Plant Hormone Cytokinin as Aggregation Modulator of Gelsolin Amyloidosis

Abstract

Amyloidosis, a self-assembly of proteins or peptides, is associated with numerous degenerative diseases, such as gelsolin amyloidosis, which remain without a cure. Gelsolin protein is an actin-binding protein, but when aggregated in a diseased state, it is a potential drug target. Specifically, gelsolin mutations, N184K and D187Y, have been linked to renal amyloidosis and systemic progressive deposition of amyloids, respectively. Understanding how such mutations mitigate gelsolin aggregation and how this process can be prevented through small molecule inhibitors is of interest. Herein, we explored the efficacies of plant-based naturally occurring cytokinin (CK) molecules as aggregation modulators in vitro. Using various biophysical methods, such as spectroscopy and microscopy, the aggregation of wild-type gelsolin peptide 184NNGDCFILDL193 and its mutants (N184K, D187Y) was investigated. The mutations significantly promoted aggregation, which is of biological significance. The CK trans-zeatin (tZ) was a more effective disaggregation promoter compared with kinetin (Kin). The experimentally determined IC₅₀ values were in the 9-20 μM range. The mode of inhibition was identified as direct non-covalent complexation between the CK and the peptides by using mass spectrometry and molecular docking studies. Data show that CKs are promising amyloid modulators, which can be easily translatable to other amyloid systems.

Keywords: aggregation; amyloid; cytokinin; gelsolin.

SCHEME 1

Crystal structure of (A) plasma gelsolin protein showing Domain 2 and NNGDCFILDL sequence; (B) Domain 2 (dark blue ribbon shows the amyloidogenic peptide region [184NNGDCFILDL193] and specific single point mutations [PBD:1KCQ and PBD:3FFN, Figures generated from Pymol software]).

FIGURE 1

(A) ThT fluorescence aggregation assay showing maximum fluorescence intensity at 510 nm for WT and mutant peptides (37°C, 12 h, [peptide] = 100 μM, all values represent means ± SD N = 3). (B) Turbidity absorbance assay for WT and mutant peptides (360 nm, 37°C, 12 h, [peptide] = 100 μM, all values represent means ± SD, N = 3). (C) TEM images of aggregated WT and mutant peptides (37°C, 12 h, scale bar = 500 nm, [peptide] = 100 μM).

FIGURE 2

(A) Schematic representation of aggregation kinetics showing nucleation phase, elongation phase, and stationary phase during fibril formation. ThT aggregation kinetics of (B) WT, (C) N184K, and (D) D187Y peptide (37°C, [peptide] = 40 μM, ThT‐based maximum fluorescence emission at 510 nm, all values represent means, N = 3, experimental data were fitted using AmyloFit 2.0).

FIGURE 3

Molecular structures of kinetin (N6‐furfuryl adenine) (Kin), trans‐zeatin ((E)‐2‐methyl‐4‐(7H‐purin‐6‐ylamino)but‐2‐en‐1‐ol) (tZ), and adenine (9H‐purin‐6‐amine).

FIGURE 4

Plot of ThT‐based maximum fluorescence emission (510 nm) for (A) WT peptide, (B) N184K, and (C) D187Y in the absence and presence of CKs (left) and TEM images (right) of (A) WT peptide, (B) N184K, and (C) D187Y in the absence and presence of CKs (right) (pH 7.4, 37°C, 12 h, [peptide] = [CK] = 100 μM, scale bar = 500 nm, all values represent means ± SD, N = 3).

FIGURE 5

Dose–response curves for (A) WT, (B) N184K, and (C) D187Y variants as a function of CK type and concentration ([peptide] = 100 μM, incubation temperature = 37°C, incubation time = 12 h, all values represent means. N = 3, experimental data were fitted using the IC₅₀ toolkit program).

FIGURE 6

Molecular docking showing WT peptide interactions with (A) Kin and (B) tZ inhibitors (top) (PDB: 6QW3). The specific intermolecular interactions between CKs and amino acids are shown using dashed and solid lines (bottom).