(B-43) Evaluation of Transmembrane Extracellular Ligand Dimerization Actuator (ELDA) Receptors Activated by Stimulatory Ligand Peptides

Introduction::

The authors believe that this abstract best fits the Bioinformatics, Computational and Systems Biology Track and Theory and Practice of Synthetic Biology Sub-Track.

Cell communication with its surrounding environment via transmembrane receptors influences a myriad of cell behaviors including division, immunomodulation, and the production of extracellular matrix components. While transmembrane receptor platforms have shown success in cancer treatment (e.g., FDA approved CAR-T cell therapy), their use has not been explored for other medical uses including tissue regeneration. Biomaterials can be designed with tailored biophysical and biochemical properties that control cell behaviors via cell-material interactions, and the hypothesis of this work is that cells programmed with transmembrane extracellular ligand dimerization actuator (ELDA) receptors can communicate with biomaterials, in 3D, via ELDA receptor-peptide ligand interactions. To test this hypothesis, human endothelial kidney (HEK293) cells were programmed with ELDA receptors that respond to two different peptide ligands (FLAG-HA and MYC-HA). Cellular responses were first evaluated between ELDA receptors and peptide ligands in their soluble form, and stimulatory responses were confirmed via the fusion of green fluorescent proteins (GFP) within activated cells. To evaluate receptor-ligand interactions within a 3D biomaterial, HEK293 cells with ELDA receptors were encapsulated in hydrogels with and without FLAG-HA peptides and we found immobilized peptides activate encapsulated cells with high specificity.

Materials and Methods::

The intracellular components of the ELDA receptor (Figure 1A) utilize a reversible leucine zipper (LZ) dimerization domain to recruit a CD3z substrate (SD) towards one half of the receptor. The intracellular region of the other half of the receptor has a kinase specific to CD3z (Binding Actuator, BA). Once phosphorylated (P), the substrate recruits a Zap70 SH2 domain. This scheme is used to bring together two split mNeonGreen halves (SFP) which then merge and fluoresce (Merging Fluorescent Protein, MFP).

The extracellular components of the ELDA receptor were designed to bind to two orthogonal epitope tag peptide groups, FLAG-HA or MYC-HA. Candidate receptors were identified de novo using computationally derived amino acid sequences. These receptors were tested in biological quadruplicate in HEK293 cells by transfecting plasmids containing the genes for each FLAG, HA, or MYC receptor half and corresponding SD, and SH2 domains connected to SFPs. To evaluate ELDA receptor activation by stimulatory peptide ligands, HEK293 cells programmed with FLAG-HA and MYC-HA ELDA receptors were exposed to their corresponding peptide ligands (100 µM for 24 hours) and interactions were confirmed by GFP fluorescence measured via flow cytometry. To evaluate peptide ligand activation within 3D materials, programmed HEK293 cells were encapsulated in norbornene-modified hyaluronic acid hydrogels with and without thiolated FLAG-HA peptides (5 mM). 24 hours post-encapsulation green fluorescence within transfected cells (as confirmed by dsRed720 fluorescence) was evaluated in the 3D hydrogels with and without stimulatory peptide ligands by analyzing images captured using 3D confocal microscopy.

Results, Conclusions, and Discussions::

Results: HEK293 cells programmed with MYC-HA ELDA receptors exposed to soluble MYC-HA peptides had a 29% increase in green fluorescence in comparison to programmed cells not exposed to the stimulatory peptide (Figure 1B, left plots). Soluble receptor-ligand interactions were much more sensitive between FLAG-HA ELDA receptor/peptide interactions, as evidenced by an over 150% increase in green fluorescence for peptide ligand-activated cells (Figure 1B, right plot). HEK293 cells programmed with FLAG-HA ELDA receptors encapsulated in hydrogels functionalized with FLAG-HA peptides were almost 700% brighter than programmed cells encapsulated in peptide-free hydrogels (Figure 1C). Representative images of single transfected HEK293 cells (red) qualitatively show more green fluorescence in the presence of tethered FLAG-HA peptides (green) (Figure 1D).

 

Discussion/Conclusion: This data suggests that ELDA transmembrane receptors selectively respond to soluble FLAG-HA and MYC-HA peptides in soluble form and to tethered FLAG-HA peptide ligands within 3D hydrogel materials. Ongoing efforts include replacing one of the kinase domains with a phosphatase domain capable of inactivating the platform in response to inhibitory peptide ligands, thus allowing for negative feedback regulation. Additionally, the split fluorescence molecules will be replaced with split transcriptional activation platforms (e.g., split dCas9) to allow for transcribing of genes of interest in response to stimulatory and inhibitory peptide ligands. This novel transcriptional activation scheme will allow for the release of molecules including cytokines and extracellular proteins with inhibitory peptide ligands to enable self-regulation of therapeutic outputs.

Acknowledgements (Optional): : The authors would like to acknowledge the Pantazes group for their contributions to this work and this work was supported by the National Science Foundation (NSF) EAGER award 2037055.

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