Novel functional assay for proteases and modulators. Application in beta-secretase studies

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Proteases are crucial for cellular functions and are implicated in various diseases.
• Secretases generating amyloid beta peptide are key targets for Alzheimer's disease research.
• Identifying and characterizing specific proteases remains a significant challenge.

Purpose of the Study:

• To develop and validate a novel functional approach for identifying proteases and their modulators.
• To demonstrate the proof of principle using beta-secretase as a model protease.
• To establish a method coupling protease activity to caspase-mediated apoptosis.

Main Methods:

• A modified caspase-3 construct incorporating a beta-secretase cleavage site was engineered.
• The construct was expressed in 293T cells to induce apoptosis.
• The effect of beta-secretase inhibitors (BACE2 and BACE2(D110A)) on cell death was assessed.

Main Results:

• A modified caspase-3 with a beta-secretase cleavage site successfully induced apoptosis in 293T cells.
• The induced apoptosis directly correlated with the susceptibility of the recognition sequence to beta-secretase.
• In vivo beta-secretase inhibitors BACE2 and BACE2(D110A) effectively prevented the cell death.

Conclusions:

• The developed functional approach provides a viable method for identifying proteases based on known recognition sequences.
• This technique can be a valuable tool for studying proteolytic cleavage and discovering protease inhibitors.
• The study validates the coupling of protease activity to caspase-mediated apoptosis for functional screening.