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dTAG system

by Dr Hadley Sheppard, The Institute of Cancer Research, London

Targeted protein degradation systems have emerged which enable pharmacological modelling of protein degradation in the absence of a chemical probe. Once such system is the dTAG system, developed by the Gray lab (formerly of Harvard and now Stanford University). The dTAG system enables degradation of a given target both in vitro and in vivo 1,2 . The system makes use of highly selective FKBP12F36V directed ligands (known as the dTAG molecule) and expression of a target protein tagged with the degron FKBP12F36V , to create a fusion protein known as the dTAG-protein (Figure 1). Once the dTAG-protein is expressed in a cell, for example by CRISPR-mediated knock in or transgenic expression, the cell-permeable dTAG molecule is added and forms a dual linker between the dTAG-protein and an endogenous E3 ligase. As a result, the dTAG-protein is ubiquitinated and targeted for proteasomal degradation.

dTAG-mediated degradation is extremely rapid and can completely remove a given target in as little as an hour. The system is reversible and washout of the dTAG molecule will enable re-expression of the dTAG-protein. To set up a dTAG system, there are a series of plasmids and sequences available from Addgene, detailed protocols , and the dTAG molecules are commercially available. There are now dTAG molecules that will recruit either the endogenous cereblon or VHL E3 ligases and both are compatible with same dTAG epitope tag on the target protein. It is important to confirm which terminus can accommodate the FKBP12F36V tag without interfering with function as well evaluate toxicity of the dTAG molecules in parental cells prior to performing degradation experiments. Once the system is established, the dTAG is ideal for target validation in the process of drug discovery. The dTAG system combines the precision of genetics with the time-dependent opportunities of small molecules.

Figure 2: Schematic of the dTAG system for degrading protein target T, through fusion with the degron, FKPB12 F36V . Addition of the dTAG molecule leads to binding with Cereblon and degradation of the target protein. dtag

References

  1. Nabet, B. et al. The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14 , 431-441, DOI:10.1038/s41589-018-0021-8 (2018).
  2. Nabet, B. et al. Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat Commun 11 , 4687, doi:10.1038/s41467-020-18377-w (2020).