Autophagy as a cell-repair mechanism: activation of chaperone-mediated autophagy during oxidative stress

Proper removal of oxidized proteins is an important determinant of success when evaluating the ability of cells to handle oxidative stress. The ubiquitin/proteasome system has been considered the main responsible mechanism for the removal of oxidized proteins, as it can discriminate between normal and altered proteins, and selectively target the latter ones for degradation. A possible role for lysosomes, the other major intracellular proteolytic system, in the removal of oxidized proteins has been often refused, mostly on the basis of the lack of selectivity of this system. Although most of the degradation of intracellular components in lysosomes (autophagy) takes place through “in bulk” sequestration of complete cytosolic regions, selective targeting of proteins to lysosomes for their degradation is also possible via what is known as chaperone-mediated autophagy (CMA). In this work, we review recent evidence supporting the participation of CMA in the clearance of oxidized proteins in the forefront of the cellular response to oxidative stress. The consequences of an impairment in CMA activity, observed during aging and in some age-related disorders, are also discussed.     

Chaperone-mediated autophagy and aging: a novel regulatory role of lipids revealed

A wide pool of cytosolic proteins is selectively degraded in lysosomes by chaperonemediated autophagy (CMA). Binding of these proteins to a receptor at the lysosomal membrane is the limiting step in CMA. Levels of this receptor are tightly regulated through changes in its degradation, multimeric organization and dynamic distribution between the lysosomal membrane and lumen. We have now reported that subcompartmentalization of the receptor in discrete lipid microdomains at the lysosomal membrane regulates its engagement in each of these processes-degradation, multimerization and membrane retrieval. Changes in the lipid composition of the membrane thus affect the dynamics of the receptor and, consequently, CMA activity. As an example of CMA dysfunction resulting from perturbation of the lipid composition of the lysosomal membrane, we discuss here a second study in which we analyzed the changes in the dynamics of the receptor during aging. CMA activity decreases with age primarily due to a decrease in the levels of the CMA receptor at the lysosomal membrane. Now we have found that age-related alterations in the lipid composition of the discrete microdomains at the lysosomal membrane are behind the reduced lysosomal levels of the receptor and, consequently, the declined CMA activity that occurs during aging.     

Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor

Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of unknown physiological function. We now demonstrate that this protease activity triggers the degradation of the lysosome-associated membrane protein type 2a (lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of lamp2a is important because its level in the lysosomal membrane is a rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a degradation, higher levels of lamp2a and higher rates of CMA. Restoration of PPCA protease activity increases rates of lamp2a degradation, reduces levels of lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the lysosomal membrane and cleaves lamp2a near the boundary between the luminal and transmembrane domains. In addition to the well-studied role of PPCA in targeting and protecting two lysosomal glycosidases, we have defined a role for the proteolytic activity of this multifunctional protein.     

Chaperone-Mediated Autophagy and Aging: A Novel Regulatory Role of Lipids Revealed

A wide pool of cytosolic proteins is selectively degraded in lysosomes by chaperone-mediated autophagy (CMA). Binding of these proteins to a receptor at the lysosomal membrane is the limiting step in CMA. Levels of this receptor are tightly regulated through changes in its degradation, multimeric organization and dynamic distribution between the lysosomal membrane and lumen. We have now reported that subcompartmentalization of the receptor in discrete lipid microdomains at the lysosomal membrane regulates its engagement in each of these processes — degradation, multimerization and membrane retrieval. Changes in the lipid composition of the membrane thus affect the dynamics of the receptor and, consequently, CMA activity. As an example of CMA dysfunction resulting from perturbation of the lipid composition of the lysosomal membrane, we discuss here a second study in which we analyzed the changes in the dynamics of the receptor during aging. CMA activity decreases with age primarily due to a decrease in the levels of the CMA receptor at the lysosomal membrane. Now we have found that age-related alterations in the lipid composition of the discrete microdomains at the lysosomal membrane are behind the reduced lysosomal levels of the receptor and, consequently, the declined CMA activity that occurs during aging.

Addendum to:

Altered Dynamics of the Lysosomal Receptor for Chaperone-Mediated Autophagy with Age

R. Kiffin, S. Kaushik, M. Zeng, U. Bandyopadhyay, C. Zhang, A.C. Massey, M. Martinez-Vicente and A.M. Cuervo

J Cell Sci 2007; 120:782-91

and

Lysosome Membrane Lipid Microdomains: Novel Regulators of Chaperone-Mediated Autophagy

S. Kaushik, A.C. Massey and A.M. Cuervo

EMBO J 2006; 25:3921-33     

Chaperone‐mediated autophagy is defective in mucolipidosis type IV

Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin‐1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two‐hybrid and co‐immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone‐mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex‐ and age‐matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal‐associated membrane protein type 2A (LAMP‐2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly‐Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV. J. Cell. Physiol. 219: 344–353, 2009. © 2008 Wiley‐Liss, Inc.     

Dietary lipids and aging compromise chaperone-mediated autophagy by similar mechanisms

Chaperone-mediated autophagy (CMA) is a selective form of autophagy whose distinctive feature is the fact that substrate proteins are translocated directly from the cytosol across the lysosomal membrane for degradation inside lysosomes. CMA substrates are cytosolic proteins bearing a pentapeptide motif in their sequence that, when recognized by the cytosolic chaperone HSPA8/HSC70, targets them to the surface of the lysosomes. Once there, substrate proteins bind to the lysosome-associated membrane protein type 2 isoform A (LAMP2A), inducing assembly of this receptor protein into a higher molecular weight protein complex that is used by the substrate proteins to reach the lysosomal lumen. CMA is constitutively active in most cells but it is maximally activated under conditions of stress.     

Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function

Chaperone-mediated autophagy (CMA), a selective mechanism for degradation of cytosolic proteins in lysosomes, contributes to the removal of altered proteins as part of the cellular quality-control systems. We have previously found that CMA activity declines in aged organisms and have proposed that this failure in cellular clearance could contribute to the accumulation of altered proteins, the abnormal cellular homeostasis and, eventually, the functional loss characteristic of aged organisms. To determine whether these negative features of aging can be prevented by maintaining efficient autophagic activity until late in life, in this work we have corrected the CMA defect in aged rodents. We have generated a double transgenic mouse model in which the amount of the lysosomal receptor for CMA, previously shown to decrease in abundance with age, can be modulated. We have analyzed in this model the consequences of preventing the age-dependent decrease in receptor abundance in aged rodents at the cellular and organ levels. We show here that CMA activity is maintained until advanced ages if the decrease in the receptor abundance is prevented and that preservation of autophagic activity is associated with lower intracellular accumulation of damaged proteins, better ability to handle protein damage and improved organ function.     

Dietary lipids and aging compromise chaperone-mediated autophagy by similar mechanisms

Chaperone-mediated autophagy (CMA) is a selective form of autophagy whose distinctive feature is the fact that substrate proteins are translocated directly from the cytosol across the lysosomal membrane for degradation inside lysosomes. CMA substrates are cytosolic proteins bearing a pentapeptide motif in their sequence that, when recognized by the cytosolic chaperone HSPA8/HSC70, targets them to the surface of the lysosomes. Once there, substrate proteins bind to the lysosome-associated membrane protein type 2 isoform A (LAMP2A), inducing assembly of this receptor protein into a higher molecular weight protein complex that is used by the substrate proteins to reach the lysosomal lumen. CMA is constitutively active in most cells but it is maximally activated under conditions of stress.     

Regulation of lamp2a levels in the lysosomal membrane

The selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy depends, at least in part, on the levels of a substrate receptor at the lysosomal membrane. We have previously identified this receptor as the lysosome-associated membrane protein type 2a (lamp2a) and showed that levels of lamp2a at the lysosomal membrane directly correlate with the activity of the proteolytic pathway. Here we show that levels of lamp2a at the lysosomal membrane are mainly controlled by changes in its half-life and its distribution between the lysosomal membrane and the matrix. The lysosomal degradation of lamp2a requires the combined action of at least two different proteolytic activities at the lysosomal membrane. Lamp2a is released from the membrane by the action of these proteases, and then the truncated lamp2a is rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is a regulated process that is inhibited in the presence of substrates for chaperone-mediated autophagy and under conditions that activate that type of autophagy. Uptake of substrate proteins also results in transport of some intact lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can be reinserted back into the lysosomal membrane. The traffic of lamp2a through the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion requires the lysosomal membrane potential and protein components of the lysosomal membrane. The distribution of lamp2a between the lysosomal membrane and matrix is a dynamic process that contributes to the regulation of lysosomal membrane levels of lamp2a and consequently to the activity of the chaperone-mediated autophagic pathway.     

Loss of hepatic chaperone‐mediated autophagy accelerates proteostasis failure in aging

Chaperone‐mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver‐specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.     

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.     

Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance

Chaperone-mediated autophagy (CMA) is a selective lysosomal pathway for the degradation of cytosolic proteins. We review in this work some of the recent findings on this pathway regarding the molecular mechanisms that contribute to substrate targeting, binding and translocation across the lysosomal membrane. We have placed particular emphasis on the critical role that changes in the lipid composition of the lysosomal membrane play in the regulation of CMA, as well as the modulatory effect of other novel CMA components. In the second part of this review, we describe the physiological relevance of CMA and its role as one of the cellular mechanisms involved in the response to stress. Changes with age in CMA activity and the contribution of failure of CMA to the phenotype of aging and to the pathogenesis of several age-related pathologies are also described.     

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.     

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.     

分子伴侣介导的细胞自噬作用机制及其功能

分子伴侣介导的细胞自噬(chaperone-mediated autophagy,CMA)是通过溶酶体途径选择性降解胞质中带KFERQ-序列的蛋白质。CMA不仅为细胞在持久饥饿状态下提供能量,还在氧化性损伤保护、维持细胞内环境稳态等方面发挥作用。此外,CMA功能障碍还与某些疾病的发生有关。该文简要综述了这方面的研究进展。     

Ketone bodies stimulate chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective lysosomal protein degradative process that is activated in higher organisms under conditions of prolonged starvation and in cell culture by the removal of serum. Ketone bodies are comprised of three compounds (beta-hydroxybutyrate, acetoacetate, and acetone) that circulate during starvation, especially during prolonged starvation. Here we have investigated the hypothesis that ketone bodies induce CMA. We found that physiological concentrations of beta-hydroxybutyrate (BOH) induced proteolysis in cells maintained in media with serum and without serum; however, acetoacetate only induced proteolysis in cells maintained in media with serum. Lysosomes isolated from BOH-treated cells displayed an increased ability to degrade both glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, substrates for CMA. Isolated lysosomes from cells maintained in media without serum also demonstrated an increased ability to degrade glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A when the reaction was supplemented with BOH. Such treatment did not affect the levels of lysosome-associated membrane protein 2a or lysosomal heat shock cognate protein of 70 kDa, two rate-limiting proteins in CMA. However, pretreatment of glyceraldehyde-3-phosphate and ribonuclease A with BOH increased their rate of degradation by isolated lysosomes. Lysosomes pretreated with BOH showed no increase in proteolysis, suggesting that BOH acts on the substrates to increase their rates of proteolysis. Using OxyBlot analysis to detect carbonyl formation on proteins, one common marker of protein oxidation, we showed that treatment of substrates with BOH increased their oxidation. Neither glycerol, another compound that increases in circulation during prolonged starvation, nor butanol or butanone, compounds closely related to BOH, had an effect on CMA. The induction of CMA by ketone bodies may provide an important physiological mechanism for the activation of CMA during prolonged starvation.