Genetic engineering approaches to constitutively or transiently eliminate a target protein within a cell are established tools in biosciences, and include widely-used gene knockouts by CRISPR/TALEN or knockdowns by RNAi. However, these approaches suffer from several limitations. For example, gene knockout is irreversible and often not viable (e.g. for essential genes), while the effect of RNAi requires long treatments, is often incomplete, and can lead to undesired off-target effects.
Recent research from the Ciulli Lab and others has led to the development of a novel approach, using small-molecules often called PROTACs (for Proteolysis targeting chimeras) to induce protein post-translational knockdown via targeted ubiquitination and subsequent proteasomal degradation (Deshaies, 2015). Small-molecule based approaches alleviate many of the limitations of genetic knockdowns, because: 1) they allow intervening directly at the protein level, rather than at the DNA or mRNA level; 2) they can achieve complete depletion of protein levels within few hours, and at very low (nM to pM) compound concentration, thus minimizing off-target effects; 3) are reversible and easy to deliver without much disturbance to cells; 4) can potentially target macromolecular complexes associated with the target protein. However, because we simply do not have a small-molecule binder for every single cellular protein, it is not possible to apply this approach generally across the genome/proteome. Toward this goal, others have achieved the degradation of non-endogenous fusion proteins using small molecules, but these approaches have suffered from limitations due to complicated genetic manipulation, leakage of induced expression or compound, and incomplete target depletion (Buckley et al., 2015; Natsume et al., 2016).
This project will combine the expertise of the Ciulli Lab in the chemical biology of PROTACs and selective protein targeting, with the expertise of the Alessi and Sapkota Labs in protein ubiquitination and genetic engineering methods, in order to develop a novel approach for targeted small-molecule induced knock-down of any protein inside cells. The student will first develop and optimize the small-molecule approach, and then validate its application for highly rapid and selective protein knockdown in live cells against targets of interest. If successful, the results of the project will provide new attractive tools for studying biological function of interesting proteins both physiologically and patho-physiologically, and a new approach for future in vivo applications to target validation in disease.
Buckley, D.L., Raina, K., Darricarrere, N., Hines, J., Gustafson, J.L., Smith, I.E., Miah, A.H., Harling, J.D., and Crews, C.M. (2015). HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins. ACS Chem Biol 10, 1831–1837.
Deshaies, R.J. (2015). Protein degradation: Prime time for PROTACs. Nat Chem Biol 11, 634–635.
Natsume, T., Kiyomitsu, T., Saga, Y., and Kanemaki, M.T. (2016). Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors. Cell Reports 15, 210–218.