Abstract
De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms β-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.
Highlights
De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants
This study demonstrates that our de novo designed Y15 peptide offers a tool for organizing supramolecular architectures with biological functions in living cells
Peptide assembly was monitored by enhancement in thioflavin T (ThT) fluorescence upon binding to amyloid-like fibrils (Fig. 1c)
Summary
De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Supramolecular nanofibers composed of designed self-assembling peptides (SAPs) are attractive biomaterials for tissue engineering, wound healing, and vaccines for the following two reasons[4,5]: First, biomaterials can provide synthetic scaffolds for these applications. De novo peptides provide promising building blocks for constructing artificial supramolecular assembly in living cells. Despite these advantages, few attempts to apply these SAP-based materials to biological tools in intracellular spaces have been reported.
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