Osmoprotectant Coated Thermostable Gold Nanoparticles Efficiently Restrict Temperature-Induced Amyloid Aggregation of Insulin

Abstract

Naturally occurring osmoprotectants are known to prevent aggregation of proteins under various stress factors including extreme pH and elevated temperature conditions. Here, we synthesized gold nanoparticles coated with selected osmolytes (proline, hydroxyproline, and glycine) and examined their effect on temperature-induced amyloid-formation of insulin hormone. These uniform, thermostable, and hemocompatible gold nanoparticles were capable of inhibiting both spontaneous and seed-induced amyloid aggregation of insulin. Both quenching and docking experiments suggest a direct interaction between the osmoprotectant-coated nanoparticles and aggregation-prone hydrophobic stretches of insulin. Circular-dichroism results confirmed the retention of insulin’s native structure in the presence of these nanoparticles. Unlike the indirect solvent-mediated effect of free osmolytes, the inhibition effect of osmolyte-coated gold nanoparticles was observed to be mediated through their direct interaction with insulin. The results signify the protection of the exposed aggregation-prone domains of insulin from temperature-induced self-assembly through osmoprotectant-coated nanoparticles, and such effect may inspire the development of osmolyte-based antiamyloid nanoformulations.

Evidence of Anti-amyloid Characteristics of Plumbagin via Inhibition of Protein Aggregation and Disassembly of Protein Fibrils

“…Under temperature-induced aggregation conditions, the population of aggregation-prone intermediate structures increases, and due to this reason, the self-assembly process is initiated. 25,38,51 In this study, the interaction between the plumbagin molecule and the proteins (BSA and insulin) seems to stabilize their native structures (Figure 1e−h and Figure 3d−f). Thermal unfolding experiments confirmed that the T m of insulin was increased by ∼3.8 °C in the presence of plumbagin (Figure 1h).…”

“…To further understand the potential of plumbagin to stabilize the native structure of insulin, fluorescence experiments were performed on guanidine hydrochloride (GnHCl)-treated insulin samples. 38 The control insulin sample showed a gradual increase in the fluorescence emission (λ max = 305 nm) of insulin with an increasing GnHCl concentration (Figure 1i), suggesting an unfolding event of the folded insulin conformation via chemical denaturation. 16 However, the extent of chemical denaturation was substantially reduced in the presence of plumbagin (Figure 1j,k), which reflects that the plumbagin molecule can stabilize the folded structure of native insulin.…”

“…Previous reports have shown that several inhibitors against insulin amyloid formation such as myricetin, eugenol, and osmoprotectant-coated nanoparticles are capable of inhibiting insulin aggregation via stabilization of its native structure under aggregation-promoting conditions. 16,26,[36][37][38]47 To visualize the morphology of insulin aggregates obtained from both the inhibited and uninhibited reactions, we performed TEM and fluorescence microscopy studies. TEM micrographs showed the formation of typical insulin amyloid fibrils as reported earlier (panel i of Figure 1l).…”

“…These data support plumbagin's inhibition effect, and similar results on the suppression of insulin amyloid aggregation in the presence of different inhibitors have been reported in previous studies. 16,26,[36][37][38]47 Plumbagin Causes Disassembly of Preformed Amyloid Fibrils of Insulin. In the next step, we examined whether plumbagin can promote disassembly of preformed amyloid aggregates.…”

The Multifaceted Roles of Proline in Cell Behavior

“…Since L-Pro is a potent and non-toxic chemical chaperone, its intracellular accumulation could be an evolutionarily conserved response aimed at inhibiting the formation of unfolded/misfolded protein aggregates. Indeed, hemocompatible gold nanoparticles coated with L-Pro inhibit both collagen fibril formation (Anand et al, 2017) and insulin aggregation (Prajapati et al, 2021), and could provide a basis for creating antifibrotic and antiamyloid formulations.…”