Ligand-based stabilization of proteins is a well-known phenomenon in protein chemistry. Stabilization of proteins through ligand binding can extend a protein's half-life in cells and result in increased protein thermostability and protection from proteolysis. However certain protein ligands can destabilize protein structure resulting in increased susceptibility to proteolysis and a shorter intracellular half-life.
Changes in protein stability upon ligand binding have been used to identify protein targets for small molecules through drug affinity responsive target stability (DARTS; Nishiya et al., Anal Biochem 2009;385:314–320). Inducing protein degradation has been achieved by linking a protein to a ligand that binds an E3 ubiquitin ligase (proteolysis targeting chimeric molecules [PROTAC]; Riana and Crews, JBC 2010;285:11057–11060) or attaching hydrophobic tags to a dehalogenase fusion protein (HaloTag™, Promega; Neklesa et al., Nat Chem Biol 2011;7:538–543). However, techniques using fusion proteins are unable to modulate the endogenous levels of a specific protein. In this article* it is shown that attaching tert-butyl carbamate-protected arginine (Boc3Arg) to a ligand results in degradation of proteins that bind the Boc3Arg-ligand.
The arginine tag targets proteins for degradation based on the N-end rule, which states that the N-terminal sequence of amino acids determines the half-life of the protein with those proteins starting with amino acids such as arginine having short (<1 h) half-lives, while those starting with hydrophobic amino acids show much longer half-lives. To initially test this tag, ethacrynic acid (EA), which could be modified with Boc3Arg, was used. The drug EA is used as a diuretic and works through covalent modification of gluthanione-S-transferases through modification of cysteine residues. The presence of a Michael acceptor in EA provides the site of covalent attachment, and the compound is of interest because it is an example of a drug that works through a covalent mechanism. Boc3Arg-EA induced degradation of GST-α1 in cell lysates and in whole cells. The authors also tagged trimethoprim (TMP), a specific inhibitor of Escherichia coli dihydrofoloate reductase (eDHFR). Whole cell assays were enabled using fusions of GST or eDHFR with GFP, which were co-expressed with a red fluorescent protein from the same mRNA (using a bicistronic construct). In this way, changes in the amount of GFP fusion protein present could be measured using the GFP/RFP ratio by FACS analysis (see Figure).