Engineering Single Pan-Specific Ubiquibodies for Targeted Degradation of All Forms of Endogenous ERK Protein Kinase.

Ubiquibodies (uAbs) are a customizable proteome editing technology that utilizes E3 ubiquitin ligases genetically fused to synthetic binding proteins to steer otherwise stable proteins of interest (POIs) to the 26S proteasome for degradation. The ability of engineered uAbs to accelerate the turnover of exogenous or endogenous POIs in a post-translational manner offers a simple yet robust tool for dissecting diverse functional properties of cellular proteins as well as for expanding the druggable proteome to include tumorigenic protein families that have yet-to-be successfully drugged by conventional inhibitors. Here, we describe the engineering of uAbs composed of human carboxyl-terminus of Hsc70-interacting protein (CHIP), a highly modular human E3 ubiquitin ligase, tethered to differently designed ankyrin repeat proteins (DARPins) that bind to nonphosphorylated (inactive) and/or doubly phosphorylated (active) forms of extracellular signal-regulated kinase 1 and 2 (ERK1/2).

Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic.

Manipulation of the ubiquitin-proteasome pathway to achieve targeted silencing of cellular proteins has emerged as a reliable and customizable strategy for remodeling the mammalian proteome. One such approach involves engineering bifunctional proteins called ubiquibodies that are comprised of a synthetic binding protein fused to an E3 ubiquitin ligase, thus enabling post-translational ubiquitination and degradation of a target protein independent of its function. Here, we have designed a panel of new ubiquibodies based on E3 ubiquitin ligase mimics from bacterial pathogens that are capable of effectively interfacing with the mammalian proteasomal degradation machinery for selective removal of proteins of interest.

A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins.

There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a genetic selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2).