E1-E2-E3 enzyme-mediated covalent attachment of ubiquitin (Ub) or ubiquitin-like proteins (Ubls) such NEDD8 is a predominant form of eukaryotic protein regulation. Ubls modify a vast number of proteins and alter their functions in a variety of ways. For example, Ub/Ubl modifications can affect the target protein's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. As a result, Ub/Ubls regulate numerous biological processes, such as the cell cycle, signal transduction, apoptosis, the immune response, autophagy, and development. Defects in Ub/Ubl pathways are widely associated with diseases, including cancers, developmental disorders, high blood pressure, neurodegenerative disorders, and cachexia. We propose to extend our expertise on Ub/Ubl pathways to mechanisms of ligation by the three largest E3 families: HECT (Homologous to E6AP C-Terminus - 28 predicted in humans), RING (Really Interesting New Gene - 570 predicted in humans) and RBR (RING1-IBR-RING2 - 13 predicted in humans). HECT and RBR E3s participate directly in catalysis, with a catalytic cysteine that forms a covalent intermediate through thioester-linkage with Ub's C-terminus via a 2-step reaction. First, the HECT or RBR E3 binds a thioester-linked E2~Ub intermediate, and Ub is transferred from the E2 catalytic cysteine to an E3 catalytic cysteine. Second, Ub is transferred from the E3 Cys to a target lysine. By contrast, RING E3s promote transfer of Ub or a Ubl from from an E2~Ub/Ubl intermediate to an associated substrate. Among the RING E3s, the largest class consists of the modular, multisubunit Cullin-RING (CRL) family. CRLs function sequentially with distinct E2s to modify distinct targets: first the RING domain binds an E2~NEDD8 intermediate, and the cullin subunit is activated by self-modification with NEDD8. Then a NEDD8-modifed CRL binds a Ub-loaded E2, which is the source of Ub to be transferred to a target. Ultimately, for all three classes of E3, repeated cycles through their enzymatic cascades can lead to polyubiquitination, with specific enzymes selectively linking a donor Ub's C-terminus to distinctive lysines on the acceptor Ub's surface. We propose a research plan focused on structural biology and biochemistry to understand mechanisms underlying Ub/Ubl ligation, determining target specificity, and modulating functions of HECT (Aim 1), CRL (Aim 2), and RBR E3s (Aim 3).
Ubiquitin-like protein (Ubl) conjugation regulates many biological processes, including cell division, the immune response, development and signal transduction, and defects in Ubl pathways have been widely associated with cancers, neurodegenerative disorders, developmental disorders, heart diseases (e.g. high blood pressure), and pathogenic infections. The recent approval of the proteasome inhibitor Bortezomib (VelcadeTM) for treatment of multiple myeloma, and current clinical trials of agents targeting other ubiquitin and Ubl pathways, underscores the therapeutic potential for targeting enzymes in the ubiquitin, and Ubl, pathways, and highlights the importance of understanding the detailed mechanisms and specificities of these enzymes. Thus, we anticipate that knowledge of the structures and mechanisms by which enzymes Ub and Ubls are transferred to specific substrate lysines as revealed by the proposed studies will be of broad significance to many human diseases, much like studies of protein kinases have influenced our knowledge of signaling pathways and how to target them therapeutically.
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