Ubiquibodies: Synthetic E3 Ubiquitin Ligases for Targeted Protein Silencing

Ubiquibodies: Synthetic E3 Ubiquitin Ligases for Targeted Protein Silencing

Protein silencing is a powerful tool for both eliciting the function of a protein and eliminating detrimental peptides from a system. Most methods for protein silencing act at the DNA and RNA levels; however, these methods cannot be used to interrogate the difference between isoforms of a single parent protein. Isoforms are generated by post-translational modifications (such as phosphorylation, cleavage, glycosylation, etc.), leading to distinct subpopulations from a single protein archetype. In order to study specific isoforms at a post-translational level, we are developing a silencing technology to function at the protein level. Cells naturally silence proteins via the ubiquitin-proteasome pathway (UPP); by hijacking this pathway, non-native substrates can be targeted to the UPP for degradation, thus silencing protein function. The canonical UPP pathway is composed of three enzymes: E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme), and E3 (ubiquitin ligating enzyme). The E3 ubiquitin ligase determines the target specificity and is the focus of our rational design. We have developed a family of hybrid E3s, termed ubiquibodies, composed of an endogenous E3 ubiquitin ligase catalytic domain fused to variable synthetic binding domains. The catalytic domain of the ubiquibody is the U-box domain of C-terminus of Hsc70-interacting protein (CHIP); U-box E3s function as either monomers or homodimers, making CHIP and an ideal scaffold for targeted protein degradation. Antibody fragments, such as short chain variable fragments (scFvs), as well as synthetic domains, such as fibronectin type three domains (FN3s) and designed ankyrin repeat proteins (DARPins), were substituted for CHIPâ??s natural binding domain, endowing the ubiquibodies with tunable specificity. We have previously shown that ubiquibodies can ubiquitinate exogenous substrates in vitro and that the same substrates can be silenced in mammalian cells expressing both the target and ubiquibody. Here, we present a case study of ubiquibody silencing of an endogenous target protein, extracellular signal-regulated kinase (ERK). Ongoing work aims to generate ubiquibodies that can differentiate between phosphorylated and non-phosphorylated isoforms of ERK. We conclude that ubiquibodies can be used as generalizable platform to silence selectable protein substrates in mammalian cells.