Structural and biophysical characterization of hereditary gelsolin amyloidosis variants: Y447H and R454C
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Abstract
Gelsolin (GSN), an actin-binding protein, undergoes alternative splicing to produce cytoplasmic and secreted plasma isoforms. In hereditary gelsolin amyloidosis (HGA), the D187N mutation in G2 impairs calcium binding, destabilizing the plasma isoform and enhancing furin cleavage to generate C68 fragments (68 kDa) that deposit systemically. This study explores the structural basis of gelsolin amyloidosis by examining full-length GSN and isolated G4 from wild-type and G4 mutants Y447H and R454C for stability and misfolding potential. Full-length and G4 GSN variants were recombinantly expressed, then evaluated for proteolysis via SDS-PAGE/Western blot and stability via far-UV CD spectroscopy, probing EDTA/Ca²⁺-dependent G4 differences. Circular dichroism (CD) spectroscopy revealed predominant α-helical content in G4, with melting temperatures (Tm) shifting from 69°C (WT, EDTA) to 81°C (Ca²⁺), 67°C to 85°C (Y447H), and 84°C to 78°C (R454C), indicating Ca²⁺-dependent stabilization. Proteolysis assays showed WT degraded rapidly, while Y447H resisted and R454C formed stable dimers, suggesting disulfide-mediated resilience. Notably, no C68 fragments were detected in supernatant up to 24 hours, contrasting with HGA’s G2-driven pathology. These findings suggest G4 mutations enhance GSN stability, shifting its energy landscape away from amyloidogenic pathways. Y447H’s Ca²⁺-bolstered fold and R454C’s oligomerization highlight variant-specific mechanisms that may mitigate misfolding, offering insights into GSN’s structural regulation and potential therapeutic targets for HGA.
Description
2025
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Attribution-ShareAlike 4.0 International