Selective Interception of Gelsolin Amyloidogenic Stretch Results in Conformationally Distinct Aggregates with Reduced Toxicity

Abstract

The pathogenesis of protein misfolding diseases is attributed to the cytotoxicity caused by amyloidogenic prefibrillar aggregates, rather than mature fibrils. The presence of one or more amyloidogenic stretches in different proteins has been proven critical for initiating fibril formation. In the present study, we show that two natural compounds, curcumin and emetine, bind tightly (Kd < 1.6 μM) to the core amyloidogenic stretch (182-192) of gelsolin (AGel). Binding happens in different structural orientations, distinctly modulating the amyloidogenic pathway of AGel. While AGel alone undergoes sigmoidal transition to thioflavin T (ThT)-responsive fibrillar aggregates with clear lag phase, the presence of curcumin or emetine abolishes the lag phase and produces starkly different, noncytotoxic end products. Atomic force microscopy revealed that while curcumin augments fibril formation, emetine arrests it at an intermediate aggregated stage with no fibrillar morphology. FTIR spectroscopy, dynamic light scattering, and ANS fluorescence experiments also suggest that these two species are distinct. Curcumin and emetine also differentially affect the preformed amyloids with the former thickening the fibrils and the latter releasing reclusive oligomers. MD simulations further provided mechanistic insights of differential interaction by the two compounds modulating amyloid formation. The results were also confirmed on the disease-associated amyloidogenic fragment of gelsolin (fAGel). Thus, our findings suggest that targeting amyloidogenic stretches in proteins could be useful in designing novel molecules against protein misfolding diseases.

DNA intercalators as amyloid assembly modulators: mechanistic insights

“…69,70 To study the interference of intercalators with amyloidogenic pathways of peptide and proteins, two different types of systems were setup; aggregation of peptide monomers to form proto-bril like oligomers and effects of intercalators on pre-formed protobrillar assembly. In rst system, eight pre-generated random conformation monomers of IAP (22)(23)(24)(25)(26)(27)(28) and AGel (186-192) were simulated separately, corresponding to their experimental aggregation conditions described elsewhere, 20,37 without applying backbone dihedral or distance constraints. In identical setups, simulations were carried out in the presence of intercalators to assess their ability in modulating assembly formation by respective peptides.…”

“…ThT uorescence exhibits a hyperchromic shi in emission maximum upon binding to b-rich brillar structures during amyloidogenesis. 37,47,48 Traversing from lag and exponential to stationary phase, amyloidogenesis generally follows sigmoidal kinetics. 49,50 In our case, reactions of proteins/peptides were prepared in the presence (increasing molar equivalents) and absence of compounds and were subjected to amyloid aggregation.…”

“…36 AGel (186-192) amyloidogenesis was carried in sodium acetate, pH 4.0 with 100 mM NaCl at 37 C without shaking. 37 Lys and PrP amyloidogenesis. For Lys amyloidogenesis, the reaction mixture contained 140 mM Lys at 37 C in acetate buffer, pH 7.2 with continuous agitation at 500 rpm.…”

“…Additionally, such associations could also interfere with lateral assembly of protobrillar intermediates, originally facilitated through p-associations, thereby impeding their progression to mature brils. 36,37 Amyloid assembly in case of PolyQ is an exception as charge interactions appears to play dominant role. However, inhibition by EGCG by virtue of its hydrophobic/H-bond interactions requires thorough investigation behind its mechanism of action.…”

Curcumin binds to the pre-fibrillar aggregates of Cu/Zn superoxide dismutase (SOD1) and alters its amyloidogenic pathway resulting in reduced cytotoxicity

“…Curcumin has been reported to inhibit the fibrillation of proteins like α-synuclein, [31] amyloid beta, [30] and prion protein, [32] involved in Parkinson's disease (PD), Alzheimer's disease (AD) and transmissible spongiform encephalopathies (TSE), respectively. However, curcumin has also been shown to augment the fibril formation in gelsolin amyloid stretch [33].…”