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

Meksiriporn, Bunyarit; Ludwicki, Morgan B.; Stephens, Erin A.; Jiang, Allen; Lee, Hyeon-Cheol; Waraho-Zhmayev, Dujduan; Kummer, Lutz; Brandl, Fabian; Plückthun, Andreas; DeLisa, Matthew P.

doi:10.1038/s41467-019-09854-y

Cited by 10 publications

(6 citation statements)

References 52 publications

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“…To evaluate the pan-specificity of EpE89–uAb in more detail, we performed an enzyme-linked immunosorbent assay (ELISA) using ERK2 and pERK2 as immobilized antigens. Consistent with the known binding specificity of unfused EpE89, , the EpE89–uAb bound avidly to both ERK2 and pERK2. The pE59–uAb and E40–uAb constructs similarly mirrored the substrate preferences of their parental DARPins, specifically binding pERK2 and ERK2, respectively, at levels that rivaled the binding activity of pan-specific EpE89–uAb for each target (Figure a and Supplementary Figure 2b).…”

Section: Resultssupporting

confidence: 70%

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

et al. 2021

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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.

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Section: Resultssupporting

confidence: 70%

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

et al. 2021

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“…To evaluate the pan-specificity of EpE89-uAb in more detail, we performed an enzyme-linked immunosorbent assay (ELISA) using ERK2 and pERK2 as immobilized antigens. Consistent with the known binding specificity of unfused EpE89 39,44 , the EpE89-uAb bound avidly to both ERK2 and pERK2. The pE59-uAb and E40-uAb constructs similarly mirrored the substrate preferences of their parental DARPins, specifically binding pERK2 and ERK2, respectively, at levels that rivaled the binding activity of pan-specific EpE89-uAb for each target (Fig.…”

Section: Resultssupporting

confidence: 69%

Engineering single pan-specific ubiquibodies for targeted degradation of all forms of endogenous ERK protein kinase

Stephens

Ludwicki

Meksiriporn

et al. 2021

Preprint

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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 proteasome for degradation. The ability of engineered uAbs to accelerate the turnover of exogenous or endogenous POIs in a posttranslational 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 comprised of a highly modular human E3 ubiquitin ligase, human carboxyl terminus of Hsc70-interacting protein (CHIP), tethered to different 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.

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“…Also, important will be the creation of designer binding domains that can reliably differentiate between posttranslationally modified proteins and their unmodified counterparts, making it possible to deplete certain protein subpopulations while sparing others and bestowing proteome editing with an additional layer of specificity. Toward this objective, several groups including ours have described methods for isolating binders that specifically recognize particular protein states such as active versus inactive conformations, mutant versus wild‐type proteins, and phosphorylated versus non‐phosphorylated isoforms …”

Section: Resultsmentioning

confidence: 99%

Proteome editing using engineered proteins that hijack cellular quality control machinery

2019

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Protein silencing is an important aspect of both scientific investigation of native protein function and therapeutic targeting of aberrant protein activity. Many techniques for silencing proteins at the DNA or RNA level exist such as CRISPR, RNAi, or TALEN. Cellular proteins can also be selectively removed at the posttranslational level using proteome editing techniques, many of which employ engineered proteins to engage natural cellular quality control machinery for accelerating the removal of otherwise stable proteins. Here, we summarize recent progress in the development of such engineered proteins, comparing them to analogous microbial-, viral-, and small molecule-based strategies for selectively degrading proteins of interest. Finally, we highlight the advantages and limitations of proteome editing technologies and discuss how the application of directed evolution could alleviate current challenges and usher in a new wave of designer proteins for diagnostic and therapeutic application.

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

Cited by 10 publications

References 52 publications

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

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

Engineering single pan-specific ubiquibodies for targeted degradation of all forms of endogenous ERK protein kinase

Proteome editing using engineered proteins that hijack cellular quality control machinery

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