Covalent attachment of ubiquitin-like proteins (UBLs) such as ubiquitin (Ub), NEDD8, and SUMO is a predominant form of eukaryotic protein regulation. UBLs modify a vast number of proteins, altering their functions in a variety of ways. UBL modifications can affect the target's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. As a result, UBLs regulate numerous biological processes, such as the cell cycle, signal transduction, apoptosis, the immune response, autophagy, and development. Defects in 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 UBL conjugation to the two largest E3 families: RING (Really Interesting New Gene - 570 predicted in humans) and HECT (Homologous to E6AP C-Terminus - 28 predicted in humans). 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 a NEDD8 E2, and the cullin subunit is activated by self-modification with NEDD8. Then a CRL binds a Ub-loaded E2, which is the source of Ub to be transferred to a target. HECT E3s utilize a distinct mechanism, in which a HECT domain catalytic Cys participates directly via a thioester-linked intermediate. First, the HECT domain binds a thioester-linked E2~Ub complex, and Ub is transferred from the E2 Cys to the HECT domain catalytic Cys. Ultimately Ub is transferred from the HECT E3 Cys to a target or Ub Lys. We propose a research plan focused on structural biology and biochemistry to understand mechanisms underlying functions of CRL E3s (Aim 1) and HECT E3s (Aim 2).

Public Health Relevance

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 viral and retroviral infections. The recent approval of the proteasome inhibitor Bortezomib (VelcadeTM) for treatment of multiple myeloma 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 mechanisms by which enzymes transfer UBLs 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 their roles in diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Gerratana, Barbara
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St. Jude Children's Research Hospital
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Scott, Daniel C; Sviderskiy, Vladislav O; Monda, Julie K et al. (2014) Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8. Cell 157:1671-84
Brown, Nicholas G; Watson, Edmond R; Weissmann, Florian et al. (2014) Mechanism of polyubiquitination by human anaphase-promoting complex: RING repurposing for ubiquitin chain assembly. Mol Cell 56:246-60
Li, Guoqiang; Ci, Weimin; Karmakar, Subhradip et al. (2014) SPOP promotes tumorigenesis by acting as a key regulatory hub in kidney cancer. Cancer Cell 25:455-68
Kamadurai, Hari B; Qiu, Yu; Deng, Alan et al. (2013) Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3. Elife 2:e00828
Kelsall, Ian R; Duda, David M; Olszewski, Jennifer L et al. (2013) TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes. EMBO J 32:2848-60
Frye, Jeremiah J; Brown, Nicholas G; Petzold, Georg et al. (2013) Electron microscopy structure of human APC/C(CDH1)-EMI1 reveals multimodal mechanism of E3 ligase shutdown. Nat Struct Mol Biol 20:827-35
Duda, David M; Olszewski, Jennifer L; Schuermann, Jonathan P et al. (2013) Structure of HHARI, a RING-IBR-RING ubiquitin ligase: autoinhibition of an Ariadne-family E3 and insights into ligation mechanism. Structure 21:1030-41
Monda, Julie K; Scott, Daniel C; Miller, Darcie J et al. (2013) Structural conservation of distinctive N-terminal acetylation-dependent interactions across a family of mammalian NEDD8 ligation enzymes. Structure 21:42-53
Werner, Achim; Disanza, Andrea; Reifenberger, Nina et al. (2013) SCFFbxw5 mediates transient degradation of actin remodeller Eps8 to allow proper mitotic progression. Nat Cell Biol 15:179-88
Tardiff, Daniel F; Jui, Nathan T; Khurana, Vikram et al. (2013) Yeast reveal a "druggable" Rsp5/Nedd4 network that ameliorates ?-synuclein toxicity in neurons. Science 342:979-83

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