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Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks.

ABSTRACT: Protease signaling and scaffold-induced control of protein-protein interactions represent two important mechanisms for intracellular signaling. Here we report a generic and modular approach to control the activity of scaffolding proteins by protease activity, creating versatile molecular platforms to construct synthetic signaling networks. Using 14-3-3 proteins as a structurally well-characterized and important class of scaffold proteins, three different architectures were explored to achieve optimal protease-mediated control of scaffold activity, fusing either one or two monovalent inhibitory ExoS peptides or a single bivalent ExoS peptide to T14-3-3 using protease-cleavable linkers. Analysis of scaffolding activity before and after protease-induced cleavage revealed optimal control of 14-3-3 activity for the system that contained monovalent ExoS peptides fused to both the N-and C-terminus, each blocking a single T14-3-3 binding site. The protease-activatable 14-3-3 scaffolds were successfully applied to construct a three-step signaling cascade in which dimerization and activation of FGG-caspase-9 on an orthogonal supramolecular platform resulted in activation of a 14-3-3 scaffold, which in turn allowed 14-3-3-templated complementation of a split-luciferase. In addition, by combining 14-3-3-templated activation of caspase-9 with a caspase-9-activatable 14-3-3 scaffold, the first example of a synthetic self-activating protease signaling network was created. Protease-activatable 14-3-3 proteins thus represent a modular platform whose properties can be rationally engineered to fit different applications, both to create artificial in vitro synthetic molecular networks and as a novel signaling hub to re-engineer intracellular signaling pathways.

SUBMITTER: Aper SJA

PROVIDER: S-EPMC6154215 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks.

Aper Stijn J A SJA den Hamer Anniek A Wouters Simone F A SFA Lemmens Lenne J M LJM Ottmann Christian C Brunsveld Luc L Merkx Maarten M

ACS synthetic biology 20180831 9

Protease signaling and scaffold-induced control of protein-protein interactions represent two important mechanisms for intracellular signaling. Here we report a generic and modular approach to control the activity of scaffolding proteins by protease activity, creating versatile molecular platforms to construct synthetic signaling networks. Using 14-3-3 proteins as a structurally well-characterized and important class of scaffold proteins, three different architectures were explored to achieve op ...[more]

PMID: 30125482

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Project description:Despite the substantial burden posed by osteoarthritis (OA) globally, difficult challenges remain in achieving early OA diagnosis and adopting effective disease-modifying treatments. In this study, we use a biomolecular approach to address these limitations by creating an inherently theranostic molecular beacon whose imaging and therapeutic capabilities are activated by early pathological changes in OA. This platform comprised (1) a peptide linker substrate for metalloproteinase-13 (MMP-13), a pathological protease upregulated in OA, which was conjugated to (2) a porphyrin moiety with inherent multimodal imaging, photodynamic therapy, and drug delivery capabilities, and (3) a quencher that silences the porphyrin's endogenous fluorescence and photoreactivity when the beacon is intact. In diseased OA tissue with upregulated MMP-13 expression, this porphyrin molecular beacon (PPMMP13B) was expected to undergo sequence-specific cleavage, yielding porphyrin fragments with restored fluorescence and photoreactivity that could, respectively, be used as a readout of MMP-13 activity within the joint for early OA imaging and disease-targeted photodynamic therapy. This study focused on the synthesis and characterization of PPMMP13B, followed by a proof-of-concept evaluation of its OA imaging and drug delivery potential. In solution, PPMMP13B demonstrated 90% photoactivity quenching in its intact form and robust MMP-13 activation, yielding a 13-fold increase in fluorescence post-cleavage. In vitro, PPMMP13B was readily uptaken and activated in an MMP-13 cell expression-dependent manner in primary OA synoviocytes without exuding significant cytotoxicity. This translated into effective intra-articular cartilage (to a 50 μm depth) and synovial uptake and activation of PPMMP13B in a destabilization of the medial meniscus OA mouse model, yielding strong fluorescence contrast (7-fold higher signal than background) at the diseased joint site. These results provide the foundation for further exploration of porphyrin molecular beacons for image-guided OA disease stratification, effective articular delivery of disease-modify agents, and OA photodynamic therapy.

Dataset Information

Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks.

Publications

Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks.

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