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

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.