Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks
Stijn J. A. AperAnniek den HamerSimone F. A. WoutersLenne J. M. LemmensChristian OttmannLuc BrunsveldMaarten Merkx
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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.Keywords:
Synthetic Biology
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Abstract Chromosome higher order structure has been an enigma for over a century. The most important structural finding has been the presence of a chromosome scaffold composed of non-histone proteins; so-called scaffold proteins. However, the organization and function of the scaffold are still controversial. Here, we use three dimensional-structured illumination microscopy (3D-SIM) and focused ion beam/scanning electron microscopy (FIB/SEM) to reveal the axial distributions of scaffold proteins in metaphase chromosomes comprising two strands. We also find that scaffold protein can adaptably recover its original localization after chromosome reversion in the presence of cations. This reversion to the original morphology underscores the role of the scaffold for intrinsic structural integrity of chromosomes. We therefore propose a new structural model of the chromosome scaffold that includes twisted double strands, consistent with the physical properties of chromosomal bending flexibility and rigidity. Our model provides new insights into chromosome higher order structure.
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Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14-3-3 dimeric protein and is controllable through the action of a small-molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small-molecule regulation of caspase-9 activity through induced dimerisation on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14-3-3 scaffold. This work provides an entry point for the long-needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemically inducible scaffolds in synthetic systems.
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Motivation: Scaffold proteins are known as crucial regulators of various cellular functions by assembling multiple proteins involved in signaling and metabolic pathways. Identification of scaffold proteins and the study of their molecular mechanisms can open a new aspect of cellular systemic regulation and the resulting application on medicine and engineering. There have been highlighted the regulatory roles of dozens of scaffold proteins, but just one computational approach tried to find scaffold proteins from interactomes until now. However, there was a limitation to find diverse types of scaffold proteins because their criteria were restricted to the classical scaffold proteins.
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Scaffold proteins operate as organizing hubs to enable high-fidelity signaling, fulfilling crucial roles in the regulation of cellular processes. Bottom-up construction of controllable scaffolding platforms is attractive for the implementation of regulatory processes in synthetic biology. Here, we present a modular and switchable synthetic scaffolding system, integrating scaffold-mediated signaling with switchable kinase/phosphatase input control. Phosphorylation-responsive inhibitory peptide motifs were fused to 14-3-3 proteins to generate dimeric protein scaffolds with appended regulatory peptide motifs. 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. In addition, a hetero-bivalent autoinhibitory platform design allowed for dual-kinase input regulation of scaffold activity. Reversibility of the regulatory platform was illustrated through phosphatase-controlled abrogation of autoinhibition, resulting in full recovery of 14-3-3 scaffold activity.
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Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process.
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Assembly of proteins into higher-order complexes generates specificity and selectivity in cellular signaling. Signaling complex formation is facilitated by scaffold proteins that use modular scaffolding domains, which recruit specific pathway enzymes. Multimerization and recombination of these conjugated native domains allows the generation of libraries of engineered multidomain scaffold proteins. Analysis of these engineered proteins has provided molecular insight into the regulatory mechanism of the native scaffold proteins and the applicability of these synthetic variants. This topical review highlights the use of engineered, conjugated multidomain scaffold proteins on different length scales in the context of synthetic signaling pathways, metabolic engineering, liquid–liquid phase separation, and hydrogel formation.
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