Chemical Biology of the E3 Ubiqutin Ligase Nedd4

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Ubiquitylation is a covalent posttranslational modification that influences a tremendous number of normal and disease-related cellular functions. The neuronal precursor cell-expressed developmentally downregulated 4 enzyme (Nedd4) is the founding member of a family of HECT-type E3 ubiquitin ligases that regulate proteostasis in various conditions including cancers and neurodegenerative disorders. In addition to its importance in cancer, Nedd4 has also recently emerged as a significant regulator of a-synuclein proteotoxicity, and may be a target for treating synucleinopathies, neurodegenerative disorders characterized by the accumulation of aggregated forms of the protein a-synuclein in both neuronal and non-neuronal cells in the brain. NAB2 is an N- arylbenzimidazole small molecule discovered from phenotypic screening that reverses a-synuclein associated toxicity in cellular models of a-synuclein-associated PD, including ER-to-Golgi trafficking disruption. Recently we elucidated the mode of action of NAB2, finding that it bound tightly to the E3 ubiquitin ligase Nedd4 (Kdapp = 42 nM) and expands the substrate specificity of Nedd4. We determined that NAB2 binding to Nedd4 triggers the ubiquitylation of new substrates, such as the trafficking-associated proteins such as TFG, TRAPP1 subunits, and RER1. We hypothesize that Nedd4 signaling influences a new pathway that regulates trafficking processes in response to a-synuclein-associated toxicity. The proteoform state of Nedd4 that responds to a-synuclein toxicity and binds NAB2 is also not known. In addition, the location of the NAB2 binding site within Nedd4 also is not known. In this proposal we wish to test the hypothesis that NAB2 acts on Nedd4 alone or as ‘molecular glue’ to facilitate communication between Nedd4 and potential substrates. We wish to understand how NAB2 interacts with NEDD4, and how this interaction affords Nedd4 a gain of function activity to ubiquitin modifiy non PxY-containing substrates like TFG. If successful, these studies will better illuminate the role Nedd4 plays in proteotoxicity signaling. In addition, the degree to which Nedd4 activity can be modulated by small molecules is important, given the important role Nedd4 ubiquitination plays in cancer pathobiology and neurodegenerative disorders. These studies will hopefully add to our fundamental knowledge of the role that E3 ligases play in proteotoxicity signaling, and may inspire the future discovery and development of therapeutics that may address some of the causes of synucleinopathies.