Description:
Intracellular pools of unconjugated ubiquitin must be regulated for the proper functions of virtually all ubiquitin-dependent signaling pathways. To date, however, only a few methods to measure unconjugated ubiquitin have been described. Typically, these methods have low precision or are difficult to implement, and none are amenable to real-time assays or can be used in living cells. To improve upon this, we developed binding proteins with high affinities and selectivity for free (i.e., unconjugated) ubiquitin. Our strategy was to engineer chimeric proteins assembled from multiple Ubiquitin Binding Domains (UBDs) that bind to distinct, non-overlapping surfaces on monoubiquitin.
Ubiqutitin’s functions depend on its covalent attachment ("conjugation") to other proteins. Despite its importance and abundance, precise measurement of the amounts of free (i.e., unconjugated) ubiquitin is difficult and, for most laboratories, not technically feasible. We have designed and produced proteins that can bind to free ubiquitin with very high affinity and specificity. Using derivatives of these binding proteins and fluorescent derivatives of ubiquitin, we have developed sensitive assays for free ubiquitin based on fluorescence anisotropy or fluorescence resonance energy transfer measurements. These assays allow measurement of ubiquitin in cell or tissue extracts, and can be configured to provide a general, real-time assay for deubiquitination enzymes. With respect to ease of implementation, precision, or specificity, other approaches described previously to assay ubiquitin are inferior to our new methods
This invention affords a practical means to measure concentrations of unconjugated ubiquitin in vitro. Approaches developed that use our invention are amenable to assays that employ complex cell or tissue extracts. These methods will be of interest to many academic and commercial research laboratories that study ubiquitin-dependent signaling processes. Several companies that currently market proteins and other reagents for ubiquitin-pathway research are likely to be interested in our sensor proteins and the associated assay methods.
Key advantages:
- Simple, practical assay for unconjugated ubiquitin to evaluate effects of proteasome inhibitors and other ubiquitin pathway inhibitors
- Provides a general method for assaying deubiqutinating enzymes
- Further development for sensors and assays for ubiquitin like proteins, such as Nedd8
