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.
Bioluminescent antibodies represent attractive detection agents in both bioanalytical assays and imaging. Currently, their preparation relies on genetic fusion of luciferases to antibodies or nonspecific chemical conjugation strategies. Here, we report a generic method to generate well-defined covalent antibody− luciferase conjugates starting from commercially available monoclonal antibodies. Our approach uses fusion proteins consisting of the bright blue light-emitting luciferase NanoLuc (NL) and an Fcbinding protein domain (Gx) that can be photo-cross-linked to the antibody using UV light illumination. Green and red color variants were constructed by tight fusion of the NanoLuc with a green fluorescent acceptor domain and introduction of Cy3, respectively. To increase the already bright NanoLuc emission, tandem fusions were successfully developed in which the Gx domain is fused to two or three copies of the NanoLuc domain. The Gx-NL fusion proteins can be efficiently photo-cross-linked to all human immunoglobulin G (IgG) isotypes and most mammalian IgG's using 365 nm light, yielding antibodies with either one or two luciferase domains. The bioluminescent antibodies were successfully used in cell immunostaining and bioanalytical assays such as enzyme-linked immunosorbent assay (ELISA) and Western blotting.
Antibody‐based molecular recognition plays a central role in today's life sciences, ranging from immunoassays to molecular imaging and antibody‐based therapeutics. Control over antibody activity by using external triggers such as light could further increase the specificity of antibody‐based targeting. Here we present bivalent peptide–DNA ligands containing photocleavable linkers as a noncovalent approach by which to allow photoactivation of antibody activity. Light‐triggered cleavage of the 3‐amino‐3‐(2‐nitrophenyl)propionic acid peptide linker converted the high‐affinity bivalent peptide–DNA lock into weakly binding monovalent ligands, effectively restoring antibody targeting of cell‐surface receptors. In this work, a proof of principle was provided with an anti‐hemagglutinin antibody, but the molecular design of the lock is generic and applicable to any monoclonal antibody for which an epitope or mimotope of sufficient affinity is available.
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