Extracellular Peptide-Ligand Dimerization Actuator Receptor Design for Reversible and Spatially Dosed 3D Cell-Material Communication. ACS Synth Biol 2025 Jul 18;14(7):2494-2513
Date
12/20/2024Pubmed ID
39705005Pubmed Central ID
PMC12281622DOI
10.1021/acssynbio.4c00482Scopus ID
2-s2.0-85212791468 (requires institutional sign-in at Scopus site) 1 CitationAbstract
Transmembrane receptors that endow mammalian cells with the ability to sense and respond to biomaterial-bound ligands will prove instrumental in bridging the fields of synthetic biology and biomaterials. Materials formed with thiol-norbornene chemistry are amenable to thiol-peptide patterning, and this study reports the rational design of synthetic receptors that reversibly activate cellular responses based on peptide-ligand recognition. This transmembrane receptor platform, termed Extracellular Peptide-ligand Dimerization Actuator (EPDA), consists of stimulatory or inhibitory receptor pairs that come together upon extracellular peptide dimer binding with corresponding monobody receptors. Intracellularly, Stimulatory EPDAs phosphorylate a substrate that merges two protein halves, whereas Inhibitory EPDAs revert split proteins back to their unmerged, inactive state via substrate dephosphorylation. To identify ligand-receptor pairs, over 2000 candidate monobodies were built in silico using PETEI, a novel computational algorithm we developed. The top 30 monobodies based on predicted peptide binding affinity were tested experimentally, and monobodies that induced the highest change in protein merging (green fluorescent protein, GFP) were incorporated in the final EPDA receptor design. In soluble form, stimulatory peptides induce intracellular GFP merging in a time- and concentration-dependent manner, and varying levels of green fluorescence were observed based on stimulatory and inhibitory peptide-ligand dosing. EPDA-programmed cells encapsulated in thiol-norbornene hydrogels patterned with stimulatory and inhibitory domains exhibited 3D activation or deactivation based on their location within peptide-patterned hydrogels. EPDA receptors can recognize a myriad of peptide-ligands bound to 3D materials, can reversibly induce cellular responses beyond fluorescence, and are widely applicable in biological research and regenerative medicine.
Author List
Recktenwald M, Bhattacharya R, Benmassaoud MM, MacAulay J, Chauhan VM, Davis L, Hutt E, Galie PA, Staehle MM, Daringer NM, Pantazes RJ, Vega SLAuthor
Mehdi Benmassaoud PhD, BS, MSc Postdoctoral Researcher in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Cell CommunicationGreen Fluorescent Proteins
Humans
Ligands
Norbornanes
Peptides
