Ubiquitin (UB) transfer through the E1-E2-E3 cascade mediates signal transduction in almost all aspects of cell biology. E3 UB ligases catalyze UB transfer from E2s to the substrate proteins and ultimately decide the targets, location, and timing of the ubiquitination reaction. Identifying the substrate proteins is the key to elucidating the biological functions of an E3. However, its has been a significant challenge to profile E3 substrate specificity because of the transient formation of E3-substrate complex and the cross-reactivity among various E3s. Our long-term goal is to elucidate the important roles of E3-catalyzed protein ubiquitination in cell biology and diseases. Working toward this goal, we developed a method that we called ?orthogonal UB transfer (OUT)? to identify the direct ubiquitination targets of an E3. In this innovative method, an engineered UB (xUB) is exclusively transferred through an engineered cascade of xE1-xE2-xE3 to the substrates of a specific E3 (?x? designates engineered enzymes free of cross creativities with native partners in the cell). We have constructed OUT cascades of HECT E3 E6AP and U-box E3 E4B and CHIP to profile their substrate specificities and validated OUT as an efficient platform to identify E3 substrates. The objective of this application is to generate OUT cascades with Ring E3s Cbl-b and Parkin to identify their substrate proteins. We will also follow the leads in the substrate profiles of E6AP and CHIP generated by OUT to establish the roles of these E3s in cell cycle and cancer cell invasion. Our central hypothesis is that the OUT cascades of E3s can identify new regulatory relationships between the E3 and substrates and elucidate the roles of E3 in cell signaling. Such a hypothesis is supported by our strong preliminary data demonstrating the feasibility of OUT in profiling the ubiquitination targets of E6AP, E4B, and CHIP. The rationale of our proposed work is that the 600 Ring E3s in the cell used a highly homologous Ring domain to bind to E2 and mediate UB transfer from E2 to their substrate proteins. Once we develop OUT platform with Ring E3s Cbl-b and Parkin, we can use similar protein engineering strategies to build the OUT platform for other Ring E3s to reveal their biological functions. We will pursue three specific aims: 1) Extending the OUT cascades to Ring E3s Cbl-b and Parkin to profile their substrate proteins; 2) Extending the OUT cascade to BRUCE to investigate its role in cytokinesis; 3) Studying the function of E6AP and CHIP based on their substrate profile generated by OUT. The expected outcome of our work is the development of the OUT platform to profile ubiquitination targets of all class of E3s including HECT, U-box and Ring types. Based on the knowledge of their substrate specificity, we will elucidate the role of Cbl-b in regulating NK cell activity against cancer cells, reveal the mechanism of Parkin in tumor suppression, and define the role the BRUCE in cytokinesis. We will establish new cellular circuits mediated by E6AP and CHIP in cell cycle control and cancer cell invasion. The positive impact of our work is that we will develop the OUT platform for other researchers to plug in their E3s of interest and map the E3s on the cell signaling network by profiling their substrate specificity.

Public Health Relevance

The proposed research is relevant to public health because the malfunctions of protein modification by ubiquitin often break down key regulatory circuits in the cell and lead to the development of cancer, neurodegeneration, and autoimmune diseases. Our work will build an efficient platform to elucidate the pathways of ubiquitin transfer in the cell. Such platform is relevant to the NIH?s mission in that it will help to reveal the cause of diseases and guide the design of effective therapies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Barski, Oleg
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Georgia State University
Schools of Arts and Sciences
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Jin, Bo; Wang, Jiayue; Liu, Xiangnan et al. (2018) Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes. Biomed Res Int 2018:6062520
Sane, Sanam; Hafner, Andre; Srinivasan, Rekha et al. (2018) UBXN2A enhances CHIP-mediated proteasomal degradation of oncoprotein mortalin-2 in cancer cells. Mol Oncol 12:1753-1777
Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng et al. (2018) Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer. Sci Adv 4:e1701393
Liu, Xianpeng; Sun, Limin; Gursel, Demirkan B et al. (2017) The non-canonical ubiquitin activating enzyme UBA6 suppresses epithelial-mesenchymal transition of mammary epithelial cells. Oncotarget 8:87480-87493
Wang, Yiyang; Liu, Xianpeng; Zhou, Li et al. (2017) Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer. Nat Commun 8:2232
Liu, Xianpeng; Zhao, Bo; Sun, Limin et al. (2017) Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes. Nat Commun 8:14286
Yin, Jun; Shen, Shihao (2016) Challenges and Innovations in Phase I Dose-Finding Designs for Molecularly Targeted Agents and Cancer Immunotherapies. J Biom Biostat 7:
Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang et al. (2015) Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities. Methods Mol Biol 1319:245-60
Zhao, Bo; Zhang, Keya; Bhuripanyo, Karan et al. (2015) Phage selection assisted by Sfp phosphopantetheinyl transferase-catalyzed site-specific protein labeling. Methods Mol Biol 1266:161-70
Zhao, Bo; Villhauer, Eric B; Bhuripanyo, Karan et al. (2014) SUMO-mimicking peptides inhibiting protein SUMOylation. Chembiochem 15:2662-6

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