The coming of age of chaperone-mediated autophagy.

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.

The pathological hallmarks of various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease prominently feature the accumulation of misfolded proteins and neuroinflammation. Chaperone-mediated autophagy (CMA) has emerged as a distinct autophagic process that coordinates the lysosomal degradation of specific proteins bearing the pentapeptide motif Lys-Phe-Glu-Arg-Gln (KFERQ), a recognition target for the cytosolic chaperone HSC70. Beyond its role in protein quality control, recent research underscores the intimate interplay between CMA and immune regulation in neurodegeneration. In this review, we illuminate the molecular mechanisms and regulatory pathways governing CMA. We further discuss the potential roles of CMA in maintaining neuronal proteostasis and modulating neuroinflammation mediated by glial cells. Finally, we summarize the recent advancements in CMA modulators, emphasizing the significance of activating CMA for the therapeutic intervention in neurodegenerative diseases.

Rationale: Protein degradation pathways play a critical role in maintaining cardiac homeostasis. Chaperone mediated autophagy (CMA) is a protein degradation pathway unique to mammalian cells, but little is known about CMA in cardiac disease. Preliminary studies in our lab showed increased CMA activity in mouse hearts subjected to MI (1.81 fold) and TAC surgery (3.5 fold), as well as, in hearts from mutant CryAB R120G transgenic mice (3 fold). Thus, we hypothesized that CMA plays a critical role in hypoxia- and proteotoxicity-induced cardiac stresses. CMA selectively targets substrate proteins for lysosomal degradation using CMA-specific lysosome membrane protein type 2a (LAMP2a) receptor. LAMP2a is both necessary and sufficient for CMA activity. Methods: An adenovirus and a siRNA were created to overexpress and silence LAMP2a levels to study the gain and loss of CMA in neonatal rat ventricular cardiomyocytes (NRVMs). Two models of cardiomyocyte stress were employed: 1) a hypoxic stress model that exposed NRVMs to CoCl 2 , a hypoxia mimetic agent; and 2) a proteotoxic model where mutant CryAB R120G , a missense mutation of αB-crystallin, was expressed causing the accumulation of insoluble protein aggregates. A more specific measure of CMA activity was also determined in lysosomal fractions of NRVMs. Results: The LAMP2a adenovirus and siRNA showed successful increase and knockdown of LAMP2a levels in NRVMs, respectively. CoCl 2 -induced hypoxia significantly increased CMA activity in NRVMs (26%). LAMP2a overexpression further augmented CoCl 2 -induced CMA activity (55%) and increased the percent of CMA-active lysosomes. LAMP2a siRNA blunted the CoCl 2 -induction of LAMP2a protein levels (65%) and silenced CMA activity in intact lysosomes. Further, CoCl 2 increased apoptosis which was rescued by enhanced CMA activity as shown by TUNEL staining. Thus, CMA is regulated by hypoxic stress and modulates cardiac cell survival. In a proteotoxic model, CryAB R120G and LAMP2a co-infection decreased CryAB R120G protein levels and insoluble aggregate pathology. Our data suggest that CMA may be cardioprotective against hypoxic and proteotoxic stresses. Impact: Defining the role of CMA in the heart could open new therapeutic pathways for the treatment of heart failure.

Chaperone-mediated autophagy (CMA) is a selective autophagic pathway that degrades soluble cytosolic proteins. It utilizes a cascade of chaperones and co-chaperones to recognize and deliver the substrates to the lysosome for degradation. Basal CMA operates in all mammalian cells in order to maintain protein homeostasis and quality control as well as performing specific functions in some cells like kidney cell growth, neuronal survival, antigen presentation and selective degradation of specific transcription factors or metabolic enzymes. CMA is induced upon stress and performs various cellular functions like recycling of raw materials under nutrient starvation, protection against oxidative stress, toxic stress and hypoxic stress. The rate limiting step in CMA is substrate binding to the lysosomal receptor lysosome-associated membrane protein-2A (LAMP-2A) followed by formation of the multimeric LAMP-2A translocation complex. It has been reported that the mTORC2/Akt1/PHLPP1 pathway regulates CMA activity by influencing the formation the LAMP-2A translocation complex. CMA is impaired during aging resulting in metabolic failure and various neurodegenerative disorders. Thus there is tremendous interest in understanding the regulation and deriving ways to regulate CMA function to harness pathological benefits. Certain evidences suggest the role of ubiquitination in CMA pathway. For instance, rat liver lysosomes depleted of CMA promoted accumulation of ubiquitin-positive aggregates, K63-ubiquitination is necessary for CMA degradation of HIF-1α and accumulation of K48 ubiquitinated protein in CMA-abolished cells, however, the exact role of ubiquitination in targeting substrates to CMA pathway remains unclear. The current study has shown the novel role of the ubiquitin system in CMA regulation by uncovering the presence of ubiquitin and a plethora of ubiquitin modifying enzymes associated with the subgroup of lysosomes specifically dedicated to perform CMA (henceforth known as CMA-active lysosomes). Based on these observations, this project aims to understand the potential modulatory role of selected lysosome associated ubiquitin modifying enzymes- E4 ligase carboxyl-terminus of Hsp70 interacting protein (CHIP) and the deubiquitinating enzyme ubiquitin C-terminal hydrolase - L1 (UCH-L1) in different aspects of CMA pathway. CHIP and UCH-L1 have been previously implicated in regulating the levels of Protein kinase B (Akt) and PH Domain and Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1), but it is not known whether this function of CHIP and UCH-L1 can regulate CMA activity. Addressing this query, the present study has revealed that CHIP is an activator of CMA while UCH-L1 inhibits CMA by regulation of Akt1 and PHLPP1 levels. Knockdown studies revealed that depletion of CHIP leads to changes in lysosomal levels of ubiquitin, CMA associated chaperones as well as accumulation of pAkt1 accompanied by a decrease in levels of PHLPP1. CHIP helps in ubiquitination of pAkt1 leading to its proteasomal degradation. In the absence of CHIP, pAkt1 accumulates which leads to downstream destabilization of LAMP-2A translocation complex resulting in CMA inhibition. In contrast, knockdown studies on UCH-L1 indicate that depletion of UCH-L1 leads to accumulation of PHLPP1 accompanied by a reduction in pAkt1 levels in the lysosomes leading to stabilization of the LAMP-2A complex and promoting CMA activity. Effectively, CHIP and UCH-L1 regulate CMA activity by manipulation of the mTOR/Akt1/PHLPP1 pathway. Subsequently, it was observed that CHIP regulates the levels of UCH-L1 at the lysosomes by ubiquitination and lysosomal degradation of UCH-L1. Thus, the two enzymes operate in the same pathway; their activities being interconnected. The evidence that the ubiquitin modifying enzymes associate with CMA-active lysosomes and regulate CMA activity could indicate a possible crosstalk between the ubiquitin proteasome system (UPS) and CMA pathway. In the current study, it was observed that upon inhibition of the proteasomal pathway, CHIP localized at the lysosomes, thus reducing lysosomal levels of UCH-L1, pAkt1 and increasing levels of PHLPP1. This could indicate a rise in CMA activity under proteasomal stress. Thus in this study, a novel mechanism of CHIP-mediated crosstalk between the UPS and CMA pathways upon conditions of proteasomal stress has been identified. This study has been advantageous in understanding the intricate connection between ubiquitination and phosphorylation pathways regulating CMA activity and can be exploited in developing therapeutic benefits by cell type specific studies as UCH-L1 is highly abundant in brain. The current study also provides an insight in understanding the crosstalk between the UPS and CMA degradation pathways both of which are implicated in various neurodegenerative diseases.

Chaperone-mediated autophagy (CMA) selectively delivers cytosolic proteins with an exposed CMA-targeting motif to lysosomes for degradation and plays an important role in protein quality control and cellular homeostasis. A growing body of evidence supports the hypothesis that CMA dysfunction may be involved in the pathogenic process of neurodegenerative diseases. Both down-regulation and compensatory up-regulation in CMA activities have been observed in association with neurodegenerative conditions. Recent studies have revealed several new mechanisms by which CMA function may be involved in the regulation of factors critical for neuronal viability and homeostasis. Here, we summarize these recent advances in the understanding of the relationship between CMA dysfunction and neurodegeneration and discuss the therapeutic potential of targeting CMA in the treatment of neurodegenerative diseases.

Two types of autophagy are better than one during hepatocyte oxidative stress

Chaperone-mediated autophagy (CMA) is a selective type of autophagy whereby a specific subset of intracellular proteins is targeted to the lysosome for degradation. These proteins are identified by a chaperone that targets them to lysosomes. There, they are translocated into the organelle lumen through a lysosomal membrane receptor/translocation complex. CMA plays an important role in maintaining cellular proteostasis by eliminating damaged and altered proteins. CMA also participates in the control of the cellular energetic balance through recycling of amino acids resulting from lysosomal proteolysis of the substrate proteins. Lastly, due to the intrinsic protein selectivity of CMA, this type of autophagy exerts regulatory functions by mediating timely degradation of key cellular proteins that participate in processes such as lipid and glucose metabolism, cell cycle, DNA repair, and cellular reprogramming, among others. Dysfunctional CMA occurs with age and has now been described in a growing list of human pathologies such as metabolic disorders, neurodegeneration, cancer, immunodeficiency, and diabetes. In this chapter, we describe current methodologies to quantitatively analyze CMA activity in different experimental models.

Chapter 19 Methods to Monitor Chaperone‐Mediated Autophagy

Proteostasis, also known as protein homeostasis, is critical for cell survival. Autophagy is a cellular process that degrades and recycles damaged or long-lived proteins, misfolded proteins, and damaged or abnormal organelles in order to preserve homeostasis. Among the three forms of autophagy, chaperone-mediated autophagy (CMA) is distinct from macroautophagy and microautophagy; it does not require the formation of vacuoles and only degrades selected individual proteins. CMA helps to maintain cellular homeostasis by regulating protein quality, bioenergetics, and substrate-associated cellular processes at the right moment. This pathway’s dysfunction has been linked to several diseases and disorders. Neurodegenerative diseases and cancer have received the most attention. In various neurodegenerative disorders, especially in their later stages, CMA activity declines. CMA has been shown to act as a tumor suppressor in cancer by destroying specific tumor promoters. Once a tumor has grown, it also helps tumor survival and the metastatic cascade. The presence of changes in CMA in these diseases disorders raises the idea of targeting CMA to restore cellular homeostasis as a potential therapeutic method. Manipulation of CMA activity may be effective therapeutic strategies for treating these diseases. Therefore, in this paper; we introduce the basic processes, regulatory mechanisms, and physiological functions of CMA; evidences supporting the role of impaired CMA function in neurodegeneration and cancer; and the potential of how targeting CMA could be a promising therapeutic method for the two diseases.

Chaperone Mediated Autophagy (CMA) is a lysosomal-dependent protein degradation pathway. At least 30% of cytosolic proteins can be degraded by this process. The two major protein players of CMA are LAMP-2A and HSC70. While LAMP-2A works as a receptor for protein substrates at the lysosomal membrane, HSC70 specifically binds protein targets and takes them for CMA degradation. Because of the broad spectrum of proteins able to be degraded by CMA, this pathway has been involved in physiological and pathological processes such as lipid and carbohydrate metabolism, and neurodegenerative diseases, respectively. Both, CMA, and the mentioned processes, are affected by aging and by inadequate nutritional habits such as a high fat diet or a high carbohydrate diet. Little is known regarding about CMA, which is considered a common regulation factor that links metabolism with neurodegenerative disorders. This review summarizes what is known about CMA, focusing on its molecular mechanism, its role in protein, lipid and carbohydrate metabolism. In addition, the review will discuss how CMA could be linked to protein, lipids and carbohydrate metabolism within neurodegenerative diseases. Furthermore, it will be discussed how aging and inadequate nutritional habits can have an impact on both CMA activity and neurodegenerative disorders.

Chaperone-mediated autophagy (CMA) represents a selective form of autophagy involved in the degradation of specific soluble proteins containing a pentapeptide motif that is recognized by a cytosolic chaperone able to deliver proteins to the lysosomes for degradation. Physiologically, CMA contributes to maintain crucial cellular functions including energetic balance and protein quality control. Dysfunctions in CMA have been associated to the pathogenesis of several neurodegenerative diseases characterized by accumulation and aggregation of proteins identified as CMA substrates. In particular, increasing evidence highlights the existence of a strong relationship between CMA defects and Parkinson’s disease (PD). Several mutations associated with familial forms of PD (SNCA, LRRK2, UCHL1 and DJ-1) have been demonstrated to block or reduce the activity of CMA, the main catabolic pathway for alpha-synuclein (asyn). CMA dysfunctions also leads to a mislocalization and inactivation of the transcription factor MEF2D that plays a key-role in the survival of dopaminergic neurons. Furthermore, reduced levels of CMA markers have been observed in post mortem brain samples from PD patients. The aim of this review article is to provide an organic revision of evidence for the involvement of CMA dysfunctions in the pathogenesis of PD. Updated findings obtained in patient’s specimens will be resumed, and results deriving from in vivo and in vitro studies will be discussed to evidence the current knowledge on the molecular mechanisms underlying CMA alterations in PD. Finally, the possibility of up-regulating CMA pathway as promising neuroprotective strategy will be considered.

Dysregulation of autophagy, the lysosomal degradation of intracellular components, has been universally described in cancer. While activity of macroautophagy changes in a context-dependent manner in cancers, the upregulation of chaperone-mediated autophagy (CMA) is well-described in all solid tumors studied to date. Upregulated CMA directly contributes to both tumor growth and protection against anti-oncogenic interventions in cancers. Selective degradation of anti-oncogenic and pro-apoptotic proteins, such as mutant p53 and PUMA, misfolded proteins, and glycolytic enzymes by CMA allows for increased survival, reduced cell death, increased stress resistance, and enhancement of the Warburg effect in cancer cells, respectively. As such, inhibition of CMA is an attractive target for anti-cancer therapeutics, but the field currently lacks protein targets specific to CMA and selective CMA inhibitors. We have previously demonstrated the regulatory effect of retinoic acid receptor alpha (RARα) and its coregulator, nuclear receptor corepressor 1 (NCoR1), on CMA through transcriptional upregulation of the CMA effector proteins (Bourdenx et al. Cell 2021, Gomez-Sintes et al, Nat. Commun. 2022). Here, we identified a high NCoR1/RARα expression ratio in human non-small cell lung cancer (NSCLC) tumors compared to healthy lung tissue. We further illustrated a dependency on NCoR1 expression and its interaction with RARα to maintain high CMA activity in NSCLC cell lines. Provided the reliance on NCoR1 repression of RARα for high CMA activity in NSCLC, we hypothesized that small molecule targeting of the druggable NCoR1/RARα interaction to disrupt the interaction may result in suppression of CMA effector proteins and CMA inhibition. Through a series of in-silico screens for predicted NCoR1/RARα targeting, and functional screens for CMA inhibition, we identified CIM7, a first-in-class potent and selective CMA inhibitor. We validated CIM7 function through binding to RARα and subsequent disruption of NCoR1 binding, resulting in alterations to CMA-related gene expression. CIM7 inhibits CMA in vivo with no observed toxicity in normal tissues. We additionally demonstrated the therapeutic potential of CIM7 through its ability to reduce tumor growth in vivo in mouse NSCLC xenografts. Our findings support the NCoR1/RARα interaction as a major regulator of CMA in NSCLC and demonstrate the potential of pharmacologic CMA inhibition as an anti-cancer therapeutic strategy. Citation Format: Mericka McCabe, Rabia R. Khawaja, Rajanya Bhattacharyya, Antonio Diaz, Thomas P. Garner, Kristen Lindenau, Rebecca Sereda, Olaya Santiago-Fernández, Ana Maria Cuervo, Evripidis Gavathiotis. Pharmacological inhibition of chaperone-mediated autophagy via NCoR1/RARα targeting is effective against non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2091.

Protein quality control (PQC) systems are crucial for eliminating misfolded or damaged proteins, and their dysfunction contributes to heart failure pathogenesis. The carboxyl terminus of Hsc70-interacting protein (Chip), a co-chaperone with E3 ligase activity, plays a central role in cardiac PQC. Chaperone-mediated autophagy (CMA) is a selective degradation process mediated by the lysosome and involved internalization through lysosome-associated membrane protein 2A (Lamp2A). For CMA to occur, the Hsc70/Chip complex recognizes a KFERQ motif on a substrate protein, will then bind to the substrate protein, and translocate it to the Lamp2A receptor. We find Lamp2A expression is downregulated in the myocardium of human heart failure with either reduced or preserved ejection fraction (HFrEF, HFpEF), suggesting impaired CMA and a potential therapeutic opportunity. However, little is known about the mechanisms regulating CMA in the myocardium or if it is possible to pharmacologically stimulate CMA in disease conditions. Here we test if phosphorylation of Chip at serine 19 by protein kinase G (PKG) stimulates CMA in cardiomyocytes. Preliminary data indicate that PKG phosphorylation of Chip provides cardioprotective effects under ischemic stress. To investigate this further, we have generated CHIP mutants, including a phosphonull (S19A) and a phosphomimic (S19E), to dissect the role of PKG-mediated regulation of CMA to enhance cardiomyocyte CMA. We find phosphorylated Chip (S19E) is more localized at the lysosome than non-phosphorylated Chip (S19A), suggesting greater CMA activity. We confirm these findings using a BioID proximity labeling system to identify protein-protein interactions and that increasing CMA via PKG/Chip S19 phosphorylation protects cardiomyocytes from proteotoxic stress. Collectively our findings suggest PKG phosphorylation of Chip at serine 19 is a new mechanism regulating CMA in the heart; which has therapeutic potential for improving myocardial PQC and function. NHLBI HL169273, AHA 20TPA35500008. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

To reason that targeting chaperone-mediated autophagy (CMA) represents a promising approach for disease therapy, we will summarize advances in researches on the relationship between CMA and diseases and discuss relevant strategies for disease therapy by targeting the CMA process. CMA is a unique kind of selective autophagy in lysosomes. Under physiological conditions, CMA participates in the maintenance of cellular homeostasis by protein quality control, bioenergetics, and timely regulated specific substrate-associated cellular processes. Under pathological conditions, CMA interplays with various disease conditions. CMA makes adaptive machinery to address stress, while disease-associated proteins alter CMA which is involved in pathogeneses of diseases. As more proteins are identified as CMA substrates and regulators, dysregulation of CMA has been implicated in an increasing number of diseases, while rectifying CMA alteration may be a benefit for these diseases. Alterations of CMA in diseases mainly including neurodegenerative diseases and many cancers raise the possibility of targeting CMA to recover cellular homeostasis as one potential strategy for therapy of relevant diseases.

Macroautophagy is required for normal cardiac function. Chaperone mediated autophagy (CMA) is distinct in that substrates are transported to lysosomes in complex with Hsc70, rather than within autophagosomes; restricted to cytoplasmic proteins; and selectively marked with a CMA-targeting motif. The role of CMA in the heart has not been assessed. We determined that CMA is induced in the heart by pressure overload with kinetics that differ markedly from those of macroautophagy in response to the same stimulus. In CMA, cargo is imported into the lysosome by LAMP2A, which is necessary, specific, and rate-limiting for this process. Accordingly, we created knockout mice lacking LAMP2A in cardiomyocytes to inactivate CMA specifically in those cells. Unexpectedly, these mice exhibited resistance to stress-induced cardiac dysfunction in both pressure overload and myocardial infarction models. Functional assessment of cardiac mitochondria from L2AKO mice and MEFs exhibited increased rates of O 2 consumption and ATP generation. Mitochondrial proteomics showed increased levels of ATP5L, a component of mitochondrial Complex V (ATP synthase). Overexpression of ATP5L phenocopied LAMP2A KO with respect to augmentation of mitochondrial function. Conversely, knockdown of ATP5L reversed increases in mitochondrial function resulting from L2AKO. We showed that ATP5L, which possesses a KFERQ motif, is a bona fide CMA substrate as it interacts with Hsc70 in a KFERQ-dependent manner; and localizes to lyososmes in a LAMP2A-dependent manner when CMA is induced. ATP5L is thought to mediate oligomerization of Complex V to enhance ATP production. In fact, we observed increases in complex V oligomerization in L2A KO mouse hearts in both basal and TAC-induced states. In summary, cardiomyocyte-specific inhibition of CMA, which renders mice resistant to stress-induced cardiac dysfunction, augments cardiac mitochondrial function through accumulation of ATP5L. Conversely, activation of CMA by cardiac stress may function as a new pathway to heart failure.