Proximity Tagging of Protein-Protein Interactions
Examples of our research and methods include
Identifying proteins that inhibit apoptosis
Ubiquitination is a common post-translational modification of more than 5,000 proteins involved in a variety of cellular processes. The final step, catalyzed by E3 ubiquitin ligases, tags proteins with ubiquitin, which labels them for rapid destruction by protein complexes called proteasomes.
Apoptosis is the programmed self-destruction of aberrant cells that is crucial to normal health. It fails to occur in cancer and can occur too much, as in neurodegenerative diseases.
A subset of E3 ligases act as inhibitors of apoptosis proteins (IAPs), targeting certain caspases, cysteine proteases that carry out the destruction of cells, as well as caspases’ numerous protein allies, which act as systemic counterbalances, preventing IAPs from binding to caspases.
To better understand the protein networks involved in cellular apoptosis, researchers here use engineered enzymes to proximity tag proteins in order to identify IAP substrates.
Our applications of such protein engineering include
Labeling weak, transient protein interactions
Re-engineering an IAP ubiquitin ligase by genetically fusing its substrate-binding domain with another enzyme that instead covalently attaches a rare ubiquitin homolog, NEDD8. This allowed more than 50 IAP natural substrates to be identified and quantified by mass spectrometry.
More generally, the fused enzyme, dubbed the NEDDylator, demonstrated a robust method for labeling weak and transient protein-protein binding partners such as those in E3 substrate interactions in ubiquitination.