Engineering Small Molecule Switches of Protein Function in Zebrafish Embryos

Abstract

Precise temporally regulated protein function directs the highly complex processes that make up embryo development. The zebrafish embryo is an excellent model organism to study development, and conditional control over enzymatic activity is desirable to target chemical intervention to specific developmental events and to investigate biological mechanisms. Surprisingly few, generally applicable small molecule switches of protein function exist in zebrafish. Genetic code expansion allows for site-specific incorporation of unnatural amino acids into proteins that contain caging groups that are removed through addition of small molecule triggers such as phosphines or tetrazines. This broadly applicable control of protein function was applied to activate several enzymes, including a GTPase and a protease, with temporal precision in zebrafish embryos. Simple addition of the small molecule to the media produces robust and tunable protein activation, which was used to gain insight into the development of a congenital heart defect from a RASopathy mutant of NRAS and to control DNA and protein cleavage events catalyzed by a viral recombinase and a viral protease, respectively.

Genetically Encoded Aminocoumarin Lysine for Optical Control of Protein–Nucleotide Interactions in Zebrafish Embryos

“…61 Most important is the fast kinetics of protein activation, which for ACK resulted in full enzymatic function within 2 min of irradiation, while smallmolecule-induced activation took about 180 min for full activation in zebrafish embryos as we reported previously (luciferase reporter). 61 Incorporation of ACK into NRAS provided higher temporal resolution, allowing us to differentiate its role during gastrulation, namely, that NRAS hyperactivity seems to disrupt the early stages of gastrulation most. Furthermore, we expect that our approach of rationally designing photocaged kinases and GTPases through sitespecific incorporation of the new caged lysine ACK can be readily applied to most enzymes in both families, making it a useful method for probing the spatiotemporal implications of these proteins in developmental processes in zebrafish.…”

“…In summary, we demonstrated precise optical control of both kinase and GTPase functions and utilized the temporal control afforded by this methodology to investigate developmental programs. Optical control of NRAS has advantages over small-molecule-induced decaging . Most important is the fast kinetics of protein activation, which for ACK resulted in full enzymatic function within 2 min of irradiation, while small-molecule-induced activation took about 180 min for full activation in zebrafish embryos as we reported previously (luciferase reporter) .…”

“…Optical control of NRAS has advantages over small-molecule-induced decaging . Most important is the fast kinetics of protein activation, which for ACK resulted in full enzymatic function within 2 min of irradiation, while small-molecule-induced activation took about 180 min for full activation in zebrafish embryos as we reported previously (luciferase reporter) . Incorporation of ACK into NRAS provided higher temporal resolution, allowing us to differentiate its role during gastrulation, namely, that NRAS hyperactivity seems to disrupt the early stages of gastrulation most.…”

Chemoselective Caging of Carboxyl Groups for On‐Demand Protein Activation with Small Molecules

“…[81] This approach has been successfully applied in a variety of human cell lines, [86] Xenopus oocytes, [87] and zebrafish embryos. [88] In the latter example, [88] Deiters and co-workers microinjected embryos with the machinery for caged UAA incorporation and showed that the resultant Angewandte Chemie proteins can be activated by adding a phosphine to the embryo water. Efforts towards exploring our carboxyl group caging strategy in zebrafish embryos are currently underway in our laboratory.…”

Chemoselective Caging of Carboxyl Groups for On‐Demand Protein Activation with Small Molecules

“…[81] This approach has been successfully applied in a variety of human cell lines, [86] Xenopus oocytes, [87] and zebrafish embryos. [88] In the latter example, [88] Deiters and co-workers microinjected embryos with the machinery for caged UAA incorporation and showed that the resultant proteins can be activated by adding a phosphine to the embryo water. Efforts towards exploring our carboxyl group caging strategy in zebrafish embryos are currently underway in our laboratory.…”