Protease-activatable scaffold proteins as versatile molecular hubs in synthetic signaling networks

Stijn J.A. Aper, Anniek den Hamer, Simone Wouters, Lenne Lemmens, Christian Ottmann, Luc Brunsveld, Maarten Merkx

Research output: Contribution to journalArticleAcademicpeer-review

17 Citations (Scopus)
172 Downloads (Pure)


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.

Original languageEnglish
Pages (from-to)2216-2225
Number of pages10
JournalACS Synthetic Biology
Issue number9
Publication statusPublished - 21 Sept 2018


  • 14-3-3 protein
  • proteases
  • protein engineering
  • self-activation
  • signaling cascade
  • synthetic biology
  • Endopeptidases/metabolism
  • Amino Acid Sequence
  • Humans
  • Peptides/chemistry
  • ADP Ribose Transferases/chemistry
  • Bacterial Toxins/chemistry
  • Peptide Hydrolases/metabolism
  • Proteolysis
  • Protein Engineering
  • Protein Binding
  • 14-3-3 Proteins/chemistry
  • Dimerization
  • Caspase 9/metabolism


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