American Society of Hematology

Figure 4.

$Figure 4. In vitro fibrillogenesis and structural analysis of Aα-chain frameshift-derived polypeptides identified in amyloid deposits in vivo. (A) ThT fluorescence assays performed on AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG (red curve) and AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNP_G (blue curve). Characteristic enhanced ThT fluorescence at 485 nm was only observed for AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG, suggesting that this peptide formed amyloid β-sheet structures. In contrast, a spectral red shift at 510 nm was recorded for AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNP_G, suggesting that it does not form typical amyloid β-sheet structures. (B) Transmission electron micrographs showed that AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG forms fibrillar aggregates, and AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNP_G spherical aggregates. (C) Fibrils formed by AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG exhibited a meridional peak at 4.72 Å, indicating the spacing between β-strands within fibrils associated with an equatorial reflection at 11.50 Å. This microdiffraction pattern confirms the presence of the characteristic amyloid cross-β architecture constituted by intermolecular β-sheets with β-strands oriented perpendicular to the fibril axis. (D) An extracted radial profile from the two-dimensional pattern shown in panel C. The noisy background is because of the small angle used for the profile extraction to avoid the intense peaks from salts in the sample. (E) Data from ThT fluorescence assays performed on ASSQIQRNPVLITLG (red curve) and ASSQIQRNP_G (blue curve), revealing that only ASSQIQRNPVLITLG induces enhanced fluorescence at 485 nm and that only the peptide containing VLITL forms aggregates with β-sheet conformation. (F) Transmission electron micrographs showed that ASSQIQRNPVLITLG forms aggregates of fibrillar morphology. (G) XRD profiles of ASSQIQRNPVLITLG fibrils displaying the typical “cross-β” microdiffraction pattern of amyloid fibrils with a spacing of 4.75 Å along the meridional direction and a periodicity of 9.90 Å in the equatorial direction. (H) The in situ XRD pattern from a cut of the pathological kidney specimen of patient II.2 with in vivo amyloid fibrils, showing meridional (4.71 Å) and equatorial (10.0 Å) reflections. (I) A ThT fluorescence assay of VLITL with increased fluorescence intensity at 485 nm, demonstrating that the dye bound to β-sheet-enriched amyloid fibrils. (J and K) Transmission electron micrographs of mature VLITL fibrils with ribbonlike structures at different magnifications. Frayed ribbons are observed at the ends of the VLITL twisted fiber structures. The scale bar represents 200 nm. (L) The VLITL fibrils exhibited strong meridional (4.65 Å) and equatorial (10.7 Å) reflections characteristic of a cross-β pattern, confirming their amyloid nature.$

In vitro fibrillogenesis and structural analysis of Aα-chain frameshift-derived polypeptides identified in amyloid deposits in vivo. (A) ThT fluorescence assays performed on AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG (red curve) and AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNP_G (blue curve). Characteristic enhanced ThT fluorescence at 485 nm was only observed for AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG, suggesting that this peptide formed amyloid β-sheet structures. In contrast, a spectral red shift at 510 nm was recorded for AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNP_G, suggesting that it does not form typical amyloid β-sheet structures. (B) Transmission electron micrographs showed that AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG forms fibrillar aggregates, and AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNP_G spherical aggregates. (C) Fibrils formed by AFFDTASTGKTFPGFFSPMLGELSVRLSLGAQNLASSQIQRNPVLITLG exhibited a meridional peak at 4.72 Å, indicating the spacing between β-strands within fibrils associated with an equatorial reflection at 11.50 Å. This microdiffraction pattern confirms the presence of the characteristic amyloid cross-β architecture constituted by intermolecular β-sheets with β-strands oriented perpendicular to the fibril axis. (D) An extracted radial profile from the two-dimensional pattern shown in panel C. The noisy background is because of the small angle used for the profile extraction to avoid the intense peaks from salts in the sample. (E) Data from ThT fluorescence assays performed on ASSQIQRNPVLITLG (red curve) and ASSQIQRNP_G (blue curve), revealing that only ASSQIQRNPVLITLG induces enhanced fluorescence at 485 nm and that only the peptide containing VLITL forms aggregates with β-sheet conformation. (F) Transmission electron micrographs showed that ASSQIQRNPVLITLG forms aggregates of fibrillar morphology. (G) XRD profiles of ASSQIQRNPVLITLG fibrils displaying the typical “cross-β” microdiffraction pattern of amyloid fibrils with a spacing of 4.75 Å along the meridional direction and a periodicity of 9.90 Å in the equatorial direction. (H) The in situ XRD pattern from a cut of the pathological kidney specimen of patient II.2 with in vivo amyloid fibrils, showing meridional (4.71 Å) and equatorial (10.0 Å) reflections. (I) A ThT fluorescence assay of VLITL with increased fluorescence intensity at 485 nm, demonstrating that the dye bound to β-sheet-enriched amyloid fibrils. (J and K) Transmission electron micrographs of mature VLITL fibrils with ribbonlike structures at different magnifications. Frayed ribbons are observed at the ends of the VLITL twisted fiber structures. The scale bar represents 200 nm. (L) The VLITL fibrils exhibited strong meridional (4.65 Å) and equatorial (10.7 Å) reflections characteristic of a cross-β pattern, confirming their amyloid nature.

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