(72c) Development and Characterization of Degron-Based Substrates Capable of E3 Ligase-Mediated Ubiquitination

Authors: 
Melvin, A., Univeristy of North Carolina
Allbritton, N., Univeristy of North Carolina
Waters, M., Univeristy of North Carolina


The ubiquitin proteasome system (UPS) is responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in the cell.  Deregulation of this critical process is the hallmark of many different forms of cancer leading to failed tumor suppression, loss of cell cycle control, and an increased metastatic response.  The specificity of this pathway comes from the >600 E3 ubiquitin ligases, each of which recognizes a specific sequence (termed a ‘degron’) on a specific protein to undergo ubiquitination.  To gain a better understanding of protein ubiquitination and E3 ligase-degron kinetics, we sought to generate and test a library of substrates based on known degrons.  In this study, we demonstrate the utility of the degron-based substrates, the variability in E3 ubiquitin ligase performance, and, ultimately identify substrates that could serve as potential reporters for E3 ubiquitin ligase-targeted chemotherapeutics.

Using solid phase peptide synthesis (SPPS), we generated small (~25-30 amino acids) substrates consisting of a degron, a proximal lysine, and a fluorescein tag.  The degrons were selected across a wide array of cellular functions from proteins associated with various cancers including β-Catenin, iNOS, TAZ, HIF-1α, p53, and others.  Substrate ubiquitination was evaluated by an in vitro HeLa S100 cytosolic-based ubiquitin pull down followed by gel electrophoresis.  We discovered that each of the nine degron-based substrates was ubiquitinated in our in vitro system, although the extent of ubiquitination varied across the library, suggesting a hierarchy of degrons could be established.  Additionally, using a methylated ubiquitin (effectively preventing ubiquitin chain formation), we found that our substrates could be mono-, di-, and tri-ubiquitinated.  To understand this observation and quantify substrate performance, we constructed a mathematical model, based on first order reaction kinetics, to evaluate kinetic rate constants for mono-, di-, and tri- substrate ubiquitination.  Our model indicates a lack of fidelity by E3 ubiquitin ligases when it comes to lysine preference proximal to the degron.  The kinetic rates constants also identified two substrates, β-Catenin and Bonger, which exhibited rapid ubiquitination when compared with the rest of the field.  The robustness of these substrates was then tested in lysates from multiple myeloma (OPM-2 and MM1.S) and leukemia (U937, THP-1, HL-60, and K562) cell lines.  Our degron-based substrates provide a robust, easy to synthesize, and highly useful tool in the study of the ubiquitin proteasome system and demonstrate a promising new technology that could be used as a reporting tool for chemotherapeutics designed to specifically target the UPS.