Achieving a rapid and efficient degradation of endogenous proteins of interest (POIs) in cells is desirable as a research toolkit to understand protein function, and for therapeutic targeting of proteins. Protein silencing through gene knockouts, for example by CRISPR/Cas9 genome editing, are irreversible, time consuming and often not feasible. Similarly, RNA interference approaches require prolonged treatments, can lead to incomplete protein depletion and are often associated with off-target effects. Direct proteolysis of POIs can potentially overcome these limitations.
The Sapkota lab has developed an affinity directed protein missile (AdPROM) system that allows for rapid and efficient destruction of endogenous target proteins in many cell lines (1, 2). The proteolytic AdPROM system fuses small, high-affinity polypeptide binders, such as nanobodies and monobodies, of specific proteins and the von Hippel-Lindau (VHL) protein to recruit the CUL2-RING E3 ligase complex. The resulting AdPROM system, when delivered in any cell, through retroviral infections, leads to efficient degradation of endogenous target proteins through the proteasome.
The Ciulli lab works on developing small molecule degraders, also known as proteolysis targeting chimeras (PROTACs), that induce specific proteins in cells to be degraded via the ubiquitin proteasome system (3). The Ciulli lab has developed PROTACs that selectively and rapidly degrade the HALO-tagged proteins from cells by recruiting them to VHL-CUL2 E3 ligase complex for proteasomal destruction of the HALO-tagged proteins. A key requirement for this approach is that the endogenous target proteins need to be tagged with a HALO-tag first in order for the HaloPROTACs to work. Introducing a HALO-tag on an endogenous protein is not only challenging but can often compromise the function of the protein as well.
This project aims to develop an effective inducible AdPROM system by fusing nano/monobodies with a HALO-tag and inducibly degrade the proteins of interest targeted by the nano/monobodies using HaloPROTACs. The advantage of this system is that it will be applicable to any cell lines where the target protein is expressed and so the function of the protein can be rapidly assessed in any cellular context. In the absence of an effective targeting nano/monobodies, CRISPR/Cas9 genome editing can be used to rapidly introduce a GFP-tag on the target protein first and use anti-GFP nanobody fused to HALO-tag as the inducible AdPROM for the degradation of the POI.
After developing a robust AdPROM technology, its efficacy to degrade a wide array of target proteins in many different cell types will be tested. Novel HaloPROTACs will be synthesized in the Ciulli Lab and tested with the goal to optimize the degradation efficiency of the system. This approach has the potential in rapidly evaluating proteolysis as a druggable approach for many so called ‘undruggable’ target proteins involved in many human diseases.
The student will receive excellent training in multidisciplinary areas: cutting-edge cell biology and biochemistry technologies from the Sapkota lab and state-of-the-art chemical biology techniques from the Ciulli lab.
1. Fulcher, L. J., Macartney, T. J., Turnbull, C., Hutchinson, L., and Sapkota, G. P. (2017) Targeting endogenous proteins for degradation through the affinity-directed protein missile system. Open biology 7: 170066.
2. Fulcher, A. J., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A., and Sapkota, G. P. (2016) An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis. Open biology 6:1602551.
3. Gadd, M. S., Testa, A., Lucas, X., Chan, K.-H., Chen, W. Lamont, D.J., Zengerle, M. and Ciulli, A. (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514–521.