If you learned that a particular protein contributes to disease, you might be interested in eliminating that protein, or at least reducing its presence. But how would you proceed? One way would be to harness the cell’s ubiquitin-proteasome pathway (UPP), which guides unneeded or damaged proteins to proteasomes, cellular complexes that break proteins apart.
To date, methods capable of harnessing the UPP have resorted to reverse genetics. These genetic engineering methods, however, are of limited utility. Each has been narrowly developed for a single protein and cannot be easily extended to other proteins of interest. But now an alternative approach is available. It is, its creators say, a generalizable protein knockout method that directly modifies a particular portion of the UPP.
The new approach relies on engineered molecules called ubiquibodies. They are protein chimeras that combine the activity of E3 ubiquitin ligases with designer binding proteins, and they can usher selected proteins down the UPP for degradation. Ubiquibodies are described in detail in a paper that will appear March 16 in the print version of the Journal of Biological Chemistry. The paper, entitled “Ubiquibodies: Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing,” is the work of chemical engineers at Cornell University.
These scientists, led by Matthew DeLisa, developed their technique by taking advantage of the modular nature of the UPP, which involves three enzymes called E1, E2, and E3. They modified a particular E3 enzyme called CHIP, removing its natural binding domain and replacing it with “a single-chain Fv intrabody or a fibronectin type III domain monobody that targets their respective antigens with high specificity and affinity.” The idea was to empower CHIP to put ubiquitin chains on any target, guided by the homing capabilities of the antibody fragment to seek out and bind to its specific target.
To prove their concept, the researchers modified CHIP with a binding protein that targets the enzyme beta-galactosidase. They introduced DNA that encoded for their beta-galactosidase target into a human cell line, along with DNA that encoded their ubiquibodies with a binding protein for the beta-galactosidase enzyme.
“Engineered ubiquibodies reliably transferred ubiquitin to surface-exposed lysines on target proteins and even catalyzed the formation of biologically relevant polyubiquitin chains,” wrote the authors. “Following ectopic expression of ubiquibodies in mammalian cells, specific and systematic depletion of desired target proteins was achieved, while the levels of one of CHIP's natural substrates were unaffected.” In other words, beta-galactosidase levels were reduced in the presence of the corresponding ubiquibodies.
“Our ability to redirect whatever protein you want to the proteasome is now made possible simply by swapping out different binding proteins with specificity for targets of interest to the researcher,” DeLisa said.
Ubiquibodies could provide a powerful way to not only completely delete a protein from a cell to study that protein’s effects, but to discover what happens if, say, only 50% of that protein is deleted. Current gene knockout technologies are all or nothing, DeLisa continued. Ubiquibodies could fine-tune research around protein deletion or reduction.
The technology could also prove useful for future drug therapies. In a cancer cell in which a certain protein has been identified as contributing to the disease, the ubiquibody could reduce or eliminate the protein from within by targeting that specific protein only. In DeLisa's lab, experiments are targeting proteins known to be present in diseases such as cancer, Alzheimer's, and Parkinson's.