Protein circuits to program intercellular signalling in mammalian cells
Protein-based circuits feature additional advantages such as fast operation, compact design, and robust context-independent performance compared to traditional transcriptional circuits. These previously described circuits only functioned in the cytosol and did not enable intercellular communication.
RELEASE (Retained Endoplasmic Cleavable Secretion) controls protein secretion by leveraging the removal of ER retention signalling motifs using synthetic protease circuits.
RELEASE can be linked to pre-existing protease circuits to realize diverse signal processing capabilities, including logic operation (AND/OR) and threshold tuning.
By linking RELEASE to additional sensing and processing circuits, we can achieve elevated protein secretion in response to “undruggable” oncogene KRAS mutants.
Check out our 2022 publication in Nature Communications for the full story.
There is a need to develop synthetic protein components that enable circuit functionality, while condensing their overall genetic payloads.
By harnessing interactions with endogenous 14-3-3 scaffolding proteins we created RELEASE-NOT, a platform to turn OFF protein secretion in response to protease activity.
With the ability to control the activation and repression of protein secretion, we created a suite of RELEASE variants known as Compact RELEASE (compRELEASE).
CompRELEASE enables functional completeness of all Boolean Logic gates and enabled analog signal filtering without the need for additional processing proteases.
We encoded entire synthetic protein circuits within polycistronic constructs under the control of a single promoter to create engineered cells with inducible control of protein secretion.
Finally, to highlight the utility of post-translational circuits, we leveraged the mRNA delivery of RELEASE to selectively express cell surface proteins only in engineered cells harbouring inducible proteases.
Check out our preprint at BioRxiv for the full story.
Tagged proteins are retained in the ER, until the ER retention motif is cleaved through activation of the protein-circuit. After the first cleavage event, the membrane-bound protein is transported to the trans-Golgi apparatus, processed into its soluble form via furin, and finally secreted.