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. 2020 Apr 15;11(8):1178-1191.
doi: 10.1021/acschemneuro.0c00069. Epub 2020 Apr 2.

Acetylation of Aβ42 at Lysine 16 Disrupts Amyloid Formation

Rashmi Adhikari  1 , Mu Yang  1 , Nabanita Saikia  2 , Colina Dutta  1 , Wafa F A Alharbi  3 , Zhiying Shan  4 , Ravindra Pandey  2 , Ashutosh Tiwari  1
Affiliations

Acetylation of Aβ42 at Lysine 16 Disrupts Amyloid Formation

Rashmi Adhikari et al. ACS Chem Neurosci. .

Abstract

The residue lysine 28 (K28) is known to form an important salt bridge that stabilizes the Aβ amyloid structure, and acetylation of lysine 28 (K28Ac) slows the Aβ42 fibrillization rate but does not affect fibril morphology. On the other hand, acetylation of lysine 16 (K16Ac) residue greatly diminishes the fibrillization property of Aβ42 peptide and also affects its toxicity. This is due to the fact that lysine 16 acetylated amyloid beta peptide forms amorphous aggregates instead of amyloid fibrils. This is likely a result of increased hydrophobicity of the K16-A21 region due to K16 acetylation, as confirmed by molecular dynamic simulation studies. The calculated results show that the hydrophobic patches of aggregates from acetylated peptides were different when compared to wild-type (WT) peptide. K16Ac and double acetylated (KKAc) peptide aggregates show significantly higher cytotoxicity compared to the WT or K28Ac peptide aggregates alone. However, the heterogeneous mixture of WT and acetylated Aβ42 peptide aggregates exhibited higher free radical formation as well as cytotoxicity, suggesting dynamic interactions between different species could be a critical contributor to Aβ pathology.

Keywords: acetylation; aggregation; amyloid fibril; amyloid β peptide; molecular dynamics; post-translational modifications; toxicity.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(A) Sequence of Aβ42 peptide (PDB entry: 1Z0Q). Atomic structures of (B) WT, (C) K16Ac, (D) K28Ac, and (E) KKAc monomer. The two acetylated positions, K16 and K28, are highlighted in ball and stick presentation.
Figure 2.
Figure 2.
Fibrillization/aggregation of WT, acetylated, and mixtured Aβ42 peptides were monitored using ThT fluorescence, bis-ANS fluorescence, and SEM. The mixture represents the 1:1 ratio of the acetylated peptides with WT Aβ42 peptides. For ThT fluorescence analysis (A–D), 10 μM peptide samples were incubated with 10 μM ThT. Peak intensities at 487 nm were plotted as a function of time. After 7 day of incubation, the structures of fibrils or aggregates were visualized using SEM (E–H and J–L). Scale bars are 1 μm for all SEM images. Structure flexibility of the fibrils or aggregates was measured by bis-ANS fluorescence (I) by incubating 10 μM peptide samples with 1 μM bis-ANS and measuring peak intensities at 484 nm. Error bars = ±SD (For A-D n = 3; for I n = 3). * represents significant differences (p < 0.05) among 7 d Aβ42 aggregated peptides.
Figure 3.
Figure 3.
Simulation snapshots depicting the structural changes in aggregation of nine strands of WT and acetylated (K16Ac, K28Ac, and KKAc) Aβ42 peptides in aqueous solution. For each structure, the predicted hydrophobic patches (yellow) were mapped on the molecular surface (violet) using SPDB software. (A) WT, (B) K16Ac, (C) K28Ac, and (D) KKAc aggregates. The water molecules are not shown for clarity.
Figure 4.
Figure 4.
Hydrophobicity of Aβ42 peptides measured by ANS fluorescence and hydrophobic bead binding assay. (A) Hydrophobicity of fresh Aβ42 peptides and their aggregates measured by ANS fluorescence by incubating 10 μM peptide samples with 10 μM ANS and measuring peak intensities at 485 nm. Mixture represents the 1:1 ratio of the acetylated peptides with WT Aβ42 peptides. Error bars = ±SD (n = 3). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.005), and (*** = p < 0.0005) among 7 d Aβ42 aggregated peptides. (B) Seven-day incubated Aβ42 peptide samples (3 μg) were further incubated with phenyl-sepharose beads overnight with gentle agitation at 25 °C. The beads were washed thrice with binding buffer, and then Aβ42 peptides bound to beads were eluted by boiling with denaturing buffer for 3 min. Eluted Aβ42 peptide samples and loading controls (WTC 1–4) were run on SDS-PAGE and visualized by staining with Coomassie blue. (C) The plot shows percentages of Aβ42 peptides bound to hydrophobic beads which were calculated with respect to WT Aβ42 peptides controls (WTC 1–4) loaded on gel.
Figure 5.
Figure 5.
The effect of fresh Aβ42 peptides and their aggregates on SH-SY5Y neuroblastoma cell health was measured by (A) MTS assay, (B) DCFH-DA assay, and (C) LDH assay. The fresh peptides, 72 h, or 7 d aggregates were added to the cells to a final concentration of 5 μM. Cells were incubated for 48 h in the presence of peptides for MTS and LDH assay and for 24 h for DCFH-DA assay. For the t-BHP control for the DCFH-DA assay, cells were incubated with 50 μM t-BHP for 4 h at 37 °C. The mixtures represent samples containing equimolar (1:1) WT and acetylated peptides. Error bars = ±SD (for A n = 6; for B n = 5; and for C n = 5). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.005), and (*** = p < 0.0005) among 7 d Aβ42 aggregated peptides.
Figure 6.
Figure 6.
The effect of fresh Aβ42 peptides and their aggregates on primary neuronal cell health was measured by (A) DCFH-DA assay and (B) LDH assay. The fresh peptides, 72 h, or 7 d aggregates were added to the cells to a final concentration of 2 μM. Cells were incubated for 24 h in the presence of peptides for both assays. For the t-BHP control for the DCFH-DA assay, cells were incubated with 50 μM t-BHP for 4 h at 37 °C. The mixtures represent samples containing equimolar (1:1) WT and acetylated peptides. Error bars = ±SD (for A n = 5; and for B n = 4). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.005), and (*** = p < 0.0005) among 7 d Aβ42 aggregated peptides.
Figure 7.
Figure 7.
Immunostaining of primary neuronal cells from rat brain. Primary neuronal cells were incubated with 2 μM 7 d WT and acetylated Aβ42 peptides for 24 h at 37 °C. Control indicates cells that are untreated and incubated under conditions identical to cells treated with Aβ42 peptides for comparison. The cells were fixed and then stained for immunofluorescence with antibodies for tubulin (green) and tau (red). The images were acquired using Leica DMIL LED at 20× magnification. Scale bar = 50 μm.
Figure 8.
Figure 8.
Suggested schematic model for WT and acetylation modified Aβ42 peptide aggregation and cytotoxicity. WT Aβ42 and K28Ac peptides show a lag and log phase of aggregation kinetics and form ordered aggregates with large surface hydrophobic patches (yellow) that can assemble as amyloid fibrils and have moderate toxicity. Peptides acetylated at K16 (K16Ac or double acetylated KKAc) show rapid aggregation kinetics and form disordered, amorphous, and flexible aggregates that have higher surface hydrophobicity and high toxicity.
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