# Non-canonical phosphoribosyl ubiquitination and de-ubiquitination by legionella effectors

> **NIH NIH R01** · CORNELL UNIVERSITY · 2021 · $320,920

## Abstract

PROJECT SUMMARY/ABSTRACT
 Ubiquitin (Ub), a 76 amino acid protein, is attached to specific proteins via a cascade of Ub activating
enzyme E1, conjugating enzyme E2, and Ub ligase E3. Ubiquitination plays an essential role in a broad
aspect of cellular processes. Aberrations in the ubiquitination system lead to a number of human diseases,
such as neurodegenerative diseases and cancers.
 A non-canonical ubiquitination pathway that acts independently of E1 and E2 enzymes was discovered
recently. The SidE family effectors from the intracellular bacterial pathogen Legionella pneumophila were
found to ubiquitinate substrates on serine residues in the presence of co-factor NAD+. This unusual SidE-
catalyzed ubiquitination involves two steps of reactions catalyzed by its mono-ADP-ribosyl transferase
(mART) and phosphodiesterase (PDE) domains, respectively. The first step is the generation of mono-
ADP-ribosylated Ub (ADPR-Ub), in which, SidE uses its mART domain to catalyze the transfer of ADP-
ribose from NAD+ to the Arg42 residue of Ub. In the second step of reaction, ADPR-Ub is conjugated to a
serine residue of substrate proteins via the PDE domain to generate serine ubiquitinated products with
the releasing of AMP.
 Our recent structural and biochemical studies, as well as results from other groups, have shed light
on the molecular mechanism underlying this novel phosphoribosyl-linked serine ubiquitination (PR-
ubiquitination). However, key questions remain unaddressed, For example: How can the mART domain
specifically recognize Ub and ADP-ribosylate the Arg42 residue of Ub? Are there any deubiquitinating
enzymes (DUBs) that can specifically de-conjugate PR-ubiquitinated species similar to the DUBs in the
canonical ubiquitination pathway? What are the specific targets of SidE family PR-Ub ligases? The
overarching goal of this proposal is to elucidate the mechanism of this novel Ub-dependent
posttranslational modification and to explore the role of PR-ubiquitination in hijacking eukaryotic cellular
processes. Specifically, we will pursue the following three aims: Aim 1: To delineate the molecular
mechanism of PR-ubiquitination mediated by SidE family effectors. Aim 2: To identify and elucidate the
mechanism of DUBs specific for PR-ubiquitinated conjugates. Aim 3: To determine the cellular targets of
SidE family effectors and their roles in the remodeling of Legionella-containing vacuoles. We expect these
exploratory studies of SidE family PR-Ub ligases will shed light on the molecular mechanism of this novel
type of posttranslational modification. More importantly, our proposed studies will also pave the way to
investigate a potential PR-ubiquitination pathway in eukaryotic species.

## Key facts

- **NIH application ID:** 10141265
- **Project number:** 5R01GM135379-02
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Yuxin Mao
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $320,920
- **Award type:** 5
- **Project period:** 2020-04-08 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10141265

## Citation

> US National Institutes of Health, RePORTER application 10141265, Non-canonical phosphoribosyl ubiquitination and de-ubiquitination by legionella effectors (5R01GM135379-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10141265. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*