Probing protein ubiquitination in live cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Protein ubiquitination is a crucial post-translational modification regulating protein function, interactions, and degradation.
• Investigating ubiquitination typically requires complex biochemical methods.
• A need exists for assays that can probe ubiquitination dynamics within living cells.

Purpose of the Study:

• To develop and validate a novel assay for studying protein ubiquitination in real-time within living cells.
• To identify novel ubiquitination targets and regulators using this assay.
• To demonstrate the assay's utility in monitoring drug effects on ubiquitination pathways.

Main Methods:

• Development of the ubiquitin fluorescent three-hybrid (ubiF3H) assay using recombinant binding domains and fluorescently tagged proteins.
• Immobilization of GFP-fused proteins and detection of ubiquitination using red fluorescent ubiquitin binders.
• Implementation of fluorescence complementation (ubiF3Hc) with split YFP to enhance signal-to-noise ratio.
• Utilizing linkage-specific ubiquitin binding domains to differentiate K48 and K63 ubiquitination.

Main Results:

• The ubiF3H assay successfully identified HP1β as a novel ubiquitination target of UHRF1.
• HP1β ubiquitination by UHRF1 was observed primarily during S phase and deubiquitination by USP7.
• The ubiF3Hc assay directly detected p53 ubiquitination and its inhibition by the anti-cancer drug Nutlin-3.

Conclusions:

• The ubiF3H assay provides a powerful tool for investigating protein ubiquitination in living cells.
• This assay facilitates the identification of specific protein ubiquitination targets, ligases, and proteases.
• The developed assay is valuable for screening small molecules and drugs that modulate ubiquitination processes, with applications in cancer research.