# A chemical strategy to unravel tumor suppression through the ubiquitin pathway

> **NIH NIH R01** · TUFTS UNIVERSITY MEDFORD · 2021 · $295,800

## Abstract

PROJECT SUMMARY
Much remains unknown about the network that controls the ubiquitin-dependent regulation of the most
frequently inactivated protein in all human cancer: the tumor suppressor p53. A complete understanding
remains elusive because current methods are unable to fully unravel how ubiquitin is led through the sequence
of E1-E2-E3 enzymes to its final target. Mounting evidence suggests that E2s have a tremendous impact on
both E3 ligase activity and the resulting ubiquitinated products. One gap in our knowledge is the identity of the
E2 ubiquitin-conjugating enzyme (or enzymes) responsible for p53 ubiquitination in vivo. Given the
combinatorial nature of interactions between ~40 human E2s and >600 E3s, this network is extraordinarily
difficult to study. As a result, there is a striking need for tools that can follow ubiquitin through the enzymatic
cascade to its target protein. The long-term goal of this proposal is to isolate and examine the network of
protein-protein interactions that regulate p53 ubiquitination in living cells. This goal will be accomplished using
a new method developed in the applicant’s lab, called targeted Charging of Ubiquitin to E2 (tCUbE), that can
track a tagged ubiquitin as it moves from an E2 enzyme to its target protein. This strategy is unique in its ability
to follow ubiquitin through the sequential E1-E2-E3 cascade to its ultimate target, and can therefore be used to
provide a systems-level view of the ubiquitin-dependent regulation of p53.
This application focuses on the questions surrounding the ubiquitin-dependent regulation of p53 activity. In Aim
1, tCUbE is used to discover the E2 network responsible for p53 ubiquitination in vivo. This method will identify
the E2(s) that lead to p53 ubiquitination in vivo, and will enable experiments in which this network is perturbed
using known molecules that inhibit p53 ubiquitination by the E3 ligases Mdm2 or MdmX. These studies will
validate tCUbE, identify previously unknown interactions that guide p53 ubiquitination, and evaluate their
impact on cancer phenotype. In Aim 2, tCUbE is used to interrogate a unique mode of p53 rescue mediated by
an E2, UbcH7, which has been shown to protect p53 from degradation in certain cell types. Using tCUbE, it will
be possible to evaluate the hypothesis that UbcH7 catalyzes the attachment different types of ubiquitin chains
on substrates, like p53, that are protected from degradation. Finally, in Aim 3, tCUbE is combined with variants
of ubiquitin that are resistant to deubiquitinase activity in order to profile the effects of deubiquitinases on the
regulation of p53 function. These studies will test the hypothesis that distinct deubiquitinases disassemble
specific ubiquitinated p53-species, and will evaluate how p53 activity can be differentially controlled through
this mechanism. This approach is unmatched in its ability to illuminate the subset of ubiquitinated products that
arise from the activity of a single E2 in livin...

## Key facts

- **NIH application ID:** 10213787
- **Project number:** 5R01GM134097-03
- **Recipient organization:** TUFTS UNIVERSITY MEDFORD
- **Principal Investigator:** Rebecca Scheck
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $295,800
- **Award type:** 5
- **Project period:** 2019-08-05 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10213787

## Citation

> US National Institutes of Health, RePORTER application 10213787, A chemical strategy to unravel tumor suppression through the ubiquitin pathway (5R01GM134097-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10213787. Licensed CC0.

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