Ubiquibodies, synthetic E3 ubiquitin ligases endowed with unnatural substrate specificity for targeted protein silencing

Abstract

The ubiquitin-proteasome pathway (UPP) is the main route of protein degradation in eukaryotic cells and is a common mechanism through which numerous cellular pathways are regulated. To date, several reverse genetics techniques have been reported that harness the power of the UPP for selectively reducing the levels of otherwise stable proteins. However, each of these approaches has been narrowly developed for a single substrate and cannot be easily extended to other protein substrates of interest. To address this shortcoming, we created a generalizable protein knock-out method by engineering protein chimeras called "ubiquibodies" that combine the activity of E3 ubiquitin ligases with designer binding proteins to steer virtually any protein to the UPP for degradation. Specifically, we reprogrammed the substrate specificity of a modular human E3 ubiquitin ligase called CHIP (carboxyl terminus of Hsc70-interacting protein) by replacing its natural substrate-binding domain with a single-chain Fv (scFv) intrabody or a fibronectin type III domain monobody that target their respective antigens with high specificity and affinity. Engineered ubiquibodies reliably transferred ubiquitin to surface exposed lysines on target proteins and even catalyzed the formation of biologically relevant polyubiquitin chains. Following ectopic expression of ubiquibodies in mammalian cells, specific and systematic depletion of desired target proteins was achieved, whereas the levels of a natural substrate of CHIP were unaffected. Taken together, engineered ubiquibodies offer a simple, reproducible, and customizable means for directly removing specific cellular proteins through accelerated proteolysis.

Keywords: Antibody Engineering; E3 Ubiquitin Ligase; Molecular Biology; Proteasome; Protein Degradation; Protein Engineering; Reverse Genetics; Synthetic Biology; Ubiquitin; Ubiquitination.

FIGURE 1.

Remodeling CHIP for ubiquitination of unnatural target proteins. a, schematic of natural and engineered UPP systems. Normally, E1, E2, and E3 mediate the transfer of ubiquitin to native substrates (S). Ubiquitin-tagged substrates are then degraded by the proteasome. In the engineered uAb pathway, a truncated E3 (E3*) is fused to a DBP, which remodels the specificity of E3* for an unnatural target protein (T). b, linear representation of CHIP, CHIPΔTPR, and R4-uAb. Numbers refer to amino acid positions from N terminus (N) to C terminus (C). The proteins are aligned vertically with the coiled-coil and U-box domains of CHIP. CHIPΔTPR is a truncated version of CHIP lacking the TPR domain. R4-uAb was designed with an additional Gly-Ser (GS) linker connecting the scFv13-R4 intrabody to CHIPΔTPR. c, Western blot analysis of cell lysates derived from E. coli strain BL21(DE3) expressing full-length CHIP, CHIPΔTPR, scFv13-R4, and several different uAb constructs. The uAbs were comprised of DBPs specific for E. coli β-gal (scFv13-uAb, R4-uAb, and R4-uAb^R272A), bacteriophage gpD (D10-uAb) and E. coli MBP (YS1-uAb). An equivalent amount of total protein was loaded in each lane. Anti-FLAG antibodies were used to detect the different proteins. d, binding activity of purified R4-uAb measured by ELISA with β-gal as antigen. The intrabody scFv13-R4 served as a positive control, whereas CHIPΔTPR and D10-uAb served as negative controls. Also tested was R4-uAb^R272A, a derivative of R4-uAb carrying a point mutation in the U-box domain that is known to block the interaction between CHIP and the E2 enzyme, UbcH5α.

FIGURE 2.

In vitro ubiquitination of β-gal by engineered ubiquibodies. a, in vitro ubiquitination of β-gal in the presence of R4-uAb. At the indicated times, reactions were stopped by boiling and immunoblotted with anti-β-gal antibodies. b, ubiquitination of β-gal was evaluated in the presence (+) or absence (−) of each ubiquitin pathway component, namely ubiquitin (Ub), E1, E2, and R4-uAb as E3. Controls included scFv13-R4, CHIPΔTPR, R4-uAb^R272A, and D10-uAb. An equivalent amount of total protein was added to each lane. Immunoblots were probed with anti-β-gal, anti-ubiquitin, anti-Lys-48, and anti-His₆ antibodies. Protein bands corresponding to β-gal, mono-ubiquitinated β-gal (*), and β-gal-ubiquitin conjugates as well as the molecular weight of the marker bands (MW) are indicated. The results are representative of at least three replicate experiments.

FIGURE 3.

Isolation of ubiquitinated β-gal. a, Coomassie-stained SDS-PAGE analysis of R4-uAb-mediated ubiquitination reactions in the presence and absence of E. coli β-gal at various times after initiation. Protein bands corresponding to unmodified β-gal, R4-uAb, various ubiquitin conjugates, and the molecular weight of the marker bands (MW) are indicated. Trypsin digest and subsequent LC-MS/MS analysis was performed on the protein bands delineated by the red box. b, mapping of ubiquitinated lysines (red spheres) onto crystal structure of a single β-gal monomer (blue). All identified lysines are solvent exposed in the homotetramer (gray) model generated in PyMOL^TM using Protein Data Bank code 1DP0. c, MS/MS spectra of a representative β-gal peptide ⁷⁷⁵KQLLTPLR⁷⁸², containing a Gly-Gly modified (ubiquitinated) lysine residue. The modified lysine residue is labeled with GG and corresponds to Lys-775 in β-gal. Fractionation of the peptides into b and y ions was performed, and the corresponding peaks are labeled on the spectra. The y axis, relative abundance, was normalized to the most abundant identified peptide fragment.

FIGURE 4.

R4-uAb-mediated proteolysis of β-gal in mammalian cells. a, immunoblots of soluble (top panels) or insoluble (bottom panel) fractions prepared from HEK293T cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per well (β-gal only) or co-transfected with pcDNA3-β-gal along with one of the following: pcDNA3-R4-uAb, pcDNA3-D10-uAb, pcDNA3-R4-uAb^R272A, or pcDNA3-scFv13-R4 (each transfected at 1.25 μg of plasmid DNA per well). The triangle indicates increasing amounts of pcDNA3-R4-uAb plasmid DNA (0.05, 0.25, 0.75, and 1.25 μg of plasmid DNA per well) used to transfect cells. The percentages of β-gal remaining in each sample were quantitated by densitometry scanning and are indicated. Blots were probed with antibodies specific for β-gal, His₆, GAPDH, and Hsp70 as indicated. An equivalent amount of total protein was loaded in each lane, as confirmed by immunoblotting with anti-GAPDH antibodies. The immunoblot results are representative of at least three replicate experiments. b, β-gal activity measured in samples described in a and in Fig. 5. The activity reported for pcDNA3-R4-uAb corresponds to the highest co-transfection level in 293T and the lowest co-transfection level in COS7 and BHK21. Data were normalized to the signal for the β-gal-only control and is expressed as the mean ± S.D. of biological triplicates.

FIGURE 5.

R4-uAb-mediated proteolysis of β-gal in different cell lines. Immunoblots of extracts prepared from BHK21 (left) and COS-7 (right) cells transfected with pcDNA3-β-gal alone at 0.05 μg of plasmid DNA per well (β-gal only) or co-transfected with pcDNA3-β-gal along with one of the following: pcDNA3-R4-uAb, pcDNA3-scFv13-R4, or pcDNA3-D10-uAb (each transfected at 1.25 μg of plasmid DNA per well). The triangle indicates increasing amounts of pcDNA3-R4-uAb plasmid DNA (0.05, 0.25, and 0.75 μg of plasmid DNA per well) used to transfect cells. The percentages of β-gal remaining in each sample were quantitated by densitometry scanning and are indicated. Blots were probed with antibodies specific for β-gal, His₆, and GAPDH as indicated. An equivalent amount of total protein was loaded in each lane, as confirmed by immunoblotting with anti-GAPDH antibodies. The immunoblot results are representative of two replicate experiments.

FIGURE 6.

Co-precipitation of ubiquitinated β-gal in HEK293T cells. Immunoblots of pulldown samples or extracts prepared from HEK293T cells transfected with pcDNA3-R4-uAb alone (R4-uAb only), pcDNA3-β-gal alone (β-gal only), or co-transfected with pcDNA3-β-gal along with one of the following: pcDNA3-scFv13-R4, pcDNA3-R4-uAb, or pcDNA3-D10-uAb. Pulldown was performed by subjecting extracts to nickel-nitrilotriacetic acid magnetic agarose beads followed by immunoblotting with antibodies specific for β-gal, ubiquitin, GAPDH, and His₆ as indicated. An equivalent amount of total protein was loaded in each lane, as confirmed by immunoblotting with anti-GAPDH antibodies. Immunoblots are representative of three replicate experiments.

FIGURE 7.

YS1-uAb-mediated proteolysis of MBP in mammalian cells. a and b, representative immunoblots of soluble or insoluble (bottom panel of a) fractions prepared from HEK293T cells transfected with pcDNA3-MBP alone (MBP only at 0.01 (a) or 0.1 μg (b) of plasmid DNA per well) or co-transfected with pcDNA3-MBP along with one of the following: pcDNA3-YS1-uAb, pcDNA3-YS1, pcDNA3-R4-uAb, or pcDNA3-D10-uAb (each transfected at 1.75 μg of plasmid DNA per well). The triangles indicate increasing amounts of pcDNA3-YS1-uAb plasmid DNA (0.25, 0.75, 1.25, and 1.75 μg of plasmid DNA per well in a and 0.5, 1.5, and 1.75 μg of plasmid DNA per well in b) used to transfect cells. The percentages of MBP remaining in each sample were quantitated by densitometry scanning and are indicated. Blots were probed with antibodies specific for MBP, His₆, Hsp70, and GAPDH as indicated. An equivalent amount of total protein was loaded in each lane, as confirmed by immunoblotting with anti-GAPDH antibodies. The immunoblot results are representative of at least two replicate experiments. c, scatter plot of densitometry analyses from five independent MBP knockdown experiments in HEK293T cells, where the level of MBP transfection was 0.05 or 0.1 μg of plasmid DNA per well as indicated. Linear regression analysis was performed on the data corresponding to the two different MBP transfection levels and R² values are shown.