Probing the molecular determinants of the activation of toll-like receptor 2/6 by amyloid nanostructures through directed peptide self-assembly.

Amyloid fibrils are proteinaceous nanostructures known for their ability to activate the innate immune system, which has been recently exploited for their use as self-adjuvanted antigen delivery systems for vaccines. Among mechanisms of immunostimulation, the activation of the heterodimeric toll-like receptor 2/6 (TLR2/TLR6) by the cross-β-sheet quaternary conformation appears important. Nonetheless, the lack of control over the process of self-assembly and the polydispersity of the resulting supramolecular architectures make it challenging to elucidate the molecular basis of TLR2/TLR6 engagement by amyloid assemblies. In this context, we harnessed the effects of N- and C-terminal modifications of a short 10-mer β-peptide derived from the islet amyloid polypeptide (I) to investigate the relationships between the morphology and physicochemical properties of amyloid assemblies and their TLR2/TLR6 activity. Chemical substitutions at the N- and C-termini of the I peptide, including addition of charged residues at the N-terminus and α-amidation of C-terminus, allowed the controlled formation of a diversity of architectures, including belt-like filaments, rigid nanorods as well as flat and twisted fibrils. These fully cytocompatible peptide nanostructures showed different potencies to activate TLR2/TLR6, which correlated with the charge exposed on the surface. These results further demonstrate the potent modulatory effect of N- and C-terminal electrostatic capping on the self-assembly of short synthetic β-peptides. This study also indicates that self-assembly into cross-β-sheet nanostructures is essential for the activation of the TLR2/TLR6 by amyloidogenic peptides, albeit the structural requirements of the engagement of this promiscuous immune receptor by the nanostructures remain challenging to precisely untangle.