A Modular Biosensor Design for Quantitative Measurement of Free Nedd8.

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Nedd8 is a post-translational modifier crucial for cellular signaling, with dysregulation linked to various diseases.
• Accurate quantification of free Nedd8 is essential for understanding its role in health and disease, but current methods are lacking.
• Existing genetically encoded biosensor designs are not optimized for quantifying free Nedd8.

Purpose of the Study:

• To develop a novel genetically encoded biosensor for the direct quantification of free Nedd8.
• To establish a modular biosensor design strategy applicable to other protein targets.
• To enable real-time monitoring of Nedd8-related enzymatic activities.

Main Methods:

• Developed a modular biosensor design employing a target binding domain and two reporter domains.
• Created a selective binder for free Nedd8.
• Integrated the binder with fluorescent or split nanoluciferase reporters for signal generation.
• Applied the biosensor to monitor deneddylation activity in real-time.

Main Results:

• Successfully developed genetically encoded biosensors that provide quantifiable and specific signals for free Nedd8.
• Demonstrated real-time monitoring of deneddylation by the DEN1 enzyme using a physiological substrate.
• Validated the sensor's ability to detect changes in Nedd8 dynamics.

Conclusions:

• The developed modular biosensor design is effective for quantifying free Nedd8.
• This biosensor facilitates real-time studies of Nedd8 dynamics and deneddylation.
• The strategy holds potential for high-throughput screening of deneddylation inhibitors for cancer therapy and can be adapted for other proteins.