A Fluorogenic-Based Assay to Measure Chaperone-Mediated Autophagic Activity in Cells and Tissues

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Medicine

Background:

• Misfolded and damaged proteins accumulate in various pathologies, including cardiovascular diseases, cancer, and neurological disorders.
• Intracellular protein degradation pathways, particularly chaperone-mediated autophagy (CMA), are vital for clearing these disease-associated proteins.
• Current methods assess CMA by measuring LAMP2A protein expression, which does not reflect actual degradation activity.

Approach:

• Synthesized a novel fluorogenic substrate, KFERQ-AMC, for specific detection of CMA activity.
• Utilized lysosomal protease inhibitor E64D to confirm substrate degradation via lysosomes.
• Validated assay specificity using negative control peptides and confirmed LAMP2A dependence in HEK and H9c2 cells.

Key Points:

• The KFERQ-AMC assay demonstrated time-dependent AMC fluorescence release, indicating CMA-mediated substrate cleavage.
• Liver tissue showed significantly higher CMA activity compared to kidney and heart tissues.
• Assay results were validated by inhibition with E64D and dependence on LAMP2A expression in cellular models.

Conclusions:

• A novel, specific fluorogenic assay for measuring chaperone-mediated autophagy (CMA) activity has been successfully developed.
• This assay provides a direct measure of CMA degradation function, overcoming limitations of previous protein expression-based methods.
• The assay is applicable across various experimental contexts for assessing CMA activity in cells and tissues.