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Intracellular artificial supramolecules based on de novo designed Y15 peptides.

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.

SUBMITTER: Miki T

PROVIDER: S-EPMC8185068 | biostudies-literature |

REPOSITORIES: biostudies-literature

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De novo designed transmembrane peptides activating the α5β1 integrin.

Project description:Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbβ3 and αvβ3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5β1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite. Peptides from each computational approach activated integrin α5β1 but not αVβ3 in human endothelial cells. Two CHAMP peptides were shown to directly associate with an α5 TM domain peptide in detergent micelles to a similar degree as a β1 TM peptide does. By solution-state nuclear magnetic resonance, one of these CHAMP peptides was shown to bind primarily the integrin β1 TM domain, which itself has a Gly-X3-Gly motif. The second peptide associated modestly with both α5 and β1 constructs, with slight preference for α5. Although the design goal was not fully realized, this work characterizes novel CHAMP peptides activating α5β1 that can serve as useful reagents for probing integrin biology.

| S-EPMC6151875 | biostudies-literature

De novo designed Hsp70 activator dissolves intracellular condensates.

Project description:Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70's ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

| S-EPMC10541127 | biostudies-literature

Metal Stabilization of Collagen and de Novo Designed Mimetic Peptides.

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| S-EPMC5335877 | biostudies-literature

De novo engineering of intracellular condensates using artificial disordered proteins.

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| S-EPMC8281385 | biostudies-literature

Artificial Diiron Enzymes with a De Novo Designed Four-Helix Bundle Structure.

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| S-EPMC5019575 | biostudies-literature

Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides.

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| S-EPMC3502156 | biostudies-literature

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| S-EPMC9495127 | biostudies-literature

Cell-Free Expression of De Novo Designed Peptides That Form β-Barrel Nanopores.

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| S-EPMC9979648 | biostudies-literature

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| S-EPMC6127164 | biostudies-literature

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| S-EPMC9202597 | biostudies-literature

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