Development and application of novel inhibitors of deubiquitinating enzymes

The function of proteins is regulated by posttranslational modifications. One type of posttranslational modification is ubiquitination, the attachment of ubiquitin (Ub) to other proteins. Polyubiquitinated proteins are degraded, and proper ubiquitination is essential for cell cycle progression and other cellular functions. Ubiquitination is reversible. Ubiquitin is removed by a large family of deubiquitinating enzymes. Deubiquitinating enzymes function in cell growth and differentiation, gene silencing, endocytosis and cancer progression. The study of deubiquitinating enzymes is of great interest. I developed active sited-directed probes for deubiquitinating enzymes to measure the activity of multiple enzymes in a crude system.; I designed active site-directed probes consisting of a ubiquitin with a C-terminal electrophilic group and a visualization/retrieval element. The electrophilic group traps the active site cysteine of a deubiquitinating enzyme. While the visualization/retrieval element permits detection of modified enzymes and their identification. I demonstrated that Ub-derived probes are specific for deubiquitinating enzymes. I then used Ub-derived probes to identify the deubiquitinating enzymes present in a mouse EL4 cell line.; Individual deubiquitinating enzymes can also be studied using Ub-derived probes. I showed that mouse USP14 associates with the 26S proteasome, a protease that degrades ubiquitinated proteins. Using Ub-derived probes, I demonstrated that only proteasome-bound USP14 has activity. Furthermore, the activity of USP14 is increased following proteasome inhibition. These observations suggest the activities of the proteasome and USP14 are coupled.; I examined the binding of Ubp6 (a yeast homolog of USP14) to purified subcomplexes of the 26S proteasome. Ubp6 binds to the base of the 19S regulatory particle of the proteasome and the ubiquitin-like domain of Ubp6 is essential for binding. A 300-fold activation of Ubp6 by binding to the 19S regulatory complex was observed. Activation of Ubp6/USP14 by binding the proteasome is the first example of a deubiquitinating enzyme whose activity is regulated by association with a binding partner and may be directed to a particular set of substrates. Taken together, this work describes the development of new tools to study deubiquitinating enzymes and underscores the importance of measuring the activity of deubiquitinating enzymes in the context of their physiological binding partners.