title: Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt.
abstract: Controlling protein function through posttranslational manipulations has emerged as an attractive complementary technology to existing genetic systems. Often these methods involve developing pharmacological agents to probe protein function without the need to generate a unique compound for each protein family. One common strategy uses small molecules that act as chemical inducers of dimerization by mediating the interaction of two proteins. Herein we report the use of a chemical inducer of dimerization for the development of a posttranslational technology for the manipulation of protein function. This system, split ubiquitin for the rescue of function (SURF), places the complementation of genetically split ubiquitin under the control of rapamycin-induced dimerization of FK506-binding protein and FKBP12-rapamycin-binding protein. Before complementation a "degron" dooms a protein of interest for destruction by the proteasome. Addition of rapamycin results in a proteolytic shunt away from degradation by inducing ubiquitin complementation and cleavage of the protein of interest from the degron. Importantly, the native protein is rescued. We characterized this system with firefly luciferase and went on to apply it to members of three important classes of proteins: proteases (caspase-3), kinases (v-Src), and transcription factors (Smad3). This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Pratt
authors.forename: Edmund C
authors.initials: EC
authors.lastname: Schwartz
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D005562:Formazans / Q000378:metabolism; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D010447:Peptide Hydrolases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D051900:Smad3 Protein / Q000502:physiology; D013778:Tetrazolium Salts / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D005562:Formazans; D051900:Smad3 Protein; D013778:Tetrazolium Salts; D053773:Transforming Growth Factor beta1; D025801:Ubiquitin; C079782:MTT formazan; D010447:Peptide Hydrolases