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(8−11) Within the context of synthetic biology, engineered scaffolds have provided substantial insight into the plasticity and molecular mechanisms of cell signaling. Scaffold proteins are particularly of interest as these serve as organizing elements that assemble two or more signaling proteins without requiring additional cellular compartmentalization and with excellent spatiotemporal control. (5−7) However, to date, only a few bottom-up synthetic protein-based signaling networks that incorporate a combination of such features have been reported, mainly because of a lack of robust scaffold protein platforms. (4) Natural signaling pathways frequently rely on enzyme-controlled covalent modifications and the recruitment and activation of proteins on scaffold proteins. (1−3) Rewiring pathways or even constructing fully artificial signaling systems offers compelling opportunities with therapeutic, diagnostic, and industrial applications. A key aspect of synthetic biology is the bottom-up construction of such signaling pathways with built-in features that allow monitoring and modulation of the underlying processes to elucidate the working principles of cell signaling. Signaling pathways are essential biological components that elegantly regulate cellular processes with high spatiotemporal control.
#SCAFFOLD PROTEIN FULL#
Reversibility of the regulatory platform was illustrated through phosphatase-controlled abrogation of autoinhibition, resulting in full recovery of 14-3-3 scaffold activity. In addition, a hetero-bivalent autoinhibitory platform design allowed for dual-kinase input regulation of scaffold activity. The availability of the scaffold for intermolecular partner protein binding could be lowered up to 35-fold upon phosphorylation of the autoinhibition motifs, as demonstrated using three different kinases. Phosphorylation-responsive inhibitory peptide motifs were fused to 14-3-3 proteins to generate dimeric protein scaffolds with appended regulatory peptide motifs. Here, we present a modular and switchable synthetic scaffolding system, integrating scaffold-mediated signaling with switchable kinase/phosphatase input control. Bottom-up construction of controllable scaffolding platforms is attractive for the implementation of regulatory processes in synthetic biology.
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Scaffold proteins operate as organizing hubs to enable high-fidelity signaling, fulfilling crucial roles in the regulation of cellular processes.