Live-cell imaging of ubiquitin-proteasome system function

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• The ubiquitin-proteasome system (UPS) is crucial for maintaining protein homeostasis.
• Dysregulation of the UPS is implicated in various diseases, driving the need for functional assays.
• Existing assays often lack the sensitivity to detect subtle changes caused by specific protein interactions or inhibitors.

Purpose of the Study:

• To develop novel assays for quantifying ubiquitin-proteasome system (UPS) function.
• To enable the measurement of UPS activity in response to alterations in protein expression, localization, or aggregation.
• To provide tools for screening chemical and protein UPS inhibitors.

Main Methods:

• Utilized cell lines stably expressing fluorescent UPS reporter substrates.
• Transfected cells with a second protein, tagged with a compatible fluorophore.
• Developed protocols to correlate changes in UPS substrate levels with the properties of the co-expressed protein.

Main Results:

• Demonstrated the ability to measure changes in UPS reporter levels.
• Correlated UPS substrate levels with alterations in the expression, localization, and aggregation state of a second protein.
• Established a quantitative link between protein properties and UPS functional output.

Conclusions:

• The described protocols provide a robust method for assessing UPS function.
• These assays are valuable for studying protein homeostasis and identifying UPS modulators.
• The methodology supports the development of therapeutics targeting UPS-related diseases.