title: A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.
abstract: Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Banaszynski
authors.forename: Ling-Chun
authors.initials: LC
authors.lastname: Chen
authors.forename: Lystranne A
authors.initials: LA
authors.lastname: Maynard-Smith
authors.forename: A G Lisa
authors.initials: AG
authors.lastname: Ooi
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D009025:Morpholines / Q000378:metabolism*; D009154:Mutation; D041681:NIH 3T3 Cells; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000235:genetics*; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
chemical_list: D008024:Ligands; D008164:Luminescent Proteins; D009025:Morpholines; D011993:Recombinant Fusion Proteins; C540198:Shield-1 compound; D046988:Proteasome Endopeptidase Complex; D022061:Tacrolimus Binding Protein 1A; D022021:Tacrolimus Binding Proteins