A Directed Approach for Engineering Conditional Protein Stability Using Biologically Silent Small Molecules

Abstract

The ability to regulate the function of specific proteins using cell-permeable molecules can be a powerful method for interrogating biological systems. To bring this type of "chemical genetic" control to a wide range of proteins, we recently developed an experimental system in which the stability of a small protein domain expressed in mammalian cells depends on the presence of a high affinity ligand. This ligand-dependent stability is conferred to any fused partner protein. The FK506-and rapamycin-binding protein (FKBP12) has been the subject of extensive biophysical analyses, including both kinetic and thermodynamic studies of the wild-type protein as well as dozens of mutants. The goal of this study was to determine if the thermodynamic stabilities (⌬⌬G U-F ) of various amino acid substitutions within a given protein are predictive for engineering additional ligand-dependent destabilizing domains. We used FKBP12 as a model system and found that in vitro thermodynamic stability correlates weakly with intracellular degradation rates of the mutants and that the ability of a given mutation to destabilize the protein is context-dependent. We evaluated several new FKBP12 ligands for their ability to stabilize these mutants and found that a cell-permeable molecule called Shield-1 is the most effective stabilizing ligand. We then performed an unbiased microarray analysis of NIH3T3 cells treated with various concentrations of Shield-1. These studies show that Shield-1 does not elicit appreciable cellular responses.Cell-permeable small molecules have long been powerful tools for interrogating biology (1). They can be used to conditionally probe biological processes, often with high temporal resolution (2-4). Interest in using perturbants of this type has grown significantly in the past decade, but one of the pressing questions remains. How can one discover a cell-permeable perturbant for any protein of interest? Nature was the source for many of the early examples, especially when the biologically relevant target of the perturbant was easily discerned. To accelerate the discovery process, many investigators are screening large libraries of small molecules either against purified proteins or against living cells using high content imaging and phenotype-based scoring to evaluate library members (5). Irrespective of the discovery process used to identify the perturbant, the question of specificity remains critical for research biology (6). When a particular reagent is used to conditionally perturb a biological process, how confident can one be that the resulting phenotype can be ascribed to the putative molecular target of the perturbant?We have chosen an alternate strategy to provide small molecule control of any protein of interest (7). Our approach relies upon one well characterized protein-ligand interaction that can be used to regulate many different proteins of interest. We have engineered small protein domains called destabilizing domains (DDs) 2 that are constitutively degraded when expressed in mammalian cells. ...

Chemical Control of FGF-2 Release for Promoting Calvarial Healing with Adipose Stem Cells

“…This may be important when access to the particular tissue is not amenable to local injection and when the systemic delivery of a protein of interest may be highly toxic or cause unwanted pathology. Notably, we observed no adverse effects of prolonged treatment with Shield-1 on the health of our mice, and we have previously demonstrated via microarray studies that Shield-1 appears to be biologically inert in mammalian cells (9).…”

“…Previous work has shown FGF-2 to bind avidly to heparin in the extracellular matrix, limiting its diffusion and protecting it from proteolysis (26 -28). The similar increase in bone formation in response to FGF-2 exposure across the different groups of animals implanted with ASCs and DD-FGF2 cells, regardless of the temporal pattern of FGF-2 exposure, is most likely in part a reflection on the pharmacokinetics of FGF-2 in vivo rather than a reflection of our delivery system in mouse models (9). Notably, we used half the number of ASCs used by Cowan et al (15) shown to regenerate skull defects, so as not to obscure the effect of chemically driven FGF-2.…”

Ligand-switchable Substrates for a Ubiquitin-Proteasome System

“…Our original screens identified dozens of FKBP mutants that display ligand-dependent stability when expressed in mammalian cells (1,49). In the work presented here, we examine a subset of these FKBP-derived DDs to study the biophysical behavior of these purified proteins in vitro as well as the intracellular stability of the same DDs when expressed in mammalian cells.…”

“…Methods to query protein function, however, have lagged behind this sequencing revolution. We have developed several general methods to conditionally control protein stability in cells using small molecules (1,2,49). Destabilizing domains (DDs) 4 are small protein domains, which, when fused to a protein of interest, promote degradation of the entire fusion protein in the non-permissive (i.e.…”

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.…”