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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