1. Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka
Alexander Froschauer, Lisa Kube, Alexandra Kegler, Christiane Rieger, Herwig O Gutzeit PLoS One. 2015 Jul 6;10(7):e0131252.doi: 10.1371/journal.pone.0131252.eCollection 2015.
Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.
2. Design and Combinatorial Development of Shield-1 Peptide Mimetics Binding to Destabilized FKBP12
Daniel Madsen, Frederik P Jørgensen, Daniel Palmer, Milena E Roux, Jakob V Olsen, Mikael Bols, Sanne Schoffelen, Frederik Diness, Morten Meldal ACS Comb Sci. 2020 Mar 9;22(3):156-164.doi: 10.1021/acscombsci.9b00197.Epub 2020 Feb 23.
On the basis of computational design, a focused one-bead one-compound library has been prepared on microparticle-encoded PEGA1900 beads consisting of small tripeptides with a triazole-capped N-terminal. The library was screened towards a double point-mutated version of the human FKBP12 protein, known as the destabilizing domain (DD). Inspired by the decoded library hits, unnatural peptide structures were screened in a novel on-bead assay, which was useful for a rapid structure evaluation prior to off-bead resynthesis. Subsequently, a series of 19 compounds were prepared and tested using a competitive fluorescence polarization assay, which led to the discovery of peptide ligands with low micromolar binding affinity towards the DD. The methodology represents a rapid approach for identification of a novel structure scaffold, where the screening and initial structure refinement was accomplished using small quantities of library building blocks.
3. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules
Laura A Banaszynski, Ling-Chun Chen, Lystranne A Maynard-Smith, A G Lisa Ooi, Thomas J Wandless Cell. 2006 Sep 8;126(5):995-1004.doi: 10.1016/j.cell.2006.07.025.
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.
