Activation of Chaperone-mediated Autophagy during Oxidative Stress

Abstract

Oxidatively damaged proteins accumulate with age in almost all cell types and tissues. The activity of chaperonemediated autophagy (CMA), a selective pathway for the degradation of cytosolic proteins in lysosomes, decreases with age. We have analyzed the possible participation of CMA in the removal of oxidized proteins in rat liver and cultured mouse fibroblasts. Added to the fact that CMA substrates, when oxidized, are more efficiently internalized into lysosomes, we have found a constitutive activation of CMA during oxidative stress. Oxidation-induced activation of CMA correlates with higher levels of several components of the lysosomal translocation complex, but in particular of the lumenal chaperone, required for substrate uptake, and of the lysosomal membrane protein (lamp) type 2a, previously identified as a receptor for this pathway. In contrast with the well characterized mechanism of CMA activation during nutritional stress, which does not require de novo synthesis of the receptor, oxidation-induced activation of CMA is attained through transcriptional up-regulation of lamp2a. We conclude that CMA is activated during oxidative stress and that the higher activity of this pathway under these conditions, along with the higher susceptibility of the oxidized proteins to be taken up by lysosomes, both contribute to the efficient removal of oxidized proteins. INTRODUCTIONAccumulation of oxidized protein is a common feature of aged cells (Dunlop et al., 2002;Grune et al., 2001Grune et al., , 2002bStadtman, 2001). Many physiological and pathological processes lead to the generation of free radicals and consequent damage of intracellular components, including proteins. In most of these oxidative events, damaged proteins are removed from the cell through degradation by proteases (Dunlop et al., 2002). The activity of different intracellular proteolytic systems decreases with age (Terman, 1995;Cuervo and Dice, 1998a;Friguet et al., 2000;Carrard et al., 2002;Donati et al., 2001;Merker et al., 2001;Friguet, 2002;Keller et al., 2002;Ward, 2002), and this impaired activity is considered responsible for the deficient removal of oxidized proteins in old organisms (Grune, 2000;Merker and Grune, 2000;Dunlop et al., 2002;Szweda et al., 2002).The susceptibility of oxidized proteins to proteases changes with the duration and degree of oxidative damage (Dunlop et al., 2002;Mehlhase and Grune, 2002). Thus, mild oxidation induces partial protein unfolding and facilitates proteolytic cleavage (Grune et al., 1995. However, persistent or extensive oxidative damage usually promotes protein aggregation, due to the exposure of patches of hydrophobic amino acids. Once a protein aggregates, it becomes less susceptible to proteolytic cleavage (Hoff et al., 1993;Davies, 2001;Demasi and Davies, 2003). Kinetics of degradation of oxidized proteins in vitro have been analyzed using different types of proteases (Merker and Grune, 2000;Dunlop et al., 2002;Szweda et al., 2002). One of the most extensively analyzed protease in this respect has be...

Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A

“…Knockout of Lamp2a or Nfe2l2 makes cells more susceptible to different stressors [8,32], indicating a crucial role in the maintenance of cell homeostasis under different circumstances. In fact, previous work supports the notion that redox status tightly controls CMA activity, presumably to eliminate proteins oxidized during mild oxidative damage [14]. Interestingly, CMA induction upon oxidative stress appears to occur through increased Lamp2a transcription [14], whereas CMA activation by nutrient deprivation relies on diminished degradation of LAMP2A and relocation of this receptor at the lysosomal membrane [33], pointing to the existence of several mechanisms of CMA regulation.…”

“…However, a more intense and diffuse fluorescent pattern throughout the cytoplasm, together with an extremely reduced number of Dendra-positive puncta per cell, were observed in nfe2l2 -KO cells, consistent with impaired basal CMA activity (the KFERQ-PS-Dendra reporter is not being properly degraded by CMA and accumulates in the cytosol). When cells were treated for 16 h with PQ, a well-characterized stimulus for CMA [14], we found a significant increase in Dendra-positive puncta per cell in Nfe2l2 -WT but not nfe2l2 -KO hepatocytes, indicating impaired induction of CMA by PQ in the absence of NFE2L2 (Figures 4g and 4h). …”

“…Activation of CMA is associated with increased levels of LAMP2A and its multimerization to form membrane translocation complexes, lysosomal enrichment in HSPA8 and relocation of lysosomes to the perinuclear region [13–15]. It has been reported that a mild oxidant environment activates the expression of Lamp2a [14] and that this process is under the transcriptional regulation of NFAT in T cells [16]. However, a generic mechanism that might regulate the expression of LAMP2A under these circumstances remains unknown.…”

“…Thus, we identified many NFE2L2-regulated genes involved in the different phases of the macroautophagy process, from cargo recognition to autolysosome clearance [22]. Considering this evidence and the role of CMA in the cellular response to oxidative stress [14], it might be feasible that NFE2L2 also participates in the regulation of CMA, but this possibility has not yet been investigated.…”

Interplay of pathogenic forms of human tau with different autophagic pathways

“…Sources of reagents and chemicals were as described before (Kiffin et al ., 2004; Massey et al ., 2006; Bandyopadhyay et al ., 2010). The antibody against mouse LAMP‐2A was developed in our laboratory (Cuervo & Dice, 1996).…”