Tumor Necrosis Factor (TNF)-family cytokine receptors, Toll-like Receptors (TLR), and other types of receptors involved in the recognition of pathogen-associated molecular patterns transduce their signals into cells via adapter proteins known as Tumor Necrosis Factor Receptor-Associated Factors (TRAFs). The TRAFs are a family of adapter proteins that bind an unusual ubiquitin-conjugating enzyme, Ubc13, which produces polyubiquitin chains linked at lysine 63 of ubiquitin rather than the canonical lysine 48. These lysine 63-linked ubiquitin polymers trigger changes in protein activity, rather than targeting proteins for proteasomal degradation. Ubiquitination by Ubc13 of TRAFs and the various protein kinases to which TRAFs bind is recognized as a critical step in signaling by TNFRs, TLRs, NLRs, and T-cell and B-cell antigen receptors (TCR/BCR) during innate and acquired immune responses. Since aberrant signaling by these receptor systems is linked to a wide variety of autoimmune, inflammatory, and infectious diseases, compounds that neutralize Ubc13 may prove useful as a novel type of immunosuppressive or anti-inflammatory agent. The goal of this proposal is to generate chemical inhibitors of Ubc13 for use as research tools for studying the role of lysine63-linked polyubiquitination in signal transduction, and as potential proof of concept reagents for exploring the suitability of Ubc13 as a possible drug target in animal models of autoimmunity and inflammation. To this end, we have devised a high throughput screening (HTS) assay that measures the biochemical activity of Ubc13 in vitro, based on the principal of time-resolved fluorescence resonance energy transfer (TR-FRET) using terbium-conjugated Ubiquitin as a donor and fluorescein-conjugated Ubiquitin as an acceptor. The HTS assay has been optimized for 384 well plate, with z' > 0.7. Importantly, Ubc13's activity is dependent on a cofactor protein Uev1a. The 3D- structure of the Ubc13-Uve1a complex suggests that the binding interface can potentially be disrupted by small molecule compounds, thus providing two sites at which to attack Ubc13: (a) active site; and (b) Uve1a interface. Downstream secondary assays have been devised that will assist with validation and characterization of hits.
Ubc13 is an enzyme that is required for the function of multiple mediators of inflammation and immunity. This proposal seeks to produce chemical inhibitors of Ubc13, which will be useful research tools for understanding more about the role of Ubc13 in immune cell function. These chemical inhibitors may also provide a starting point for developing future medicines aimed at ameliorating a wide variety of acute and chronic inflammatory and autoimmune disorders, including sepsis, inflammatory bowel diseases, arthritis, lupus, and organ rejection. ? ? ?
| Madiraju, Charitha; Welsh, Kate; Cuddy, Michael P et al. (2012) TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors. J Biomol Screen 17:163-76 |
