Research ArticleMOLECULAR NEUROSCIENCE

Copper-induced structural conversion templates prion protein oligomerization and neurotoxicity

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Science Advances  01 Jul 2016:
Vol. 2, no. 7, e1600014
DOI: 10.1126/sciadv.1600014
  • Fig. 1 PrP constructs used in the experiments.

    Two constructs of PrP were used in the experiments. (A) Full-length human PrP [PrP(23–231) or PrP(23–230)]. The unstructured N-terminal domain (orange) has four metal binding octapeptide repeats. The C-terminal domain is globular and rich in α helices (blue). Red dots indicate cysteine-179 and cysteine-214, which were used to tether PrP to the surface. (B) PrP without unstructured N-terminal region [PrP(90–231) or PrP(90–230)]. (C) SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot of purified PrP(23–230) [molecular weight (MW), 25,200] and PrP(90–230) (MW, 18,500). SDS-PAGE was stained by Krypton fluorescent protein stain. The anti-prion antibodies POM1 and SAF32 were used to recognize the globular domain of PrP and the octapeptide repeats, respectively.

  • Fig. 2 Monitoring protease resistance in single PrP monomers.

    PrP(90–231) or PrP(23–231) was biotinylated and covalently immobilized on a substrate functionalized with a PEG monolayer. (A to C) The immobilized PrP was incubated (A) either with or without 1 mM Cu2+/Mn2+/Ni2+ [PrP(90–231)], (B) either with or without 1 mM Mn2+/Ni2+ [PrP(23–231)], or (C) with 1mM Cu2+ [PrP(23–231)]. Following incubation, the immobilized PrP was digested with PK; undigested PrP was detected by labeling with fluorescent streptavidin. Structural conversion to a PK-resistant conformation resulted in a fluorescence signal. (D and E) Measured fluorescence from (D) PrP(23–231) and (E) PrP(90–231) after PK digestion for different time courses normalized by the fluorescence measured from undigested PrP under each condition. Only PrP(23–231) incubated in Cu2+ converted to a stable PK-resistant conformation. PEG substrates functionalized with either fluorescently labeled DNA or biotin-conjugated BSA were used as positive and negative controls, respectively. The data in (D) were acquired from a total of 32,652, 17,638, 4776, and 29,207 molecules in the absence and presence of Cu2+, Mn2+, and Ni2+, respectively. The data in (E) were acquired from a total of 9634, 12,438, 10,680, and 8869 molecules in the absence and presence of Cu2+, Mn2+, and Ni2+, respectively. Error bars correspond to the SE calculated using a bootstrap with replacement protocol.

  • Fig. 3 Binding probabilities of full-length PrP and truncated PrP measured using single-molecule AFM.

    (A and B) Probability of binding was measured for (A) PrP(23–231) bound to both the AFM tip and the substrate and (B) PrP(90–231) bound to both the AFM tip and the substrate. (C and D) Representative force versus tip-substrate separation when opposing PrPs (C) do not interact or (D) interact with each other. In the case of PrP interaction, the PEG tether is stretched as an FJC before PrP unbinding. (E) Interaction probabilities were measured for PrP only on the AFM tip (red), for PrP only on the substrate (green), and for PrP on both the AFM tip and the substrate (blue). Red and green bars indicate the probability of nonspecific interactions. Only PrP(23–231) on both the tip and the substrate shows a higher binding rate than nonspecific interactions. (F) Binding probability (orange) and relative on-rate (blue) between opposing PrP(23–231) and binding probability (cyan) between opposing PrP(90–231) in the absence and presence of 1 mM Mn2+, 1 mM Ni2+, and 1 mM Cu2+. The total number of PrP(23–231) measurements was 62,523, 20,440, 24,315, and 19,070 in the absence of divalent metal ions and in the presence of Mn2+, Ni2+, and Cu2+, respectively. The total number of PrP(90–231) measurements was 22,048, 18,866, 8293, and 8368 in the absence of divalent metal ions and in the presence of Mn2+, Ni2+, and Cu2+, respectively. Error bars of binding probability indicate the SE and were estimated using a bootstrap with replacement protocol. Error bars of relative on-rates indicate the SE and were propagated from the SE of binding probability.

  • Fig. 4 Dissociation rate of PrP(23–231) dimers measured using single-molecule dynamic FS.

    (A) Plots of the measured rupture forces and loading rates in the absence of divalent ions. Colored open squares correspond to individual unbinding events at different loading rates (556 events). Black circles representing the most probable rupture forces were fit to the Bell-Evans model (black dashed line) to determine the off-rates (koff) and energy barrier width (xβ). Error bars indicate the SD of forces. Ninety percent confidence interval was calculated using a bootstrap with replacement protocol. (B to D) Similar analysis for PrP(23–231) in (B) 1 mM Mn2+ (709 events), (C) 1 mM Ni2+ (1133 events), and (D) 1 mM Cu2+ (606 events).

  • Fig. 5 Seeding activity and neurotoxicity of PrP(23–230) measured using RT-QuIC and organotypic slice cultures.

    (A) In the RT-QuIC assay, enhancement of ThT fluorescence is measured upon binding to amyloid fibrils. For nucleated polymerization, duration of the Tth increases when the initial amount of seeds decreases. (B) For templated aggregation, the duration of the Tth is predicted to be inversely correlated to the seed concentration. (C) RT-QuIC traces for 15 μg of human recombinant PrP(23–230) substrate seeded with PrP(23–230) that had been pre-exposed to 10 μM Cu2+. Fluorescence signals from PrP aggregates were averaged across five replicates and were baseline-corrected. Duration of the Tth was determined to be the time point where ThT fluorescence intensity first increased beyond a predetermined threshold (five times the SD of blank samples without PrP seeds and substrate). rfu, relative fluorescence units. (D) Log-log plot of Tth duration for different amounts of PrP(23–230) seeds formed in 10 μM Cu2+. The linear decrease in Tth duration with increasing seed concentration is suggestive of an inverse correlation as predicted by theory. (E and F) Similar analysis using seeds formed in (E) 10 μM Mn2+ and (F) 10 μM Ni2+ ions. In contrast to seeds formed in 10 μM Cu2+, the threshold time does not decrease with increasing seed concentration, indicating that aggregation in (E) and (F) is not templated. Error bars correspond to the SE of the mean. (G) Representative Western blot of protein markers for neuroinflammation and neurodegeneration after organotypic slice cultures were exposed to PrP(23–230) seeds. (H) Quantification of Western blot band intensities shows increased GFAP, PKC-δ, and Bax protein expression upon exposure to copper-induced prion amyloids. Each group is represented by the mean ± SEM from at least four separate measurements.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/7/e1600014/DC1

    Supplementary Methods

    Supplementary Results

    fig. S1. Fluorescence images with different amounts of fluorescent streptavidin on the substrate.

    fig. S2. Calibration plot of the number of streptavidin versus fluorescence intensity.

    fig. S3. Histogram of unbinding force for PrP(23–231) measured in the absence of divalent ions.

    fig. S4. Histogram of unbinding force for PrP(23–231) measured in 1 mM Mn2+.

    fig. S5. Histogram of unbinding force for PrP(23–231) measured in 1 mM Ni2+.

    fig. S6. Histogram of unbinding force for PrP(23–231) measured in 1 mM Cu2+.

    fig. S7. Formation of PrP(23–230) seeds monitored in real time using ThT fluorescence intensity.

    fig. S8. No seeds were formed when PrP(90–230) was incubated with divalent metal ions for 84 hours.

    fig. S9. Seeding activity of PrP(23–230) seeds generated in 10 μM Mn2+ measured using RT-QuIC.

    fig. S10. Seeding activity of PrP(23–230) seeds generated in 10 μM Ni2+ measured using RT-QuIC.

    fig. S11. Seeding activity of PrP(23–230) seeds generated in 10 μM Cu2+ measured using RT-QuIC.

    fig. S12. Formation of PrP(23–230) seeds using 1 μM Cu2+ and its corresponding seeding activity.

    fig. S13. Upon addition of protein seeds to brain slice cultures, residual copper does not increase levels of PKC-δ and Bax.

    fig. S14. Biotinylation of PrP and functionalization with PEG tethers do not alter sensitivity to PK digestion.

    fig. S15. PrP(23–230) and PrP(90–230) remain in a native conformation after reduction of disulfide bond and functionalization with PEG tethers.

    fig. S16. One-dimensional 1H-NMR spectra of PrP(23–231) show that the protein is in a natively folded conformation.

    fig. S17. One-dimensional 1H-NMR spectra of PrP(90–231) show that the protein is in a natively folded conformation.

    fig. S18. One-dimensional 1H-NMR spectra of PrP(23–230) show that the protein is in a natively folded conformation.

    fig. S19. One-dimensional 1H-NMR spectra of PrP(90–230) show that the protein is in a natively folded conformation.

    fig. S20. One-dimensional 1H-NMR spectra of PrP(23–230) after disulfide bond reduction show that the protein remains in a natively folded conformation.

    fig. S21. One-dimensional 1H-NMR spectra of PrP(90–230) after disulfide bond reduction show that the protein remains in a natively folded conformation.

    fig. S22. One-dimensional 1H-NMR spectra of PrP(23–230) linked to PEG tethers show that the protein remains in a natively folded conformation.

    fig. S23. One-dimensional 1H-NMR spectra of PrP(90–230) linked to PEG tethers show that the protein remains in a natively folded conformation.

    fig. S24. Functionalization with PEG tethers does not alter the thermal stabilities of PrP(23–230) and PrP(90–230).

    fig. S25. Reduction of disulfide bond and functionalization with PEG tethers do not cause aggregation of PrP(23–230) and PrP(90–230).

    fig. S26. Protein age and the presence of N-terminal His tag do not cause aggregation of PrP(23–230) and PrP(90–230).

    fig. S27. Protein age and the presence of N-terminal His tag do not alter the secondary structure of PrP(23–230) and PrP(90–230).

    table S1. Surface density of PrP in PK digestion experiments.

    table S2. Off-rate, relative on-rate, and relative association constant (KA) for PrP(23–231) binding.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • Supplementary Results
    • fig. S1. Fluorescence images with different amounts of fluorescent streptavidin on the substrate.
    • fig. S2. Calibration plot of the number of streptavidin versus fluorescence intensity.
    • fig. S3. Histogram of unbinding force for PrP(23–231) measured in the absence of divalent ions.
    • fig. S4. Histogram of unbinding force for PrP(23–231) measured in 1 mM Mn2+.
    • fig. S5. Histogram of unbinding force for PrP(23–231) measured in 1 mM Ni2+.
    • fig. S6. Histogram of unbinding force for PrP(23–231) measured in 1 mM Cu2+.
    • fig. S7. Formation of PrP(23–230) seeds monitored in real time using ThT fluorescence intensity.
    • fig. S8. No seeds were formed when PrP(90–230) was incubated with divalent metal ions for 84 hours.
    • fig. S9. Seeding activity of PrP(23–230) seeds generated in 10 μM Mn2+ measured using RT-QuIC.
    • fig. S10. Seeding activity of PrP(23–230) seeds generated in 10 μM Ni2+measured using RT-QuIC.
    • fig. S11. Seeding activity of PrP(23–230) seeds generated in 10 μM Cu2+ measured using RT-QuIC.
    • fig. S12. Formation of PrP(23–230) seeds using 1 μM Cu2+ and its corresponding seeding activity.
    • fig. S13. Upon addition of protein seeds to brain slice cultures, residual copper does not increase levels of PKC-δand Bax.
    • fig. S14. Biotinylation of PrP and functionalization with PEG tethers do not alter sensitivity to PK digestion.
    • fig. S15. PrP(23–230) and PrP(90–230) remain in a native conformation after reduction of disulfide bond and functionalization with PEG tethers.
    • fig. S16. One-dimensional 1H-NMR spectra of PrP(23–231) show that the protein is in a natively folded conformation.
    • fig. S17. One-dimensional 1H-NMR spectra of PrP(90–231) show that the protein is in a natively folded conformation.
    • fig. S18. One-dimensional 1H-NMR spectra of PrP(23–230) show that the protein is in a natively folded conformation.
    • fig. S19. One-dimensional 1H-NMR spectra of PrP(90–230) show that the protein is in a natively folded conformation.
    • fig. S20. One-dimensional 1H-NMR spectra of PrP(23–230) after disulfide bond reduction show that the protein remains in a natively folded conformation.
    • fig. S21. One-dimensional 1H-NMR spectra of PrP(90–230) after disulfide bond reduction show that the protein remains in a natively folded conformation.
    • fig. S22. One-dimensional 1H-NMR spectra of PrP(23–230) linked to PEG tethers show that the protein remains in a natively folded conformation.
    • fig. S23. One-dimensional 1H-NMR spectra of PrP(90–230) linked to PEG tethers show that the protein remains in a natively folded conformation.
    • fig. S24. Functionalization with PEG tethers does not alter the thermal stabilities of PrP(23–230) and PrP(90–230).
    • fig. S25. Reduction of disulfide bond and functionalization with PEG tethers do not cause aggregation of PrP(23–230) and PrP(90–230).
    • fig. S26. Protein age and the presence of N-terminal His tag do not cause aggregation of PrP(23–230) and PrP(90–230).
    • fig. S27. Protein age and the presence of N-terminal His tag do not alter the secondary structure of PrP(23–230) and PrP(90–230).
    • table S1. Surface density of PrP in PK digestion experiments.
    • table S2. Off-rate, relative on-rate, and relative association constant (KA) for PrP(23–231) binding.

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