ABSTRACT
The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A. thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.
Subject(s)
Light , Optogenetics/methods , Recombinant Fusion Proteins/metabolism , Amino Acid Motifs , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cryptochromes/chemistry , Cryptochromes/genetics , Cryptochromes/metabolism , HEK293 Cells , Half-Life , Humans , Low Density Lipoprotein Receptor-Related Protein-6/chemistry , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Confocal , Mutation , Plasmids/genetics , Plasmids/metabolism , Recombinant Fusion Proteins/genetics , Wnt Signaling Pathway , Red Fluorescent ProteinABSTRACT
Fragment antigen-binding domains (Fabs) from anti-Frizzled and anti-LRP6 monoclonal antibodies were conjugated using SpyTag-SpyCatcher chemistry via a one-pot reaction. The resulting synthetic heterodimeric agonist outperformed the natural ligand, Wnt-3a, in activating canonical Wnt signaling in mammalian cells. This approach should be broadly applicable to activate receptor-mediated cellular signaling.
Subject(s)
Antibodies, Monoclonal/pharmacology , Wnt3A Protein/agonists , Antibodies, Monoclonal/chemistry , Dimerization , Humans , Ligands , Molecular Structure , Wnt Signaling Pathway/drug effectsABSTRACT
Multivalent interactions in which multiple ligands on one object bind to multiple receptors on another are commonly found in natural biological systems. In addition, these interactions can lead to increased strength and selectivity when compared to the corresponding monovalent interaction. These attributes have also guided the design of synthetic multivalent ligands to control biological interactions. This review will highlight the recent literature describing the use of multivalent ligand display in the design of vaccines, immunomodulators, cell signaling effectors, and vehicles for targeted drug delivery.
Subject(s)
Drug Delivery Systems/methods , Drug Design , Immunologic Factors/immunology , Vaccines/immunology , Animals , Cell Line, Tumor , Humans , Immunologic Factors/chemical synthesis , Ligands , Signal Transduction/drug effects , Vaccines/chemical synthesisABSTRACT
Despite extensive research on the canonical Wnt signaling pathway, the mechanism by which this signal downregulates the activity of destruction complexes and inhibits ß-catenin degradation remains controversial. In particular, recent attention has focused on two main competing mechanisms-inhibition of phosphorylation and inhibition of ubiquitination. Our combined experimental and theoretical analysis demonstrates that the disassembly of a fraction of the intracellular destruction complexes results in the partial inhibition of both ß-catenin phosphorylation and ubiquitination. This inhibition is spatially patterned, consistent with the relocalization of some destruction complexes to the cellular membrane upon Wnt stimulation. Moreover, in contrast to the generally accepted view that the destruction complex is highly processive, our analysis supports a distributive model, in which ß-catenin can dissociate from the complex between sequential phosphorylation events. Understanding the fundamental mechanism by which Wnt signaling is regulated provides a rational basis for tuning the pathway for scientific and therapeutic purposes.
