Abstract
Protein-based biosensors or activators have been engineered to visualize molecular signals or manipulate cellular functions. Here we integrate these two functionalities into one protein molecule, an integrated sensing and activating protein (iSNAP). A prototype that can detect tyrosine phosphorylation and immediately activate auto-inhibited Shp2 phosphatase, Shp2-iSNAP, is designed through modular assembly. When Shp2-iSNAP is fused to the SIRPα receptor which typically transduces anti-phagocytic signals from the ‘don’t eat me’ CD47 ligand through negative Shp1 signaling, the engineered macrophages not only allow visualization of SIRPα phosphorylation upon CD47 engagement but also rewire the CD47-SIRPα axis into the positive Shp2 signaling, which enhances phagocytosis of opsonized tumor cells. A second SIRPα Syk-iSNAP with redesigned sensor and activator modules can likewise rewire the CD47-SIRPα axis to the pro-phagocytic Syk kinase activation. Thus, our approach can be extended to execute a broad range of sensing and automated reprogramming actions for directed therapeutics.
Highlights
Protein-based biosensors or activators have been engineered to visualize molecular signals or manipulate cellular functions
We have adopted a modular assembly approach to develop a general class of integrated sensing and activating protein (iSNAP) that can sense specific biochemical events and activate the reprogramming of cellular functions (Supplementary Fig. 1a, b)
Among the peptide sequences that we have tested (Supplementary Table 1), a BTAM peptide derived from the CD47 receptor SIRPα with two tyrosines led to an iSNAP that undergoes both fluorescence resonance energy transfer (FRET) change (Fig. 1b, c) and protein tyrosine phosphatase (PTP) activation (Fig. 1d) in vitro upon phosphorylation by Src, a kinase known to phosphorylate SIRPα in response to various mitogens[15]
Summary
Protein-based biosensors or activators have been engineered to visualize molecular signals or manipulate cellular functions. Synthetic proteins that can undergo dimerization or allosteric conformational change upon stimulation by radio wave, light, chemical compounds or cell–cell interactions have been designed to activate signal cascades and control cellular behavior[2,3,4,5] These two separate approaches have not been integrated into one platform to engineer proteins with both sensing and activating functions. We have developed a novel approach of using integrated sensing and activating proteins (iSNAPs) to surveil the intracellular space, and to immediately trigger designed molecular actions upon detection and visualization of specific signals, with the consequences of modulating the downstream signaling cascades and cellular functions. We integrate protein modules with sensing and actuating functions to engineer iSNAPs capable of detecting tyrosine phosphorylation events and activating desired enzymatic functions We apply these iSNAPs to rewire the ‘don’t eat me’ CD47 signaling, leading to significantly enhanced phagocytic capabilities of engineered macrophages against tumor cells
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