# Expanding Mechanistic Insights into Protein Ubiquitylation

> **NIH NIH R35** · UNIVERSITY OF WASHINGTON · 2024 · $612,984

## Abstract

PROJECT SUMMARY
Covalent attachment of ubiquitin (Ub) to other proteins is among the most widespread and
diverse modes of eukaryotic cellular regulation. The modification occurs on practically every
protein in a cell at some point in its lifetime and is itself highly diverse. The type of ubiquitylation
determines a product’s fate and a protein may undergo different modes of ubiquitylation
depending on cellular circumstances. The origins of this diversity stem from the protein
machinery responsible for Ub attachment. A trio of enzymes, E1, E2, and E3 coordinate the
process, with several E1s, dozens of E2s, and many hundreds of E3s encoded in the human
genome. Over the past 20 years, we have asked fundamental questions about how E2s and
E3s work and have contributed to the structural, biochemical, and mechanistic understandings
of the field. Our work began with the breast cancer tumor suppressor, BRCA1/BARD1 that was
among the earliest RING-type E3 ligases to be identified. Over the years, we have expanded to
study numerous E3s and E2s, making many unexpected discoveries along the way.
The wide reach of protein ubiquitylation in cellular function means that dysfunction of
components is associated with myriad human diseases and developmental issues. Such
associations make the Ub system attractive for therapeutic targeting. Direct targeting of the
ubiquitylation machinery as well as efforts to re-engineer protein ubiquitylation machinery to
selectively target a specific cellular protein are both proving to be powerful strategies. Such
translational efforts rely implicitly on mechanistic understanding and reveal the power of well-
grounded structure/function research. Despite the apparent maturity of the field, there is still
much we do not understand at a fundamental level. We do not know the full range of
biochemical reactions carried out by the ~30 human E2s as fully one-quarter of these are
uncharacterized. Existing data reveal that not all E2s carry out the presumed reaction that
attaches Ub to lysine sidechains, implying the existence of ubiquitylated species that have yet to
be investigated in cells. Second, understanding of how E2/E3s carry out mono-ubiquitylation is
lacking. Unlike poly-ubiquitylation, attachment of a single Ub (mono-Ub) tends to occur in a site-
selective manner implying that substrates to be mono-ubiquitylated are handled differently from
those destined to have chains built upon them. Third, lack of knowledge regarding how mono-
Ub attachment affects the structure and function of proteins limits understanding of how the
modification regulates critical cellular processes including transcription, translation, and DNA
damage response, among others.

## Key facts

- **NIH application ID:** 10877803
- **Project number:** 5R35GM144127-03
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Rachel E Klevit
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $612,984
- **Award type:** 5
- **Project period:** 2022-08-15 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10877803

## Citation

> US National Institutes of Health, RePORTER application 10877803, Expanding Mechanistic Insights into Protein Ubiquitylation (5R35GM144127-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10877803. Licensed CC0.

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