Ubiquibodies, synthetic E3 ubiquitin ligases endowed with unnatural substrate specificity for targeted protein silencing.
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.
From the Department of Biomedical Engineering.
Alyse D
AD
Portnoff
Erin A
EA
Stephens
Jeffrey D
JD
Varner
Matthew P
MP
DeLisa
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D019556:COS Cells; D002522:Chlorocebus aethiops; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D025821:Polyubiquitin / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D020033:Protein Isoforms / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D054875:Ubiquitination
D000906:Antibodies; D020033:Protein Isoforms; D025801:Ubiquitin; D025821:Polyubiquitin; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine
Antibody Engineering; E3 Ubiquitin Ligase; Molecular Biology; Proteasome; Protein Degradation; Protein Engineering; Reverse Genetics; Synthetic Biology; Ubiquitin; Ubiquitination