Rates of protein degradation decrease with age, contributing to the cytosolic accumulation of altered proteins in old cells. We have identified a decline with age in chaperone-mediated autophagy (CMA), one of the mechanisms responsible for the degradation of both normal and damaged cytosolic proteins inside lysosomes. CMA substrates are targeted by cytosolic chaperones to a receptor protein in the surface of the lysosomes. Assisted by an intralysosomal chaperone, substrates are then translocated and completely degraded by lysosomal proteases. We have found that substrate binding and uptake are the CMA steps primarily altered during aging, and have identified an age-related decrease in the levels of the lysosomal receptor. However, because restoration of normal receptor levels in senescent cells only recovers CMA activity partially, it is likely that other still unidentified CMA components, might also change with age and contribute to this impaired function. We will analyze age-related qualitative and quantitative changes in lysosomal membrane and matrix protein components and will identify the ones responsible for the declined CMA activity during aging. Because no single separation technology is applicable to all proteins, we will use a complementary dual approach to generate the CMA-related lysosome subproteome and to track changes with age in this subproteome: 1) differential two-dimensional electrophoresis of lysosome associated membrane proteins and of lysosomal matrix proteins isolated from different age rodents, and 2) multidimensional chromatography and mass spectrometry of trypsinized lysosomal membranes isolated from early and late passage fibroblasts, differentially labeled in culture with stable isotope- labeled amino acids (SlLAC). We have previously defined six different conditions, other than aging, resulting in changes in CMA activity. Therefore, we expect to be able to discriminate changes in the lysosomal components that may affect CMA activity from the other age-related changes. Changes altering CMA activity will be the target of future corrective interventions aimed to restore normal lysosomal activity in aging and to thus, facilitate the elimination of abnormal proteins accumulated in old tissues.
