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

Abstract

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). Two of the resulting uAbs were found to be global ERK degraders, pan-specifically capturing all endogenous ERK1/2 protein forms and redirecting them to the proteasome for degradation in different cell lines, including MCF7 breast cancer cells. Taken together, these results demonstrate how the substrate specificity of an E3 ubiquitin ligase can be reprogrammed to generate designer uAbs against difficult-to-drug targets, enabling a modular platform for remodeling the mammalian proteome.

Intracellular Protein Delivery: Approaches, Challenges, and Clinical Applications

“…While protein stability varies, proteins are generally more stable than mRNA, both in storage and in biological media [ 41 ]. Finally, it has been reported that some therapeutically interesting proteins are poorly expressed as transgenes despite facile purification from E. coli cultures [ 42 ]. Thus, for some applications, nucleic acids may be precluded entirely as a treatment modality, and direct protein delivery could be the only method for introducing potent therapeutics into the cytoplasm.…”

Design of Peptide-Guided Protein Degraders with Structure-Agnostic Language Models

“…All purified uAb constructs, and unfused CHIPΔTPR were obtained from cultures of E. coli BL21(DE3) cells carrying pET28a-based plasmids encoding the SnP_7 the SnP_8 uAbs or CHIPΔTPR. 26 Cells were grown in Luria-Bertani (LB) medium according to protocols described previously. 26 Briefly, protein expression was induced with 1M isopropyl β-D-1-thiogalactopyranoside (IPTG) when the culture density, determined by optical density at 600 nm (OD 600 ), reached 0.5-0.7 and proceeded for 12-16 h at 37°C.…”

“…26 Cells were grown in Luria-Bertani (LB) medium according to protocols described previously. 26 Briefly, protein expression was induced with 1M isopropyl β-D-1-thiogalactopyranoside (IPTG) when the culture density, determined by optical density at 600 nm (OD 600 ), reached 0.5-0.7 and proceeded for 12-16 h at 37°C. Following expression, cells were harvested by centrifugation at 10,000 x g for 10 min at 4 °C.…”

“…ELISA was performed according to previously published protocols. 26 Briefly, 96-well plates (MaxiSorp; Nunc Nalgene) were incubated with 1 μg/mL of β-catenin (Biomatik, Cat # RPU40704) diluted in PBS, pH 7.4, 50 µL/well, at 4 °C overnight. Plates were incubated with 200 µL blocking buffer (5% (w/v) nonfat dry milk (Carnation) in PBS) overnight at 4°C, then washed three times with 200 µL PBS-T (PBS, 0.1% (v/v) Tween 20) per well.…”

Photoswitchable binders enable temporal dissection of endogenous protein function

“…Previous studies showed that the E40 DARPin binds to unphosphorylated ERK 43,44 and prevents phosphorylation and activation by MEK 43 . However, as E40 had previously been shown only to prevent phosphorylation of chimeric exogenous ERK 43 , we first established that E40 can inhibit endogenous ERK activity in a live-cell assay.…”