Development and Characterization of Degron-Based Substrates Capable of E3 Ligase-Mediated Ubiquitination

Tuesday, October 18, 2011: 1:54 PM
L100 E (Minneapolis Convention Center)
Adam Melvin, Marcey Waters and Nancy Allbritton, Chemistry, Univeristy of North Carolina, Chapel Hill, NC

The ubiquitin-proteasome pathway is responsible for the degradation of misfolded, damaged, or tightly regulated proteins.   A protein is marked for degradation by the transfer, and subsequent polymerization, of the small molecule ubiquitin.  Ubiquitination is governed by a series of enzymatic steps culminating in the recognition of a specific protein by the E3 ubiquitin ligases.  An E3 ligase can target a protein through the identification of a specific amino acid degradation sequence, or degron.  Improper regulation of the ubiquitin-proteasome pathway has been implicated in the development of cancers such as multiple myeloma and acute myeloid leukemia.  In fact, targeting this pathway is one of the leading directions in cancer chemotherapeutics; however there are no clinically available biochemical assays that measure protein ubiquitination and proteasome activity in single primary tumor cells before and after treatment.  In this study we sought to design and characterize a series of reporter substrates, based on degrons, capable of intracellular ubiquitination, as a first step in the generation of single cell reporters of E3 ligase and proteasome-targeted therapeutics.

Using solid phase peptide synthesis (SPPS), we developed small (~25-30 amino acids) reporters consisting of four essential components: the E3-ligase specific degron, a proximal lysine residue to be ubiquitinated, a linker to avoid steric hindrance issues between the lysine and the degron, and a fluorescein tag to serve as a visual marker.  The degrons were selected across a wide array of cellular functions from protein targets associated with various cancers including β-Catenin, SRC3, iNOS, HIF-1α, TAZ and others.  Reporter ubiquitination is being studied in a HeLa S100 cytosolic lysate-based assay, a system consisting of a wide range of E3 ligases and an ideal choice for studying an array of potential targets.  The S100-based assay is being optimized for a well studied degron, β-Catenin, by determining the proper assay concentrations and components.  We will report the extent of ubiquitination of all reporters using gel and capillary electrophoresis (CE), effectively separating the reporters into 'bins' based on the extent of ubiquitination, calculated based on ubiquitinated reporter relative to parent reporter.  Our intent is that these results will provide the groundwork for the development of a ubiquitinatable substrate, which we will then incorporate into a more complex, single-cell reporter of E3 ligase and proteasome-targeted therapeutics.

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