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. 2022 Jun 15;13(12):1714-1718.
doi: 10.1021/acschemneuro.2c00077. Epub 2022 May 24.

A Chemical Mutagenesis Approach to Insert Post-translational Modifications in Aggregation-Prone Proteins

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A Chemical Mutagenesis Approach to Insert Post-translational Modifications in Aggregation-Prone Proteins

Ying Ge et al. ACS Chem Neurosci. .

Abstract

Neurodegenerative diseases are a class of disorders linked to the formation in the nervous system of fibrillar protein aggregates called amyloids. This aggregation process is affected by a variety of post-translational modifications, whose specific mechanisms are not fully understood yet. Emerging chemical mutagenesis technology is currently striving to address the challenge of introducing protein post-translational modifications, while maintaining the stability and solubility of the proteins during the modification reaction. Several amyloidogenic proteins are highly aggregation-prone, and current modification procedures can lead to unexpected precipitation of these proteins, affecting their yield and downstream characterization. Here, we present a method for maintaining amyloidogenic protein solubility during chemical mutagenesis. As proof-of-principle, we applied our method to mimic the phosphorylation of serine-26 and the acetylation of lysine-28 of the 40-residue long variant of amyloid-β peptide, whose aggregation is linked to Alzheimer's disease.

Keywords: Alzheimer’s disease; amyloid-β; chemical mutagenesis; post-translational modification.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Experimental strategy. (1) SD–Aβ40 with a single cysteine was expressed as a His6-tagged fusion protein in E. coli BL21(DE3) cells. (2) The fusion protein was purified from the cell lysate by Ni-NTA affinity chromatography. (3) Purified fusion protein was reacted with DBHDA to convert the cysteine residue into a Dha. (4) Dha was then converted into a phosphocysteine or acetyllysine mimic by thia-Michael addition with sodium thiophosphate (4a, R = PO3) or N-acetylcysteamine (4b, R = C4H8NO), respectively. (5) The SD was removed via proteolysis by the addition of TEV protease, and the digestion mixture was subjected to size-exclusion chromatography to remove the SD and TEV and to obtain monomeric Aβ. Structure of the SD (PDB 4FBS) was rendered as cartoon using BioRender.
Figure 2
Figure 2
Deconvoluted mass spectra of SD–Aβ40-K28C (A) before modification, (B) after introduction of the Dha intermediate, (C) after introduction of the acetyllysine mimetic, and (D) as a monomeric peptide. Cal., calculated mass; Obs., observed mass.
Figure 3
Figure 3
(A) Representative SDS-PAGE showing the Aβ40 (or PTM variants) as a fusion protein (lane 1), after reaction with TEV (lane 3), and after purification by size exclusion chromatography (lanes 4–6). (B) ThT aggregation assay of 5 μM recombinant Aβ40 (black), Aβ40-Ac(S)K28 (red), and Aβ40-Epi(S)K28 (blue). Fluorescence of 20 μM ThT in buffer alone was recorded in parallel and subtracted as baseline. Error bars show standard deviation (N = 5). Data were normalized and fitted to a sigmoidal curve in Prism.
Figure 4
Figure 4
TEM images showing fibrils of (A) Aβ40, (B) Aβ40-Ac(S)K28, and (C) Aβ40-Epi(S)K28 taken during the plateau phase of aggregation.

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