Proteolysis-inducing Peptides as Tools for Chemical Genetics

Type of Award: Catalyst
Date Awarded: January 2009
Award End Date: December 2010
Amount Awarded: $ 199,333.00
PI(s): Stephen Kron, PhD, UChicago; Brian Kay, PhD, UIC; Stephen Kent, PhD, UChicago;

Abstract: In this Catalyst proposal, an interdisciplinary team consisting of Stephen Kent and Stephen Kron at The University of Chicago and Brian Kay at the University of Illinois at Chicago will work together to investigate a new approach to testing protein function in cells. They will pursue development of ProTaPs, Proteolysis Targeting Peptides, as a tool for chemical manipulation of cellular proteins. In detail, ProTaPs consist of three functionalities: a targeting peptide that binds to specific protein target, a domain that binds to an E3 ubiquitin ligase to induce polyubiquitination and a cell penetrating peptide to deliver the ProTaP to its site of action. These three functionalities can be synthesized independently as peptide modules and then linked together by native chemical ligation. This modular approach allows great flexibility, so that a large number of ProTaPs can be synthesized and tested in parallel. Treating cells with a ProTaP will rapidly induce ubiquitin-proteasome dependent destruction of the target protein. We hope to validate ProTaPs alongside knockouts and knockdowns as a tool for analysis of gene functions, but also as a novel route to validating proteins as targets for therapeutics. This new tool will be used to analyze chromatin modification and protein assembly at DNA double strand breaks. We will identify a small number of key target proteins and exploit phage display to discover high affinity and high specificity binding peptides that will tether a ProTaPs to each of these proteins. Because ProTaPs can be used much like a drug, they will allow determination of the requirements for normal responses to DNA damage, such as modification of histone proteins, recruitment and assembly of DNA damage signaling and repair proteins, and for assembly of the characteristic protein complex at the double strand break site. They will exploit a GFP fusion to 53BP1, a protein that rapidly localizes to DNA breaks, as a fluorescent reporter for changes in chromatin modification or protein assembly. By examining changes in the characteristic relocalization of GFP-53BP1 from diffuse nuclear distribution to discrete foci at double strand break sites and then tracking whether the persistence of the foci is normal, we will be able to determine whether a ProTaPs has disrupted a key function in the DNA damage response. This work will identify new mechanisms and targets in DNA damage response and provide useful data on the approach of targeting chromatin as a route to radiosensitization. Developing a new tool to help understand protein function in the response to DNA damage may have broad consequences. The new drug-like molecules to be studied here may provide leads for a new class of drugs that will enhance the effects of radiation on tumors, with the potential for major impact on the treatment of metastatic cancer.