# Regulation by post-translation modifications in response to stress

> **NIH NIH R35** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2023 · $736,661

## Abstract

Abstract
Over a thousand human genes have dedicated roles in the ubiquitin pathway, making it one of the most
complex signaling systems. About 650 of these genes encode ubiquitin ligases (E3s), which recognize
substrates and target them for degradation. Our laboratory has developed several methods, based on both cell
biological and affinity approaches, to identify substrates of ubiquitin ligases, and have used these to identify
over two dozen substrates of five ligases. However, like phosphorylation, in many cases loss of ubiquitination
has no obvious phenotype. We have used the R35 mechanism to completely re-configure ourselves into a
mammalian cell laboratory and undertaken a large screen to identify phenotypes for poorly understood human
E3s/DUBs. We created a CRISPR library against human E3s/DUBs and performed a pooled CRISPR-Cas9
screen combined with chemical inhibition of 41 compounds targeting genome integrity, cell cycle progression,
transcription, RNA processing, translation, mitochondrial function, protein folding, metabolic pathways,
transport, cytoskeleton, etc. By probing a diverse set of biological processes for E3/DUB involvement, we
were able to assess the specificity of these interactions. Overall, we identified one or more specific interactions
for 161 E3/DUBs (about 25% of those examined), many of which were previously unstudied. Some genes,
such as FBXW7, showed interactions with more than one-third of the compounds. Others showed interactions
only with a single compound or a set of related compounds. We are focusing our efforts on following up four
sets of ligases: mutants in the poorly studied RING ligase RNF25 were extremely sensitive to alkylating
agents, but not other forms of DNA damage; mutants in the unstudied CUL5 adaptor WSB2 were exquisitely
sensitive to inhibitors of nuclear export; the CUL4 adaptor DCAF7 has a role in maintaining viability during a
CDK4/6 arrest; and the poorly-characterized F-box protein FBXO42 has clear roles in mitosis. We will use both
genetic screens and our established mass spec approaches to identify the relevant substrates of these ligases.
In addition, we will continue with our analysis of ubiquitin linkages. We have carried out a large genetic
interaction screen in yeast to identify roles of individual ubiquitin linkages. We will expand upon this screen and
will carry out complementary mass spectrometry approaches to identify relevant substrates.

## Key facts

- **NIH application ID:** 10609884
- **Project number:** 5R35GM118104-08
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** David Paul Toczyski
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $736,661
- **Award type:** 5
- **Project period:** 2016-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10609884

## Citation

> US National Institutes of Health, RePORTER application 10609884, Regulation by post-translation modifications in response to stress (5R35GM118104-08). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10609884. Licensed CC0.

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