A new tool for rapid protein degradation in live animals

Understanding how genotype links to phenotype is essential for understanding the biology of disease. One method for this is altering the function of a gene in a model system – cells in a dish, organoids or animal models. Generally this has been achieved through manipulating of DNA or RNA in the cell but these methods have limitations.

Andrew Wood, Group Leader at the MRC Human Genetics Unit and lead of the MRC National Mouse Genetics Network’s (NMGM)  Degron Tagging cluster has provided a proof-of-principle for a new way of studying protein function in mouse models of development and disease.

Methods that could drive degradation of a target protein in cells and tissues could work much faster than those that prevent production of new protein, and may also limit compensatory mechanisms that arise due to gene deletion.  Genetic changes are also hard to reverse, so a method that can remove protein and then restore it to normal levels could be of great benefit to our understanding of the roles of proteins at different stages. Therefore, techniques that drive degradation of proteins should aim to be rapid, reversible, and targeted.

The method takes advantage of a chemical genetic tool known as auxin-inducible degrons. It is based on a plant system for protein degradation, where a plant hormone, auxin, binds a protein, Tir1, which then targets proteins containing a ‘degron’ sequence. These are subsequently flagged for degradation by the mammalian proteasome complex. The system has already revolutionised functional studies of proteins in mammalian in vitro systems, but it has not yet been possible to degrade tagged endogenous proteins in adult tissues of living animals.

In their research just published in eLife, Andrew’s group have derived novel transgenic mouse lines that use this degron system to rapidly (<2 hours) and nearly completely (>90%) degrade two protein subunits of condensin complexes, condensin I and condensin II. These proteins are essential for chromosome formation and segregation during mitotic cell division. The researchers found that both protein subunits are needed for cell division in precursor lymphocytes, but not in differentiated peripheral lymphocyte derivatives, where cells can divide with only one subunit present. Although degradation was seen in most cell-types tested, issues such as absent components of the ubiquitin proteasome system or poor transit of auxin across barriers within tissues meant that degradation was not possible in a minority of cell-types.

Importantly the team did not see any degradation in the absence of auxin administration, suggesting that the system, in this case, is not leaky. The amount of target protein, auxin and Tir1 all determined how fast proteins could be degraded. This may make it challenging to use the system for proteins which are present in very high quantities in cells.

Being able to rapidly reduce protein levels is really important for studying processes which occur quickly like mitosis, the focus of our lab’s research. As well as helping us understand protein function, the system should also be useful for modelling the mechanism and effects of drugs in disease models, as most drugs target proteins.

 Andrew Wood, Group Leader

Rapid removal of proteins is key to studying essential cell cycle proteins such as condensins, as the later changes arising from abnormal cell division can quickly obscure the primary effects of loss of the degraded protein. As most drugs target proteins, these tools could also provide a useful way to model the effects of drugs and other therapeutic interventions, alongside their use for functional studies of proteins in disease models. The Degron Tagging Cluster also including Asier Unciti Broceta, at Edinburgh Cancer Research, Joe Marsh at MRC HGU, Gopal Sapkota and Roland Wolf, at the University of Dundee aim to build on this work to develop a suite of degron models. They will build genetic reporters to evaluate and compare different degron systems across tissues and disease models. They will use these to test existing ligands and synthesise novel derivatives to identify compounds with favourable pharmacokinetic and pharmacodynamic properties in vivo. 

This study was performed in collaboration with Dr Bin Gu (Michigan State University) and Professor Janet Rossant (Sick Kids, Toronto) and supported by UKRI MRC, Wellcome Trust, and Canadian Institutes of Health Research.

Links

• Original article: Macdonald, L, Taylor, GC, Brisbane, JM, Christodoulou, E, Scott, L, von Kriegsheim, A, Rossant, J, Gu, B, Wood, AJ. Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons. eLife (2022) 11:e77987 https://doi.org/10.7554/eLife.77987
• Andrew Wood Research Group
• UKRI National Mouse Genetic Network https://nmgn.mrc.ukri.org/