title: Ligand-switchable substrates for a ubiquitin-proteasome system.
abstract: Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.
authors.affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305, USA.
authors.forename: Emily L
authors.initials: EL
authors.lastname: Egeler
authors.forename: Lorenz M
authors.initials: LM
authors.lastname: Urner
authors.forename: Rishi
authors.initials: R
authors.lastname: Rakhit
authors.forename: Corey W
authors.initials: CW
authors.lastname: Liu
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D008024:Ligands; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D055550:Protein Stability; D058767:Protein Unfolding; D013379:Substrate Specificity; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D022021:Tacrolimus Binding Proteins; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D008024:Ligands; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D022061:Tacrolimus Binding Protein 1A; D022021:Tacrolimus Binding Proteins