Enzymatic Regulation of Protein-Protein Interactions in Artificial Cells
© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.
| Veröffentlicht in: | Advanced materials (Deerfield Beach, Fla.). - 1998. - 35(2023), 29 vom: 05. Juli, Seite e2300947 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2023
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| Zugriff auf das übergeordnete Werk: | Advanced materials (Deerfield Beach, Fla.) |
| Schlagworte: | Journal Article artificial cells coacervates phosphorylation scaffold proteins synthetic signaling 14-3-3 Proteins Peptides |
| Zusammenfassung: | © 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH. Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein-protein or protein-nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein-protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains |
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| Beschreibung: | Date Completed 31.07.2023 Date Revised 31.07.2023 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1521-4095 |
| DOI: | 10.1002/adma.202300947 |