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.

Integrating the biological characteristics of oncolytic viruses and immune cells can optimize therapeutic benefits of cell-based delivery

“…This might be achieved by the use of tissue or tumor specific promoters driving the transgene (so limiting transgene expression to cells within the tumor), 44 or else, the use of inducible or repressible systems to control viral gene expression may be incorporated. 45 These have an additional advantage that transgene expression may subsequently be switched off if necessary to prevent adverse events.…”

A small molecule‐directed approach to control protein localization and function

“…In considering this work, it is perhaps most important to bear in mind that our yeastbased mislocalization system represents a variation on a simple and expandable theme: protein heterodimerization is linked to a chemical stimulus, and a single well-characterized compound, such as rapamycin, may be used to regulate the activity of a wide variety of gene products. Related systems have been employed previously in metazoans and cell lines to regulate protein stability (Stankunas et al, 2003;Liu et al, 2007;Stankunas et al, 2007;Janse et al, 2004;Banaszynski et al, 2006a;Maynard-Smith et al, 2007) and in both metazoans and in yeast to regulate protein-protein interactions (Gruber et al, 2006;Karpova et al, 2005;Mallet et al, 2002;Welm et al, 2002;Pownall et al, 2003;Gallagher et al, 2007;Schwartz et al, 2007). We expect future modifications of this dimerization approach to advance not only this system (e.g.…”

Xenopus: An ideal system for chemical genetics

“…We have previously used this system to regulate GSK-3b in vivo (Liu et al, 2007). In this approach, the peptide moiety, derived from the FK506/ rapamycin binding (FRB) domain, has been engineered to be unstable in the absence of drug (Banaszynski et al, 2006;Stankunas et al, 2003). The relevant drug then acts as a ''chemical chaperone'' which restores stability and function of the fusion protein (Fig.…”

“…3a). Two additional systems have been developed using directed evolution (error-prone PCR) to generate unstable variants of FKBP-12 (FKBP*) and dihydrofolate reductase (DHFR*), (Banaszynski et al, 2006). These systems have not yet been tested, and pharmacokinetics and pharmacodynamics will need to be assessed.…”

“…To avoid off-target effects, analogs of rapamycin and FK506 have been developed (MaRap and Shld-1, respectively). Neither MaRap nor Shld-1 can bind to the natural targets of the parental drugs (Banaszynski et al, 2006;Stankunas et al, 2003). We have also validated this approach in Xenopus using expression of a chemically dependent luciferase protein and found that stabilization of the target protein was both dose-dependent, temperature-dependent and reversible (Liu et al, 2006).…”