A General Chemical Method to Regulate Protein Stability in the Mammalian Central Nervous System

SummaryThe ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. We have developed a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of the Escherichia coli dihydrofolate reductase (ecDHFR) were engineered to be degraded, and, when this destabilizing domain is fused to a protein of interest, its instability is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. A small-molecule ligand trimethoprim (TMP) stabilizes the destabilizing domain in a rapid, reversible, and dose-dependent manner, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood-brain barrier enables the tunable regulation of proteins expressed in the mammalian central nervous system

Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands

We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell , 2003 , 12 , 1615). To determine the precise residues that cause instability, we calculated the folding free energy (Δ G ) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilised by up to 6 kcal mol −1 relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (COS) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Δ G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Δ G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56088/1/1162_ftp.pd

A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules

SummaryRapid 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

Imaging the Impact of Chemically Inducible Proteins on Cellular Dynamics In Vivo

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters

Smoking and reconstruction of the anterior cruciate ligament

We sought to determine whether smoking affected the outcome of reconstruction of the anterior cruciate ligament. We analysed the results of 66 smokers (group 1 with a mean follow-up of 5.67 years (1.1 to 12.7)) and 238 non-smokers (group 2 with a mean follow-up of 6.61 years (1.2 to 11.5)), who were statistically similar in age, gender, graft type, fixation and associated meniscal and chondral pathology. The assessment was performed using the International Knee Documentation Committee form and serial cruciometer readings. Poor outcomes were reported in group 1 for the mean subjective International Knee Documentation Committee score (p < 0.001), the frequency (p = 0.005) and intensity (p = 0.005) of pain, a side-to-side difference in knee laxity (p = 0.001) and the use of a four-strand hamstring graft (p = 0.015). Patients in group 1 were also less likely to return to their original level of pre-injury sport (p = 0.003) and had an overall worse final 7 International Knee Documentation Committee grade score (p = 0.007). Despite the well-known negative effects of smoking on tissue healing, the association with an inferior outcome after reconstruction of the anterior cruciate ligament has not previously been described and should be included in the pre-operative counselling of patients undergoing the procedure. ©2006 British Editorial Society of Bone and Joint Surgery