A fluorescence-based assay to measure chaperone-mediated autophagy in cell and tissue lysates

Intracellular proteolytic mechanisms are important in clearing aberrant proteins causal for different pathologies including cardiovascular diseases, cancer, and neurological disorders. Chaperone-mediated autophagy (CMA) is a protein degradation pathway that employs chaperones to bind proteins bearing a unique KFERQ (Lys-Phe-Asp-Arg-Gln)-like motif and delivers them to lysosomes for degradation via a CMA-specific receptor, namely, Lysosome Associated Membrane Protein 2A (LAMP2A). Several assays are available to monitor CMA function. However, most of these methods are laborious, expensive and cannot be performed on frozen samples. We have developed a straightforward and inexpensive fluorogenic assay to assess CMA activity in both fresh and frozen samples. The CMA substrate, KFERQ-AMC (7-amino-4-methylcoumarin), was synthesized by Solid-Phase Peptide Synthesis. Mechanistically, AMC fluorescence remains quenched when the fluorophore is conjugated to KFERQ. Upon lysosomal hydrolysis of KFERQ-AMC, free AMC is released, and its fluorescence levels serve as an indicator for CMA activity. Lysate from fresh or frozen mouse tissue and cells when incubated with the KFERQ-AMC peptide displayed a time-dependent increase in CMA activity (0-15 hours for tissues and 0-6 hours for cells). KFERQ-AMC fluorescence was markedly reduced in cultured cells following transfection with Lamp2a siRNA, indicating suppression of CMA activity. In contrast, overexpression of Lamp2a via adenoviral delivery enhanced CMA and promoted KFERQ-AMC degradation. Notably, inhibition of proteasomal or macroautophagic pathways had no effect on KFERQ-AMC degradation, underscoring the assay's specificity for CMA. This novel fluorescence-based approach provides a valuable tool for elucidating the role of CMA across diverse experimental systems.