# Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity

> **NIH NIH R01** · GEORGIA STATE UNIVERSITY · 2020 · $4,172

## Abstract

Project Summary / Abstract
 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 ...

## Key facts

- **NIH application ID:** 10146131
- **Project number:** 3R01GM104498-07S2
- **Recipient organization:** GEORGIA STATE UNIVERSITY
- **Principal Investigator:** HIROAKI KIYOKAWA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $4,172
- **Award type:** 3
- **Project period:** 2013-01-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10146131

## Citation

> US National Institutes of Health, RePORTER application 10146131, Orthogonal Ubiquitin Transfer to Profile E3 Substrate Specificity (3R01GM104498-07S2). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10146131. Licensed CC0.

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