figure 2d). The higher intensity and the redshift of the signal at 24 h may reflect gradual suspension of thepeptide into the aqueous medium and decreased solvent accessibilityupon aggregation.40 It should be notedthat the spectra of Figure 2d are dominatedby the structural features of Aβ1–42 whichare present at a large molar excess (90%). At 1:1 molar ratio, the1-h spectrum shows a minimum at 209 and a shoulder at 223 nm (Figure 2e), implying α-helix structure, possibly includinga β-sheet component, as in the case of pure AβpE3-42 (cf. blue spectra in Figure 2c and e). At24 h, the spectrum has a β-sheet minimum at 216 nm and a shoulderat 227 nm, likely generated by a turn structure. It is remarkablethat AβpE3-42 exerts a dominant structuraleffect, especially at the early stages of fibrillogenesis. Thus, consistentwith the TEM data, CD results indicate that (a) Aβ1–42 and AβpE3-42 evidently follow distinct structuralpathways of fibrillogenesis and (b) AβpE3-42 is able to divert the overall path toward less β-sheet andmore α-helical intermediates.

Bottom Line: The amyloid hypothesis causatively relates the fibrillar deposits of amyloid β peptide (Aβ) to Alzheimer's disease (AD).Pyroglutamylated Aβ (pE-Aβ) is present in AD brains and exerts augmented neurotoxicity, which is believed to result from its higher β-sheet propensity and faster fibrillization.Circular dichroism and FTIR data indicate that while the unmodified Aβ readily forms β-sheet fibrils in aqueous media, pE-Aβ displays increased α-helical and decreased β-sheet propensity.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Graduate Program, University of Central Florida , 4000 Central Florida Boulevard, Orlando, Florida 32816, United States.

ABSTRACT
The amyloid hypothesis causatively relates the fibrillar deposits of amyloid β peptide (Aβ) to Alzheimer's disease (AD). More recent data, however, identify the soluble oligomers as the major cytotoxic entities. Pyroglutamylated Aβ (pE-Aβ) is present in AD brains and exerts augmented neurotoxicity, which is believed to result from its higher β-sheet propensity and faster fibrillization. While this concept is based on a set of experimental results, others have reported similar β-sheet contents in unmodified and pyroglutamylated Aβ, and slower aggregation of pE-Aβ as compared to unmodified Aβ, leaving the issue unresolved. Here, we assess the structural differences between Aβ and pE-Aβ peptides that may underlie their distinct cytotoxicities. Transmission electron microscopy identifies a larger number of prefibrillar aggregates of pE-Aβ at early stages of aggregation and suggests that pE-Aβ affects the fibrillogenesis even at low molar fractions. Circular dichroism and FTIR data indicate that while the unmodified Aβ readily forms β-sheet fibrils in aqueous media, pE-Aβ displays increased α-helical and decreased β-sheet propensity. Moreover, isotope-edited FTIR spectroscopy shows that pE-Aβ reverses β-sheet formation and hence fibrillogenesis of the unmodified Aβ peptide via a prion-like mechanism. These data provide a novel structural mechanism for pE-Aβ hypertoxicity; pE-Aβ undergoes faster formation of prefibrillar aggregates due to its increased hydrophobicity, thus shifting the initial stages of fibrillogenesis toward smaller, hypertoxic oligomers of partial α-helical structure.

Show MeSH
Related in: MedlinePlus