Over the past two decades, biomaterials research has made significant progress, moving from traditional biomaterials to structurally controlled and dynamically functional biomaterials. A number of building blocks have been explored for the development of biomaterials through self-assembly, but SAPs have received particular attention due to their adaptability and potential use in various applications such as tissue engineering, wound healing. wounds and vaccinations. Despite these advantages, the SAP-based approach is less explored in the intracellular context.
Fortunately, a team of scientists from the Tokyo Institute of Technology (Tokyo Tech), led by assistant professor Takayuki Miki, reported a de novo peptide, Y15, which easily forms secondary structures to allow the upward synthesis of functional protein assemblies. in living cells. Their findings are published in Nature Communication. Assistant Professor Miki explains: “The cellular environment is a dense medium in which macromolecules occupy a considerable volume; it is difficult to develop SAPs that interact in such a raw environment. Fortunately, we were able to create a peptide de novo – that is, from scratch – which shows a strong propensity to assemble in intracellular spaces. “
To take advantage of the propensity for self-assembly, scientists designed and synthesized four Yn peptides of varying length – Y9, Y11, Y13, and Y15 – along with the negative control peptide Y15 (K9P). Y15 clearly outperformed the others and self-assembled quickly at low buffer concentrations, creating leaf structures. Y15 forms an amphiphilic strand with a hydrophobic face composed of tyrosine residues (Tyr) and a hydrophilic face composed of an alternation of glutamic acid (Glu) and lysine (Lys) residues. Thus, neighboring β strands can connect via hydrophobic and aromatic interactions between Tyr residues as well as electrostatic interactions between negatively charged Glu residues and positively charged Lys residues.
Scientists realized that Y15 could be a key motif in the formation of protein assemblies in liquid solution. To assess the mechanism of assembly, Y15 was genetically tagged with a model protein, the superfolder green fluorescence protein (sfGFP). They found that it could form fibrous structures in test tubes. In addition, when Y15-sfGFP were expressed in living cells, they retained their self-assembly affinity by forming clusters. Several tests have corroborated the clustering of Y15-sfGFP in living cells, confirming that these peptides can facilitate protein assembly.
The researchers were also able to build artificial micro-scale structures by fusing Y15 to Azami-Green, a tetrameric fluorescent protein. Thus, Y15-based constructs could also be adorned with functional proteins. In addition, the researchers reconstructed clusters of Nck (an adapter protein) by incorporating Y15-tagged Nck into Y15-based supramolecules. This resulted in the polymerization of actin mediated N-WASP (Wiskott-Aldrich neural syndrome protein), which in turn plays an essential role in cellular locomotion. The Y15-based construct helped assess the impact of Nck domain valence and density dependence, thus serving as a platform for cell reconstitution studies.
Y15 SAPs are much smaller than the previously reported protein-based tags. Likewise, using an SAP-based system makes sense and is straightforward, and it can be genetically tagged to optimize operation. Assistant Professor Miki concludes: “We believe that SAPs will pave the way for a range of applications, such as clustering with responsiveness to stimuli, artificial constitution of phase separations, reconstitution of natural protein complexes, and much more. others. We hope that this engineering of the supramolecular assembly will pave the way for future intracellular research. “
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