# Di-ubiquitin modification of ubiquitin ligase adaptors in membrane protein downregulation

> **NIH NIH R01** · CORNELL UNIVERSITY · 2022 · $410,689

## Abstract

PROJECT SUMMARY/ABSTRACT
 Post-translational modification by ubiquitin is an essential mechanism to alter protein function in
eukaryotes. Ubiquitin, a 76 amino acid protein, is attached to specific proteins via a cascade of ubiquitin
activating enzyme E1, conjugating enzyme E2, and ubiquitin ligase E3. Ubiquitination plays an essential
role in a broad aspect of cellular processes, including transcription, DNA repair, signal transduction,
autophagy, cell cycle, immune response, and membrane trafficking. Aberration in the ubiquitination
system leads to a number of human diseases, such as neurodegenerative diseases and cancers. Within
the ubiquitination cascade, E3s primarily dictate the specificity of the ubiquitination system and thus are
often the focal points of research and attractive therapeutic targets. The Nedd4 family E3s are an essential
family of HECT-type E3s, members of which contain an N-terminal C2 domain followed by 2-4 WW
domains and the C-terminal HECT domain. Nedd4 E3s recognize substrates carrying a “PPxY” motif
through their WW domains. However, most substrates lack such a motif but engage with the ligase through
“PPxY” motif-containing adaptors. We recently discovered that in yeast, the ubiquitin E3 ligase adaptor
protein Art1 is primed with di-ubiquitination and the attachment of the di-Ub chain to a specific lysine
residue in Art1 is warranted for its full activity. In this proposal, we plan to investigate the physiological
function of adaptor di-ubiquitination and to elucidate the molecular mechanisms of the modular
ubiquitination platform form with Nedd4 E3 ligase and di-ubiquitinated adaptors. Specifically, we will
pursue the following aims: Aim 1: To investigate the mechanism of Nedd4 E3 adaptor di-ubiquitination.
Aim 2: To determine the role of di-ubiquitinated adaptor-E3 complexes in substrate ubiquitination. Aim 3:
To elucidate the molecular architecture of di-ubiquitinated adaptors with the Nedd4 E3 ligases. Uncovering
the physiological role of Need4 E3 adaptors di-ubiquitination will be of critical importance to understand
the molecular basis of how E3 adaptors specifically recognize substrate proteins and efficiently present
the substrates for ubiquitination by HECT E3 ligases. We expect the successful implementation of this
proposal will not only make significant contributions to the understanding of the molecular mechanisms
underlying the Nedd4 E3 ligase/adaptor mediated ubiquitination, but also shed light on the mechanism of
protein quality control governed by targeted ubiquitination.

## Key facts

- **NIH application ID:** 10521677
- **Project number:** 1R01GM144452-01A1
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** SCOTT D EMR
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $410,689
- **Award type:** 1
- **Project period:** 2022-08-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10521677

## Citation

> US National Institutes of Health, RePORTER application 10521677, Di-ubiquitin modification of ubiquitin ligase adaptors in membrane protein downregulation (1R01GM144452-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10521677. Licensed CC0.

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