An inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells

Conditional inactivation or depletion of proteins is a powerful method for determining gene function and central to our understanding of complex biological systems. Protein expression is frequently controlled at the DNA level by disruption of the coding sequence of the gene or at the level of the mRNA by employing methodologies for suppressing mRNA accumulation, especially with RNA interference (RNAi). However, in both cases protein depletion is indirect and the rapidity of loss is dependent on the stability of the protein, resulting in very slow loss for long-lived proteins. This serves as a major limitation for all but the shortest lived proteins, since different severities of phenotypes are typically observed at differing levels of depletion, especially in the case of proteins which have multiple functions. Additionally, neither gene inactivation nor RNAi silencing are readily reversible. RNAi-mediated mRNA degradation often also suffers from incomplete silencing and/or off target effects. 

 

To overcome these limitations a variety of systems have been developed that allow the post-translational degradation of proteins using cell-permeable small molecules. Recently, a system for inducible-protein depletion was developed which relies on the transplantation of a ligand-induced degradation system found in plants (Nishimura et al., Nat Med, 2009). All eukaryotes possess Skp1, Cullin and F-Box protein (SCF) ubiquitin ligases, which are comprised of three core subunits and a variable F-box that is responsible for substrate recruitment to the SCF complex. F-box proteins associate with the SCF complex through an interaction of their F-box domain with the Skp1 protein. In plants, auxin hormones promote the interaction between the F-box protein TIR1 and proteins containing an auxin-inducible degron (AID). Orthologs of TIR1 and AID are only found in plant species; however, due to the high degree of conservation of Skp1 in eukaryotes, ectopically expressed TIR1 can associate with Skp1 in animal cells and form an SCFTIR1 complex. SCFTIR1 can then promote auxin-inducible degradation of proteins tagged with an AID in human cells.

 

The properties of this inducible destruction system are remarkable.

 

We demonstrated that an AID-tagged protein can functionally replace an endogenous protein depleted by RNAi, leading to an inducible null phenotype rapidly after auxin addition. The AID system was shown to be capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 minutes and proceeding to completion with first order kinetics. Auxin-inducible, degron-mediated instability was demonstrated to be rapidly reversible and induced degradation initiated and continued in all cell cycle phases, including mitosis. This system is especially useful for identifying the function(s) of proteins of interest during specific points in the mammalian cell cycle (Holland and Fachinetti et al., PNAS, 2012). We are currently combining the auxin-inducible destruction system with endogenous gene targeting in human somatic cells to address outstanding questions about centrosome duplication and mitotic regulation.