Chaperone-mediated autophagy: selectivity pays off

“Three different mechanisms for delivery of autophagic cargo to lysosomes co-exist in most mammalian cells: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) [6,8] (Box 1).”

“I bet you just pictured a garbage container, didn’t you? This organelle has been associated with cellular cleaning almost since its description more than 50 years ago by Christian de Duve [1].”

“Three different mechanisms for delivery of autophagic cargo to lysosomes co-exist in most mammalian cells: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) [6,8] (Box 1).”

“The use of blocking antibodies revealed that substrate proteins bind to the lysosomal membrane through the cytosolic tail of LAMP-2A [18].”

“Lastly, CMA is also likely to be altered in Danon’s disease, a type of vacuolar myopathy resulting from mutations in the LAMP2 gene [52].”

“For example, the alterations in macroautophagy and in lysosomal biogenesis observed in Lamp2 null mice [33] and after knocking-out or knocking-down all LAMP-2 isoforms in cultured cells [34,35], cannot be reproduced by blocking only LAMP-2A in cultured cells [25].”

“Alphasynuclein can be degraded by the ubiquitin proteasome system, CMA and macroautophagy [43,44].”

“those containing HSC70 in their lumen, through still unknown mechanisms [37].”

“It is likely that LAMP-2A participates in other cellular functions common to all LAMP-2 protein variants with which LAMP-2A shares a common luminal region [61].”

“Instead, pathogenic alpha-synucleins remain tightly bound to the CMA receptor, preventing the translocation of other CMA substrates into lysosomes [43,45].”

“A mutant cleaved form of Tau, the protein that organizes into toxic fibrillar structures in Alzheimer’s disease (AD), is also delivered to lysosomes via CMA, and similar to the abnormal alpha-synucleins, mutant Tau fails to translocate properly [47].”

“For example, some motifs located in the core of the protein will only be exposed upon unfolding [28]; other motifs placed in regions of protein/protein interaction will become accessible when the protein is no longer part of a complex [60]; lastly, in some enzymes the motifs are close to their catalytic site and only…”

“For most of these functions, HSC70 binds to stretches of hydrophobic residues in the substrates; however, to date, the KFERQ-like motif is the only region recognized by HSC70 in CMA substrates [12].”

“For example, RNA interference against LAMP-2A, which blocks CMA results in constitutive activation of macroautophagy and vice versa; however, both pathways are non-redundant, and the lack of the defective pathway becomes evident in response to an additional stressor [25,27].”

“Similarly, the age-dependent decline in CMA activity could be an aggravating factor in most of these neurodegenerative disorders [39].”

“We developed a bi-transgenic mouse model expressing an additional copy of LAMP-2A in liver that can be activated in middle life to supplement the reduced levels of endogenous protein [38].”

“The first function proposed for CMA was to provide an alternative source of amino acids under conditions of prolonged starvation [9,10,24].”

“HSC70 is a multifunctional chaperone that also contributes to the disassembly of clathrin from endocytic pits, folding of newly synthesized proteins, refolding of unfolded proteins and, as a partner of HSP90, to the stabilization of proteins in transient unstable conformations [14].”

“For example, RNA interference against LAMP-2A, which blocks CMA results in constitutive activation of macroautophagy and vice versa; however, both pathways are non-redundant, and the lack of the defective pathway becomes evident in response to an additional stressor [25,27].”

“Some recent studies propose that the carboxyl terminus of HSP70-interacting protein (CHIP), another cytosolic chaperone that contributes to the degradation of cytosolic proteins by the proteasome, might also target proteins for lysosomal degradation [15].”

“In professional antigen presenting cells, CMA participates in the presentation of cytoplasmic peptides by class II molecules contributing to organism autoimmunity [30].”

“Recently, malfunctioning of CMA has been connected to a growing number of human disorders [42].”

“Blocking antibodies against luminal HSC70 prevented substrate translocation, supporting that this chaperone might act by “pulling” the substrates inside the lysosomal lumen as they cross the membrane [22].”

“Formation of the limiting membrane also requires the interaction between a protein and a specific lipid molecule (phosphatidyl ethanolamine), also regulated by conjugating enzymes [7].”

“Furthermore, not only pathogenic forms of alphasynuclein, but even high levels (overexpression) of the wild type form of this protein has recently been found to hinder the degradation of neuronal proteins by CMA [31].”

“Luminal HSC70 is necessary for CMA, and in fact, the subset of lysosomes able to perform CMA is often identified in cells by the presence of HSC70 in their lumen [23].”

“Conditions associated with enhanced CMA always require high LAMP-2A levels, which can be attained by transcriptional upregulation of the Lamp2 gene [28], but more often through changes in the degradation and lysosomal distribution of LAMP-2A [19].”

“Numerous reviews have summarized the recent findings on the coordinated orchestration of macroautophagy, the mechanisms that regulate this process, and its important and growing number of physiological functions [5-8].”

“For example, the alterations in macroautophagy and in lysosomal biogenesis observed in Lamp2 null mice [33] and after knocking-out or knocking-down all LAMP-2 isoforms in cultured cells [34,35], cannot be reproduced by blocking only LAMP-2A in cultured cells [25].”

“Tracking of individual proteins undergoing selective lysosomal degradation and analyzing their sequences led to the identification of both a targeting motif in all of the substrates for this pathway and a chaperone able to recognize this motif [11,12].”

“Chimeras of CMA substrates with a protein unable to unfold can bind to the lysosomal membrane but do not translocate, suggesting that unfolding occurs after binding but before translocation [17].”

“The connections between CMA and PD have further expanded with the discovery that mutant forms of the ubiquitin carboxyl-terminal esterase L1 (UCHL-1), another pathogenic protein in PD, interact abnormally with LAMP-2A leading to CMA blockage [46].”

“Enhanced recruitment of LAMP-2A to these areas of degradation at the lysosomal membrane reduces levels of this receptor and downregulates CMA [21].”

“Instead, the stability of the CMA receptor is markedly compromised once it reaches the lysosome in old organisms because a large percentage of lysosomal LAMP-2A remains in the lysosomal lumen where it undergoes massive unregulated degradation [40].”

“In contrast to macro- and microautophagy, delivery of cargo via CMA does not require formation of intermediate vesicle compartments, membrane fusion or membrane deformity of any kind [57,58].”

“Degradation is initiated through dual cleavage by a pair of lysosomal proteases, cathepsin A and a yet unidentified metalloprotease, followed by the release of the truncated form of LAMP-2A in the lumen and its complete degradation by resident hydrolases [50].”

“Macroautophagy is activated in most cells during the first hours of starvation, reaches its maximum activity at about 6 hours and then gradually declines [25,26].”

“Interestingly, increased levels of oxidized proteins might also contribute to keeping CMA upregulated during prolonged starvation, because the increased levels of circulating ketone bodies in these conditions promotes protein oxidation [29].”

“A recent study reported abnormal upregulation of macroautophagy in mice with accelerated aging that might contribute to their systemic degeneration [41].”

“For example, some motifs located in the core of the protein will only be exposed upon unfolding [28]; other motifs placed in regions of protein/protein interaction will become accessible when the protein is no longer part of a complex [60]; lastly, in some enzymes the motifs are close to their catalytic site and only…”

“The observed changes in CMA activity in the affected cells could be secondary to the changes in pH; however, because their lysosomes contain lower amounts of cytosolic chaperones, it is also possible that TRPML1 might participate in lysosomal docking of CMA chaperones [51].”

“Early studies in cultured primary fibroblasts revealed that the pool of cytosolic proteins whose lysosomal degradation was accelerated upon serum removal was degraded at slower rates as cells become senescent [36].”

“protein also implicated in the pathogenesis of this disease, undergoes CMA [48].”

“Decreased CMA activity under these conditions leads to high levels of the nuclear transcription factor Pax2, essential in regulating cellular growth in the kidney [54].”

“Modulation of kidney growth is a specialized function attributed to CMA through the degradation of a specific protein, the transcription factor Pax2, in renal tubular cells [32].”

“Most of the early studies on autophagy were performed in rat liver, tracking the degradation of the soluble pool of cytosolic protein or measuring changes in the size and volume of the lysosomal system [2-4].”

“The first function proposed for CMA was to provide an alternative source of amino acids under conditions of prolonged starvation [9,10,24].”

“To date, only one membrane protein, the epidermal growth factor receptor, has been proposed to undergo lysosomal degradation in a manner dependent on the receptor for CMA [59]; however, the requirement for protein unfolding, essential to define this process as CMA, has not yet been determined for the epidermal growth…”

“For example, the alterations in macroautophagy and in lysosomal biogenesis observed in Lamp2 null mice [33] and after knocking-out or knocking-down all LAMP-2 isoforms in cultured cells [34,35], cannot be reproduced by blocking only LAMP-2A in cultured cells [25].”

“Lysosomal storage disorders (LSD) are a group of genetic diseases in which the lack of enzymatic activity in lysosomes leads to massive accumulation of the substrate of that enzyme, resulting in lysosomal engorgement [49].”

“To date, three autophagy-related pathways have been described in higher eukaryotes: macroautophagy (MA), chaperone-mediated autophagy (CMA), andmicroautophagy (Cuervo, 2010; Mizushima et al., 2008).”

“In the third common type of autophagy, CMA, cargo is not sequestered but is instead selectively recognized by a complex of cytosolic chaperones that mediates its delivery to a receptor at the lysosomal membran”

“Strong autophagy induction in associationwith neurodegeneration and autophagic cell death is so far uncommon compared to neurological responses associated with autophagy deficiency (Nixon, 2006; Wong and Cuervo, 2010).”

“Both HSC70 and LAMP2A are critical components in the CMA pathway (Cuervo, 2009).”

“It has been reported that posttranslation modifications can regulate this process (Cuervo, 2010).”

“This selective degradation pathway contributes to protein quality control and maintenance of cellular homeostasis particularly under stress conditions (Cuervo 2010; Dice 2007; Kaushik and Cuervo 2012).”

“CMA dysfunction in proteinopathies Dysfunction of CMA activity has been described in a growing number of pathologies (reviewed in [6,35,36]).”

“Alterations in autophagy in these disorders spread across problems in autophagosome formation, cargo recognition, autophagosome mobilization toward lysosomes, autophagosome/lysosome fusion or in the degradation of the autophagic cargo once delivered to lysosomes (Wong and Cuervo 2010).”

“CMA is a highly selective form of autophagy that requires unfolding of the protein before internalization into the lysosome for degradation (Dice, 2007; Cuervo, 2010).”

“Interested readers are referred to more focused reviews [2,3,8,29,54] for a comprehensive description of the molecular apparatus executing these different autophagic pathways.”

“CMA is a uniquely selective form of autophagy by which specific cytosolic proteins are transported one-by-one across the lysosomal membrane for degradation [10,11].”

“CMA is not involved in ferritin degradation by knocking down Lamp2A in MEFs, which is consistent with the observation that ferritin delivery to the lysosome in iron-replete MEFs was inhibited by 3-MA, since CMA is insensitive to 3-MA (17).”

“Mutation-based impairment of chaperone proteins [9], as well as proteins involved with the ubiquitin proteasomal [10, 11] and the autophagy lysosomal [12, 13] systems, has been described.”

“Among the different types of coexisting autophagic pathways, this study focuses on chaperone-mediated autophagy (CMA) (1), in which specific cytosolic proteins directly cross the lysosomal membrane for degradation inside.”

“In contrast, the selective pool of cytosolic proteins degraded by CMA is directly translocated across the lysosomal membrane (Cuervo, 2010; Dice, 2007).”

“Some level of CMA activity can be detected under basal conditions in tissues such as liver, kidney, brain and in different cell types in culture, but cells maximally activate this pathway in response to stress 3 .”

“Analyses of cellular conditions that activate CMA, substrates degraded under these conditions and the consequences of blocking this pathway in cultured cells have helped to understand the cellular functions of CMA (Dice, 2007; Cuervo, 2010).”

“Introduction In chaperone-mediated autophagy (CMA) (Cuervo, 2010; Kaushik & Cuervo, 2012), cytosolic proteins bearing a targeting motif biochemically related to the pentapeptide KFERQ (Dice, 1990) are recognized by the chaperone hsc70 that delivers them one-by-one to lysosomes for degradation (Chiang et al.”

“HSC70, associated with cochaperones, participates in CMA by recognizing a motif related to the pentapeptide KFERQ and directing it to lysomal degradation[11].”

“Chaperone-mediated autophagy (CMA): An autophagic process in mammalian cells by which proteins containing a particular pentapeptide motif related to KFERQ are transported across the lysosomal membrane and degraded.(72,73) The translocation process requires the action of the integral membrane protein LAMP-2A and both c…”

“The binding of substrate to monomeric form of LAMP-2A is followed by substrate unfolding possibly assisted by hsc70 and its co-chaperones [10].”

“Upregulation of chaperonemediated autophagy (CMA), the other stress-induced autophagic pathway in mammals (Cuervo, 2010), has been previously described in vitro in macroautophagy-deficient cell lines (Kaushik et al.”

“…to experimental blockage in macroautophagy by upregulating chaperone-mediated autophagy (CMA) (Kaushik et al., 2008), a type of autophagy conducive to degradation of cytosolic proteins in lysosomes through direct translocation across the membrane of these organelles (Dice, 2007; Cuervo, 2010).”

“We next considered whether chaperone-mediated autophagy (CMA), an alternative degradation pathway by which client proteins are targeted by heat-shock protein Hsc70 and its co-chaperones for lysosomal destruction (Figure 6A)(Cuervo, 2010), could be involved.”

“Autophagy Degrades CRY1 in a Time-Dependent Manner Three distinct mechanisms deliver cargo to lysosomes: macroautophagy (or autophagy) (He and Klionsky, 2009), chaperonemediated autophagy (CMA) (Cuervo, 2010), and endosomal microautophagy (eMI) (Sahu et al.”

“It is well known that blockage of macroautophagy can lead to a compensatory upregulation of CMA [26], an alternative lysosome-mediated degradation pathway wherein cytosolic components are directly translocated across the lysosomal membrane [36].”

“The autophagosome fuses with the lysosome to create the autophagolysosome in which the luminal content is degraded [132-135].”

“Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd chaperone-substrate complex to the lysosomal-associated membrane protein 2 (LAMP2A), the receptor for CMA, followed by translocation of the unfolded substrate in the lysosomal lumen for degradation…”

“In contrast to micro- and macroautophagy, where whole regions of the cytosol are sequestered and delivered to lysosomes, CMA targets single cytosolic proteins to the lysosomal surface and mediates their one-by-one translocation across the lysosomal membrane for degradation [4,5].”

“Chaperone-mediated autophagy (CMA) is a process of chaperondependent selection of cytosolic proteins that are translocated into the lysosome for degradation.”

“Selectivity thus became one of the hallmarks of CMA [51].”

“The CMA-targeting motif is degenerate and allows a series of amino acid combinations (Cuervo, 2010), and, consistently, an analysis with relaxed stringency revealed that almost 79% of all statistically decreased proteins had potential a CMA targeting motif, including p53 and Hsc70, which have been previously reported…”

“…to the lumenal side of the lysosomal membrane by HSP90 Hsc70 with lysosomal markers (Immunofluorescence); LAMP2A (western blot); radiolabeling Cuervo, 2010; Parzych and Klionsky, 2014; Patel and Cuervo, 2015; Arias, 2017; Wu et al., 2019 Abbreviations: LAMP2A: lysosome-associated membrane…”

“There is increasing evidence that members of the Hsp70 family may be directly implicated in protein degrading processes via shuttling their substrates to the degradation machinery, Cuervo has reported for example that Hsc70 is involved in chaperonemediated autophagy [15].”

“them into the lysosomes via LAMP2A (lysosomal associated membrane protein 2A) [9].”

“Its phosphorylation status determines whether the cell will bypass the “restriction point” of G1 phase committing the cell to progress to S phase without the requirements of extracellular stimulants [112-333].”

“Likewise, the lysosome not only handles internalized cargoes from the cell surface, but is also a crucial partner of the autophagosome pathway that engulfs a wide range of misfolded cytosolic proteins and dysfunctional organelles [29,33].”

“Notably, this motif is a pattern recognition motif associated with the hydrophobicity and charge of amino acid residues, rather than 100 % compliance with specific amino acid residues [26].”

“Macroautophagy and chaperone-mediated autophagy (CMA) are 2 well-characterized autophagic pathways in mammalian cells.(4,5) CMA is a selective process mediated in particular by HSPA8 and LAMP2A; HSPA8 can recognize and interact”

“are bound by an Hsc70-containing chaperone complex and translocated directly into lysosomes without involvement of autophagosomes [55].”

“…degree of oxidation still allows for the protein to be unfolded, which is a requirement for entry into the narrow catalytic chamber of the proteasome or the translocation channel that mediates substrate internalization into lysosomes via CMA (Dunlop et al., 2009; Cuervo, 2010; David et al., 2010).”

“In fact, failure to perform this cellular surveillance as a result of impaired autophagic function seems to underlie the basis of common protein conformational disorders, including severe neurodegenerative disorders and myopathies (Wong and Cuervo 2010).”

“Activation of CMA during other types of stress also mediates the elimination of proteins damaged by the stressor [2].”

“It can target degradation of selective cytosolic proteins (36, 37).”

“Chaperone-mediated autophagy is responsible for the degradation of single soluble cytosolic proteins that are recognized by affinity binding of a chaperone to a pentapeptide motif in their sequence ( Cuervo 2010a; Kon and Cuervo 2010; Orenstein and Cuervo 2010).”

“proteins in the lysosome via chaperone-mediated autophagy (CMA) [14,15] through recognition of a CMAtargeting motif, a pentapeptide sequence biochemically related to KFERQ present within 25–30% of all cytosolic proteins [16].”

“The now-established details of the pathway can be found in recent review”

“Chaperone-mediated autophagy (Figure 2), a more selective autophagic mechanism than macroautophagy which has been identified only in mammals, does not involve vesicle formation (Cuervo, 2010).”

“Like the proteasome system, autophagy is a vital pathway for the refolding or removal of abnormal and aggregated proteins, and thus, acts as a cytoprotective response, particularly under stress or injury conditions (Cuervo, 2010; Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006).”

“RNA interference of Lamp2a reduced CMA activity and decreased Lamp2a in dopaminergic cell lines, prolonging the half-life of α-synuclein [31, 32].”

“The translocation complex comprised of LAMP2a and a luminal HSC70 within the lysosomes (lys-HSC70) forms an integral part of the CMA machinery [81, 82].”

“…directly into the lysosome through invagination of the lysosomal membrane (Cai et al., 2012 Nixon and Yang, 2011), while CMA involves a selective translocation of cytoplasmic proteins containing a KFERQ peptide motif into the lysosomal lumen (Cuervo, 2010; Dice, 2007; Klionsky et al., 2010).”

“Finally, CMA is a uniquely selective form of autophagy that degrades a wide range of substrate proteins after transporting them one by one into the lysosome [51].”

“CMA degrades a wide range of substrate proteins, including certain glycolytic enzymes, transcription factors and their inhibitors, calcium and lipid binding proteins, proteasome subunits, and proteins involved in vesicular trafficking [31].”

“It is apparent that autophagic degradation is a preferential pathway for degradation of protein in the disease state due to progressive proteasomal impairment (Kaushik, Singh, & Cuervo, 2010).”

“In 2 of these mechanisms, macroautophagy and microautophagy, the substrates are engulfed or sequestered in bulk, whereas in CMA, the substrates are selectively transported across the lysosomal membrane on a one-by-one basis.(19) During CMA, protein substrates containing peptide regions similar to Lys-Phe-Glu-Arg-Gln (…”

“In macroautophagy, large regions of the cytosol are sequestered, and is widely seen in eukaryocytes.(1,3,4,6,13) Chaperonemediated autophagy is a process in which single, soluble proteins are selectively sequestered and delivered to lysosomes, and is only seen in mammals.”

“Whereas autophagy and microautophagy can degrade organelles and proteins, CMA is essentially involved in protein degradation [123].”