Amyloid fibrils are highly ordered protein aggregates characterized by a conserved cross-β-sheet architecture despite originating from structurally diverse precursor proteins. Growing evidence suggests that interactions between different amyloidogenic proteins can modulate aggregation pathways through heterologous cross-seeding; however, the influence of seed polymorphism on the structure and biological properties of cross-seeded fibrils remains poorly understood. Here, we investigated the cross-seeding of native human insulin by two structurally distinct polymorphs of hen egg-white lysozyme (HEWL): flexible fibrils (FFs) and rigid fibrils (RFs). Native insulin remained stable under physiological conditions and underwent spontaneous fibrillation only under acidic conditions. In contrast, both HEWL polymorphs efficiently induced insulin aggregation at physiological pH, bypassing the nucleation barrier. Thioflavin T fluorescence, circular dichroism spectroscopy, and transmission electron microscopy revealed that lysozyme FFs templated the formation of insulin flexible fibrils (IFFs), whereas lysozyme RFs produced insulin rigid fibrils (IRFs), demonstrating that the structural characteristics of the parental HEWL polymorphs were propagated during heterologous cross-seeding. The toxicity of the resulting insulin fibrils was evaluated in SH-SY5Y neuronal cells and CCF-STTG1 astrocytes. IFFs exhibited minimal cytotoxicity and only subtle morphological alterations, whereas IRFs caused modest reductions in cell viability accompanied by more pronounced cellular damage. These findings demonstrate that the structural polymorphism of HEWL fibrils governs both the architecture and biological activity of cross-seeded insulin fibrils, highlighting amyloid polymorphism as an important determinant of heterologous amyloid propagation and a potential design principle for engineering functional amyloid-based biomaterials and protein delivery platforms.
S. Metkar, Vijay Eerati, A. Ramamoorthy· bioRxiv· 0 citations
Apolipoprotein A-I mimetic 4F, an 18-residue amphipathic α-helix, can self-assemble with lipids to form peptide nanodiscs, yet the molecular determinants governing their assembly and stability remain poorly understood. Here, using coarse-grained molecular dynamics (CG-MD), we capture the de novo formation of 4F nanodiscs with DMPC and reveal a multistep assembly pathway involving nucleation, fusion, and ellipse-to-disc maturation. All-atom back-mapping shows that the nanodisc rim is structurally heterogeneous and stabilized by aromatic-acyl interactions, Lys headgroup anchoring, and inter-peptide electrostatic contacts. Lipid composition and temperature critically regulate nanodisc integrity: DMPC supports continuous peptide belts and long-term stability, whereas DPPC below its main phase transition temperature suppresses fusion and yields fragmented, non-uniform rims. These findings validate the ability of CG-MD to resolve nanodisc assembly mechanisms. Experimental measurements corroborate the simulations, demonstrating that 4F nanodiscs exhibit lower thermal resilience than MSP nanodiscs while retaining structural integrity at moderate temperatures. As a functional benchmark, MSP nanodiscs suppress the amyloid-binding thioflavin-T fluorescence signal associated with Aβ (1-40) fibrillar assembly, consistent with our previously reported findings for 4F nanodiscs and supporting the ability of amphipathic nanodisc rims to delay Aβ (1-40) aggregation. Together, these results establish a mechanistic framework and design principles for single-helix peptide nanodiscs and delineate the conditions under which they converge with or diverge from MSP-based scaffolds.
Bikash R. Sahoo, B. Krishnarjuna, Thirupathi Ravula et al.· Journal of Colloid and Inter...· 0 citations
Lipid nanodiscs have emerged as a versatile and promising tool for drug delivery due to their biocompatibility, structural flexibility, and ability to mimic native cell membrane environments. These nanoscale assemblies, composed of lipid bilayers stabilized by scaffold amphipathic proteins, peptides, synthetic polymers, or saponins, offer a stable membrane mimetic system for encapsulating hydrophobic drugs and membrane proteins. Ongoing research continues to expand the diversity of nanodisc formulations, each with distinct advantages and limitations. Their tunable size, surface functionality, dynamic lipid exchange, and ability to incorporate various lipids and membrane components make them suitable for targeted delivery and controlled drug release under physiological conditions. Recent advances underscore their potential in cancer therapy, antimicrobial delivery, and vaccine development, areas where conventional carriers often fall short. This review discusses the recent developments of lipid nanodiscs for drug delivery, focusing on design strategies, functionalization methods, and key challenges for potential clinical translation.
Thirupathi Ravula, C. Obi, A. Ramamoorthy· Colloids and Surfaces B: Bio...· 0 citations
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