Imaging proteolytic activity in live cells and animal models

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Proteases regulate crucial cellular processes, including signal transduction and apoptosis.
• Dysregulation of protease activity is implicated in various diseases like cancer and inflammation.
• Existing methods for studying protease activity have limitations in live-cell and in vivo applications.

Purpose of the Study:

• To develop and characterize a novel luciferase biosensor for quantifying protease activity in real-time.
• To demonstrate the utility of the biosensor in studying apoptosis regulation and screening for pharmacologically active compounds.
• To enable the investigation of protease roles in both cellular and whole-animal models of disease.

Main Methods:

• Development of a luciferase biosensor (GloSensor Caspase 3/7) for detecting proteolytic activity.
• Validation of the biosensor's signal-to-noise ratio and dynamic range.
• Application of the biosensor in high-throughput screening campaigns and in genetically engineered mouse models.

Main Results:

• The GloSensor Caspase 3/7 biosensor exhibits a robust signal-to-noise ratio (50-100 fold) and dynamic range.
• The biosensor successfully quantifies protease activity in live cells and mouse models.
• Demonstrated utility in studying caspase activity related to apoptosis and in screening for drug candidates.

Conclusions:

• The developed luciferase biosensor provides a sensitive and versatile tool for studying protease function.
• This technology facilitates research into protease-mediated diseases and the discovery of novel therapeutics.
• The biosensor design principles can be extended to study other proteases involved in health and disease.