Versatile and cost-effective assay to accurately monitor steady-state CMA and flux in cells and tissues.

Many pathologies such as cardiovascular disease, cancer, and neurological disorders are caused by the accumulation of misfolded proteins. Intracellular protein degradation mechanisms play a critical role in maintaining a balance between protein synthesis and degradation, and are thus integral to normal cellular function and health. Chaperone-mediated autophagy (CMA) is a protein degradation pathway that selectively degrades specific substrate proteins, including those that are mutant or damaged. A major limitation to measuring CMA function is the lack of assays or tools to monitor CMA flux, the rate of autophagic degradation.

Researchers at the University of Utah have developed and validated a fluorescence-based assay to measure steady-state CMA as well as CMA flux. Flux is an important indicator of how effectively the CMA pathway can degrade the substrate proteins.

To assess CMA function, KFERQ-AMC, a fluorogenic CMA substrate comprised of a small peptide attached to an AMC group, is cleaved via lysosomal hydrolysis causing AMC to release and fluoresce. The amount of AMC cleavage directly corresponds to CMA activity in the samples. Flux is determined by treating a parallel set of the same samples with a lysosomal inhibitor that blocks KFERQ-AMC degradation. The protocol can be readily adapted to identify different CMA activators/inhibitors as potential drugs for the prevention or treatment of a variety of pathologies.

Publication

• As a complement to steady-state CMA, monitoring CMA flux provides information on autophagic activity over time.

• Safe and efficient compared to radioactivity and microscopy-based assays: no radiation hazard, long counting times, lag, or high waste disposal costs.

• Does not rely on live cell/animal models – can be performed on fresh and frozen cells and tissues, including those of human origin.

Jonnavithula A et al. A Fluorogenic-Based Assay to Measure Chaperone-Mediated Autophagic Activity in Cells and Tissues. BioRxiv 571785 [Preprint]. December 15, 2023. Available from: https://doi.org/10.1101/2023.12.14.571785.

Lucia Irazabal
lu.irazabal@utah.edu