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.
Immunohistochemically detected p53 protein accumulation was an independent marker of shortened survival and was seen more often in familial than in sporadic carcinomas. Our findings also suggest a correlation between p53 protein accumulation and p53 gene mutation.
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. ...
The FKBP-derived destabilizing domains are increasingly being used to confer small moleculedependent stability to many different proteins. The L106P domain confers instability to yellow fluorescent protein when it is fused to the N-terminus, the C-terminus, or spliced into the middle of yellow fluorescent protein, however multiple copies of L106P do not confer greater instability. These engineered destabilizing domains are not dominant to endogenous degrons that regulate protein stability.It is generally appreciated that cell-permeable small molecules, typically ligands for cellular proteins, can be useful as conditional perturbants of biological processes. 1 However, the discovery and characterization process for new molecules to perturb a protein of interest can be lengthy and uncertain. Our approach to develop a general "chemical genetic" strategy to regulate any protein of interest involved two elements. We first required a small protein domain that was unstable when expressed in cells. Further, we required that this instability be faithfully transmitted to any fused partner protein. Secondly, we desired cell-permeable ligands for these protein domains that bind with high affinity and protect these protein domains from being targeted for degradation. We call these FKBP mutants "destabilizing domains" (DDs), and we have used both unbiased screening and data-driven design to identify protein-ligand pairs that display the desired ligand-dependent stability when expressed in mammalian cells. 2-4The human FKBP12 protein is the parent molecule for our early studies, and we have shown that a variety of different FKBP ligands can be used to stabilize destabilizing domains derived from this protein fold. This technology can be used to regulate protein levels in cultured mammalian cells as well as in living mice and other organisms. 5-7 In this manuscript we report several lines of investigation that significantly expand the utility of this regulatory technique for the research biology community.The L106P FKBP mutant was one of the most potent DDs identified during our initial screening process, and Shield-1 is the ligand that stabilizes this domain. We fused L106P to the Nterminus of yellow fluorescent protein (L106P-YFP) and used a MMLV-based retroviral expression system to stably transduce NIH3T3 cells. When these cells are cultured in the absence of Shield-1 the YFP levels fall to only 1-2% of levels in the presence of Shield-1. Addition of Shield-1 stabilizes L106P-YFP in a dose-dependent fashion, and full stability is achieved with 1 μM Shield-1. 2 When L106P is fused to the C-terminus of YFP, transduced into cells, and cultured in the absence of Shield-1, YFP is expressed at approximately 10% of maximum fluorescence levels observed. 2Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultin...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.