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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

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Published on: May 10, 2015

A cell-permeable Ub-Dha probe for profiling E1-E2-E3 enzymes in live cells.

Qiong Xia¹, Xianbin Meng², Yu Wang¹

¹School of Food and Biological Engineering, Engineering Research Center of Bio-process, Ministry of Education, Hefei University of Technology, Hefei 230009, China. ymli@hfut.edu.cn.

Chemical Communications (Cambridge, England)

|March 28, 2024

Summary

Researchers developed new cell-penetrating ubiquitin probes (Ub-ABPs) for studying E1-E2-E3 enzyme activity in live cells. These probes enable tracking ubiquitin enzyme function within intact cells, overcoming previous limitations of cell lysate studies.

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Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Published on: September 20, 2016

Area of Science:

Biochemistry
Cell Biology
Chemical Biology

Background:

Activity-based ubiquitin probes (Ub-ABPs) are crucial for studying E1-E2-E3 enzyme functions.
Existing Ub-ABPs are often limited to cell lysates, hindering in vivo studies.
There is a need for tools that allow real-time monitoring of ubiquitin enzyme activity in live cells.

Purpose of the Study:

To develop the first cell-penetrating ubiquitin probes (Ub-Dha) for live-cell imaging and activity profiling.
To demonstrate the efficacy of these probes in delivering into cells and labeling target enzymes.
To identify and quantify active E1-E2-E3 enzymes in live cells using mass spectrometry.

Main Methods:

Design and synthesis of thiazolidine-protected cysteine-based ubiquitin probes (Ub-Dha).
Incorporation of an N-terminal fluorophore for visualization and confirmation of cellular delivery via live-cell fluorescence microscopy.
Application of Ub-Dha probes for labeling E1-E2-E3 enzymes in live cells, followed by enrichment and label-free quantification (LFQ) mass spectrometry.

Main Results:

Successful development and validation of cell-penetrating Ub-Dha probes.
Demonstrated efficient delivery of probes into live cells, confirmed by fluorescence microscopy.
Identified and quantified a total of 18 E1-E2-E3 enzymes in live cells, providing insights into their activity.
Established a novel method for studying ubiquitin enzyme activity and function in a native cellular environment.

Conclusions:

The novel cell-penetrating Ub-Dha probes represent a significant advancement in studying ubiquitin enzyme activity.
These probes overcome the limitations of cell lysate-based methods, enabling real-time analysis in live cells.
This technology offers new avenues for understanding the ubiquitin system's role in cellular processes and disease.