| Description |
Misfolded and damaged proteins are usually checked by protein degradation pathways which are essential to maintain proteostasis. Chaperone-mediated autophagy (CMA) is a protein degradation pathway unique to mammalian cells. CMA uses protein type 2a (LAMP2A) receptors, which are specific to CMA, to target proteins that contain a KFERQ-like motif for lysosomal degradation. LAMP2A protein expression has been used previously to assess CMA function. In this project, we have established a fluorescent-based CMA activity assay to measure CMA in cells and tissues. We first extract intact lysosomes from adult mouse heart, liver, and kidney and cultured mouse embryonic fibroblasts. We then aim to demonstrate the use of KFERQ-AMC (Lys-Phe-Asp-Arg-Gln-AMC) fluorogenic substrate to measure CMA activity. AMC when attached to the substrate remains in the quenched state and does not fluoresce. The cleavage of KFERQ-AMC due to lysosomal hydrolysis causes free AMC to release which is excited at 355 nm and fluoresces at 460 nm. By measuring the cleavage, we can measure the levels of CMA activity. To determine the flux, we used another set of samples treated with E64D, (L-trans- Epoxy-succinyl-leucylamido(4-guanidino)butane), a cathepsin inhibitor, which would serve as controls. Until now, we found that mouse liver shows the highest CMA activity (6-fold) followed by kidney (2.4-fold) and heart (0.3-fold) at 5 hours. The levels of KFERQ-AMC degradation in the different tissues positively correlated to the LAMP2A protein expression. We also found that KFERQ motif is specific for CMA degradation, as the negative control peptides showed no fluorescence. Further KFERQ-AMC cleavage was blocked by E64D treatment, which allowed us to measure CMA flux. We believe that the CMA activity assay would allow us to dynamically monitor CMA flux in a host of cells and tissues and help to identify and validate various CMA activators as potential drugs for the prevention or treatment of different pathologies. |
