Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks. Aper, S. J A, den Hamer, A., Wouters, S. F A, Lemmens, L. J M, Ottmann, C., Brunsveld, L., & Merkx, M. ACS Synth Biol, 7(9):2216–2225, August, 2018. abstract bibtex 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.
@ARTICLE{Aper2018-qv,
title = "{Protease-Activatable} Scaffold Proteins as Versatile Molecular
Hubs in Synthetic Signaling Networks",
author = "Aper, Stijn J A and den Hamer, Anniek and Wouters, Simone F A and
Lemmens, Lenne J M and Ottmann, Christian and Brunsveld, Luc and
Merkx, Maarten",
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.",
journal = "ACS Synth Biol",
volume = 7,
number = 9,
pages = "2216--2225",
month = aug,
year = 2018,
keywords = "14-3-3 protein; proteases; protein engineering; self-activation;
signaling cascade; synthetic biology",
language = "en"
}
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J M","Ottmann, C.","Brunsveld, L.","Merkx, M."],"bibdata":{"bibtype":"article","type":"article","title":"Protease-Activatable Scaffold Proteins as Versatile Molecular Hubs in Synthetic Signaling Networks","author":[{"propositions":[],"lastnames":["Aper"],"firstnames":["Stijn","J","A"],"suffixes":[]},{"propositions":["den"],"lastnames":["Hamer"],"firstnames":["Anniek"],"suffixes":[]},{"propositions":[],"lastnames":["Wouters"],"firstnames":["Simone","F","A"],"suffixes":[]},{"propositions":[],"lastnames":["Lemmens"],"firstnames":["Lenne","J","M"],"suffixes":[]},{"propositions":[],"lastnames":["Ottmann"],"firstnames":["Christian"],"suffixes":[]},{"propositions":[],"lastnames":["Brunsveld"],"firstnames":["Luc"],"suffixes":[]},{"propositions":[],"lastnames":["Merkx"],"firstnames":["Maarten"],"suffixes":[]}],"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. 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Here we report a generic and modular\n approach to control the activity of scaffolding proteins by\n protease activity, creating versatile molecular platforms to\n construct synthetic signaling networks. Using 14-3-3 proteins as\n a structurally well-characterized and important class of scaffold\n proteins, three different architectures were explored to achieve\n optimal protease-mediated control of scaffold activity, fusing\n either one or two monovalent inhibitory ExoS peptides or a single\n bivalent ExoS peptide to T14-3-3 using protease-cleavable\n linkers. Analysis of scaffolding activity before and after\n protease-induced cleavage revealed optimal control of 14-3-3\n activity for the system that contained monovalent ExoS peptides\n fused to both the N-and C-terminus, each blocking a single\n T14-3-3 binding site. The protease-activatable 14-3-3 scaffolds\n were successfully applied to construct a three-step signaling\n cascade in which dimerization and activation of FGG-caspase-9 on\n an orthogonal supramolecular platform resulted in activation of a\n 14-3-3 scaffold, which in turn allowed 14-3-3-templated\n complementation of a split-luciferase. 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