Chaperone-mediated Autophagy Components Research Articles

LUBAC promotes angiogenesis and lung tumorigenesis by ubiquitinating and antagonizing autophagic degradation of HIF1α

Hypoxia-inducible factor 1 (HIF1) is critically important for driving angiogenesis and tumorigenesis. Linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin ligase capable of catalyzing protein linear ubiquitination to date, is implicated in cell signaling and associated with cancers. However, the role and mechanism of LUBAC in regulating the expression and function of HIF1α, the labile subunit of HIF1, remain to be elucidated. Herein we showed that LUBAC increases HIF1α protein expression in cultured cells and tissues of human lung cancer and enhances HIF1α DNA-binding and transcriptional activities, which are dependent upon LUBAC enzymatic activity. Mechanistically, LUBAC increases HIF1α stability through antagonizing HIF1α decay by the chaperone-mediated autophagy (CMA)-lysosome pathway, thereby potentiating HIF1α activity. We further demonstrated that HIF1α selectively interacts with HOIP (the catalytic subunit of LUBAC) primarily in the cytoplasm. LUBAC catalyzes linear ubiquitination of HIF1α at lysine 362. Linear ubiquitination shields HIF1α from interacting with heat-shock cognate protein of 70 kDa and lysosome-associated membrane protein type 2 A, two components of CMA. Consequently, linear ubiquitination confers protection against CMA-mediated destruction of HIF1α, increasing HIF1α stability and activity. We found that prolyl hydroxylation is not a perquisite for LUBAC’s effects on HIF1α. Functionally, LUBAC facilitates proliferation, clonogenic formation, invasion and migration of lung cancer cells. LUBAC also boosts angiogenesis and exacerbates lung cancer growth in mice, which are greatly compromised by inhibition of HIF1α. This work provides novel mechanistic insights into the role of LUBAC in regulating HIF1α homeostasis, tumor angiogenesis and tumorigenesis of lung cancer, making LUBAC an attractive therapeutic target for cancers.

Navigating the cellular pathways: Chaperone-mediated autophagy as a targeted approach for management of parkinson\\\'s disease

Parkinson's disease (PD) is a common neurodegenerative condition marked by the degeneration of dopaminergic neurons and the amassing of α-synuclein protein in Lewy bodies. Chaperone-mediated autophagy (CMA), a selective form of autophagy, has been implicated in the development of PD. Mutant GBA1, α-synuclein, UCHL1, VPS35, and LRRK2 are affected proteins in PD that impair the CMA process. CMA Dysfunction cause accumulation of PD-associated proteins such as α-synuclein and many other, including DJ-1, MEF2D, PARK7,etc resulting in mitochondrial dysfunctioning and apoptosis. The impact of gene mutations associated with PD on CMA has been observed, along with dysregulation of miRNAs targeting CMA components. Toxicant-induced PD models demonstrate that impaired CMA increases α-synuclein aggregates and neurotoxicity. Understanding the molecular mechanisms of CMA has identified potential therapeutic targets, including increasing LAMP2A levels. Several compounds and substances have shown promise in enhancing CMA and reducing α- synuclein aggregates, such as 6-aminonicotinamide, geldanamycin, metformin, and natural compounds like trehalose and caffeine. Pharmacological modulation of CMA, such as through retinoic acid derivatives, has demonstrated positive effects on reducing protein aggregates in neurodegenerative diseases. However, the specific effects of inhibiting CMA on macroautophagy remain uncertain. Overcoming challenges in studying CMA, such as developing suitable models and monitoring methods, is crucial for advancing our understanding of CMA's role in neurodegenerative diseases and developing effective therapeutic strategies. Overall, CMA emerges as a key player in the pathogenesis of PD, and targeting this selective autophagy pathway holds promise for developing novel therapies to combat neurodegenerative disorders.

LAMP2A regulates the balance of mesenchymal stem cell adipo-osteogenesis via the Wnt/β-catenin/GSK3β signaling pathway.

Chaperone-mediated autophagy (CMA) plays multiple roles in cell metabolism. We found that lysosome-associated membrane protein type 2A (LAMP2A), a crucial protein of CMA, plays a key role in the control of mesenchymal stem cell (MSC) adipo-osteogenesis. We identified a differentially expressed CMA gene (LAMP2) in GEO datasets (GSE4911 and GSE494). Further, we performed co-expression analyses to define the relationships between CMA components genes and other relevant genes including Col1a1, Runx2, Wnt3 and Gsk3β. Mouse BMSCs (mMSCs) exhibiting Lamp2a gene knockdown (LA-KD) and overexpression (LA-OE) were created using an adenovirus system; then we investigated LAMP2A function in vitro by Western blot, Oil Red staining, ALP staining, ARS staining and Immunofluorescence analysis. Next, we used a modified mouse model of tibial fracture to investigate LAMP2A function in vivo. LAMP2A knockdown in mMSCs decreased the levels of osteogenic-specific proteins (COL1A1 and RUNX2) and increased those of the adipogenesis markers PPARγ and C/EBPα; LAMP2A overexpression had the opposite effects. The active-β-catenin and phospho-GSK3β (Ser9) levels were upregulated by LAMP2A overexpression and downregulated by LAMP2A knockdown. In the mouse model of tibial fracture, mMSC-overexpressing LAMP2A improved bone healing, as demonstrated by microcomputed tomography and histological analyses. In summary, LAMP2A positively regulates mMSC osteogenesis and suppresses adipo-osteogenesis, probably via Wnt/β-catenin/GSK3β signaling. LAMP2A promoted fracture-healing in the mouse model of tibial fracture. KEY MESSAGES: • LAMP2 positively regulates the mBMSCs osteogenic differentiation. • LAMP2 negatively regulates the mBMSCs adipogenic differentiation. •LAMP2 regulates mBMSCs osteogenesis via Wnt/β-catenin/GSK3β signaling pathway. •LAMP2 overexpression mBMSCs promote the fracture healing.

Diving into the Evolutionary History of HSC70-Linked Selective Autophagy Pathways: Endosomal Microautophagy and Chaperone-Mediated Autophagy.

Autophagy is a pleiotropic and evolutionarily conserved process in eukaryotes that encompasses different types of mechanisms by which cells deliver cytoplasmic constituents to the lysosome for degradation. Interestingly, in mammals, two different and specialized autophagic pathways, (i) the chaperone-mediated autophagy (CMA) and (ii) the endosomal microautophagy (eMI), both rely on the use of the same cytosolic chaperone HSPA8 (also known as HSC70) for targeting specific substrates to the lysosome. However, this is not true for all organisms, and differences exist between species with respect to the coexistence of these two autophagic routes. In this paper, we present an in-depth analysis of the evolutionary history of the main components of CMA and eMI and discuss how the observed discrepancies between species may contribute to improving our knowledge of these two functions and their interplays.

Chaperone Mediated Autophagy Substrates and Components in Cancer.

Chaperone-mediated autophagy (CMA) represents a specific way of lysosomal protein degradation and contrary to macro and microautophagy is independent of vesicles formation. The role of CMA in different physiopathological processes has been studied for several years. In cancer, alterations of the CMA principal components, Hsc70 and Lamp2A protein and mRNA levels, have been described in malignant cells. However, changes in the expression levels of these CMA components are not always associated with changes in CMA activity and their biological significance must be carefully interpreted case by case. The objective of this review is to discuss whether altering the CMA activity, CMA substrates or CMA components is accurate to avoid cancer progression. In particular, this review will discuss about the evidences in which alterations CMA components Lamp2A and Hsc70 are associated or not with changes in CMA activity in different cancer types. This analysis will help to better understand the role of CMA activity in cancer and to elucidate whether CMA can be considered as target for therapeutics. Further, it will help to define whether the attention of the investigation should be focused on Lamp2A and Hsc70 because they can have an independent role in cancer progression beyond of their participation in altered CMA activity.

Chaperone Mediated Autophagy Degrades TDP-43 Protein and Is Affected by TDP-43 Aggregation.

TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver’s CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain’s CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.

Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy.

Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA.IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1α contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1α. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1α caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1α to target HNF-1α for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1α. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.

MP92-15 SUPPRESSION OF CHAPERONE-MEDIATED AUTOPHAGY: A NOVEL MECHANISM OF ACTION OF SILIBININ AGAINST BLADDER AND RENAL CANCER

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2016MP92-15 SUPPRESSION OF CHAPERONE-MEDIATED AUTOPHAGY: A NOVEL MECHANISM OF ACTION OF SILIBININ AGAINST BLADDER AND RENAL CANCER Jin Zeng, Wei Liu, Feng Li, Yi Sun, Lei Li, Xinyang Wang, and Dalin He Jin ZengJin Zeng More articles by this author , Wei LiuWei Liu More articles by this author , Feng LiFeng Li More articles by this author , Yi SunYi Sun More articles by this author , Lei LiLei Li More articles by this author , Xinyang WangXinyang Wang More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2648AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chaperone-mediated autophagy (CMA) contributes to cellular quality control through the selective degradation of cytosolic proteins in lysosomes. Dysregulation of CMA activity occurs in age-related disorders and cancers. Chemical regulation of CMA is not currently possible, owing to the lack of information on the signaling mechanisms. A bulk of evidence from others and us suggests that silibinin, a natural flavonoid from milk thistle, exerts strong pleiotropic anticancer capabilities in various cancers. However, underlying mechanisms are poorly understood. The aim of this study was to investigate the role of CMA in silibinin-induced cell death in bladder cancer (BCa) and renal cell carcinoma (RCC). METHODS Human BCa 5637 and T24 cell lines and RCC ACHN and 769-P cell lines served as the model system in vitro and in vivo. Cell viability was determined by MTS assay. Lysosomes were isolated from cells by iodixanol-based density gradient centrifugation. The expression of two key CMA components heat shock-cognate chaperone of 70 kDa (hsc70) and lysosome-associated membrane protein type 2a (LAMP2a) on lysosomes were examined by western blot after silibinin treatment. Co-immunoprecipitation was used to determine the interaction between hsc70 and LAMP2a. CMA activity was monitored by in vitro lysosome binding and uptake assay. RNase and GAPDH, two well-characterized CMA substrates, were used in this functional assay allow tracking CMA activity. Immunohistochemistry analysis was performed for evaluating hsc70 and LAMP2a expression in BCa and RCC xenografts after oral silibinin in vivo. RESULTS Exposure of BCa and RCC cells to silibinin resulted in significant inhibition of cell growth. Downregulation of hsc70 and LAMP2a on lysosomes were observed after silibinin treatment. Overexpression of exogenous hsc70 or LAMP2a attenuated silibinin-induced cell death, while knocking down hsc70 or LAMP2a sensitized cells to death by silibinin. Furthermore, silibinin inhibited the interaction between hsc70 and LAMP2a on lysosome in both BCa and RCC cells. Additionally, silibinin treatment decreased the ability of substrates to be taken up by the lysosomes and inhibited the lysosomal degradation of substrates, suggesting the suppression of CMA activity. Oral silibinin suppressed the growth of BCa and RCC xenografts in vivo, which were accompanied with downregulation of hsc70 and LAMP2a. CONCLUSIONS This study reveals suppression of CMA as a novel mechanism for silibinin′s anti-proliferative effect, and suggests targeting CMA pathway may have antitumor potential against BCa and RCC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1167 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Jin Zeng More articles by this author Wei Liu More articles by this author Feng Li More articles by this author Yi Sun More articles by this author Lei Li More articles by this author Xinyang Wang More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Chaperone-mediated autophagy regulates proliferation by targeting RND3 in gastric cancer

ABSTRACTLAMP2A is the key protein of chaperone-mediated autophagy (CMA), downregulation of LAMP2A leads to CMA blockade. CMA activation has been implicated in cancer growth, but the exact mechanisms are unclear. Elevated expression of LAMP2A was found in 8 kinds of tumors (n=747), suggesting that LAMP2A may have an important role in cancer progression. Unsurprisingly, LAMP2A knockdown in gastric cancer (GC) cells hindered proliferation, accompanied with altered expression of cell cycle-related proteins and accumulation of RND3/RhoE. Interactomic and KEGG analysis revealed that RND3 was a putative CMA substrate. Further study demonstrated that RND3 silencing could partly rescue the proliferation arrest induced by LAMP2A knockdown; RND3 was increased upon lysosome inhibition via both chemicals and LAMP2A-shRNA; Furthermore, RND3 could interact with CMA components HSPA8 and LAMP2A, and be engulfed by isolated lysosomes. Thus, constant degradation of RND3 by CMA is required to sustain rapid proliferation of GC cells. At last, the clinical significance of LAMP2A was explored in 593 gastric noncancerous lesions and 173 GC tissues, the results revealed that LAMP2A is a promising biomarker for GC early warning and prognosis of female GC patients.

Maintenance of chaperone-mediated autophagy activity in cultured cells expressing mutant huntingtin

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by mutant huntingtin (Htt) with an expanded polyglutamine tract. It has been reported that Htt regulates autophagy. However, it remains unclear whether mutant Htt affects chaperone-mediated autophagy (CMA). Our study aimed to investigate the effect of mutant Htt on CMA activity in cultured HEK293T cells. A HEK293T cell model of HD was produced by transient transfection of wild-type (20Q) or mutant (120Q) Htt plasmids. The effect of mutant Htt on two CMA components, lysosomal-associated membrane protein 2a (Lamp2a) and heat-shock cognate protein 70 (Hsc70), was determined by western blotting and immunofluorescent staining. We observed that mutant Htt did not significantly alter the expression of Lamp2a and Hsc70 when compared to normal Htt. These findings suggest that mutant Htt does not reduce CMA activity and that enhancing CMA activity to clear mutant Htt may be a novel strategy for the management of HD.

Statins induce microautophagy in RAW 264.7 cell line. (APP3P.107)

Abstract Autophagy is process by which cells recycle cellular components to overcome starvation. This process is constitutive in antigen presenting cells such as dendritic cells and macrophages. Among the various autophagy pathways, microautophagy (Mi) has a unique significance as it is implicated in the biogenesis of exosomes which are relevant to the design of therapeutics. Statins have been shown to up regulate macroautophagy and alleviate the effect of LPS apart from inhibiting cholesterol synthesis. However their role in microautophagy has not been reported. In the present study we investigated the effect of statins on microautophagy using the RAW264.7 cell line as a model system. Treatment with statins resulted in an up regulation of the genes for MA as well as Mi as determined by qPCR. Further studies showed that the levels of proteins TSG101 and VPS4 were increased during statin treatment. Pretreatment with statins led to a lowering of LAMP2A levels and decreased LAMP2A on the late endosomes, mimicking a LAMP2A knock down. Pretreatment with statins not only lowered the transcription of iNOS and TNF-alpha genes but also prevented the effect of LPS by decreasing initial levels of LAMP2A and increasing levels of TSG101. We propose that Mi can be a surrogate for Chaperone mediated autophagy (CMA) under conditions where the components of CMA are rendered non-functional due to disruption in normal cellular metabolism.

Chaperone‐mediated autophagy components are upregulated in sporadic inclusion‐body myositis muscle fibres

Sporadic inclusion-body myositis (s-IBM) is an age-associated degenerative muscle disease. Characteristic features are muscle-fibre vacuolization and intramuscle-fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome-associated membrane protein type 2A (LAMP2A) and the heat-shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge. We studied CMA components by immunocytochemistry, immunoblots, real-time PCR and immunoprecipitation in: (a) 16 s-IBM, nine aged-matched normal and nine disease control muscle biopsies; and (b) cultured human muscle fibres (CHMFs) with experimentally inhibited activities of either the 26S proteasome or autophagy. Compared with age-matched controls, in s-IBM muscle, LAMP2A and Hsc70 were on a given transverse section accumulated as aggregates in approximately 5% of muscle fibres, where they (a) colocalized with each other and α-synuclein (α-syn), a CMA-targeted protein; and (b) were bound to each other and to α-syn by immunoprecipitation. By immunoblots, LAMP2A was increased sevenfold P < 0.001 and Hsc70 2.6-fold P < 0.05. LAMP2A mRNA was increased 4.4-fold P < 0.001 and Hsc70 mRNA 1.9-fold P < 0.05. In CHMFs inhibition of either the 26S proteasome or autophagy induced CMA, evidenced by a significant increase of both LAMP2A and Hsc70. Our study demonstrates, for the first time, up-regulation of CMA components in s-IBM muscle, and it provides further evidence that altered protein degradation is likely an important pathogenic aspect in s-IBM.

The role of chaperone-mediated autophagy in huntingtin degradation.

Huntington Disease (HD) is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt). HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA) components, heat shock protein cognate 70 (Hsc70) and lysosome-associated protein 2A (LAMP-2A) in degradation of Htt fragment 1-552aa (Htt-552). A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.

Constitutive upregulation of chaperone-mediated autophagy in Huntington's disease.

Autophagy contributes to the removal of prone-to-aggregate proteins, but in several instances these pathogenic proteins have been shown to interfere with autophagic activity. In the case of Huntington's disease (HD), a congenital neurodegenerative disorder resulting from mutation in the huntingtin protein, we have previously described that the mutant protein interferes with the ability of autophagic vacuoles to recognize cytosolic cargo. Growing evidence supports the existence of cross talk among autophagic pathways, suggesting the possibility of functional compensation when one of them is compromised. In this study, we have identified a compensatory upregulation of chaperone-mediated autophagy (CMA) in different cellular and mouse models of HD. Components of CMA, namely the lysosome-associated membrane protein type 2A (LAMP-2A) and lysosomal-hsc70, are markedly increased in HD models. The increase in LAMP-2A is achieved through both an increase in the stability of this protein at the lysosomal membrane and transcriptional upregulation of this splice variant of the lamp-2 gene. We propose that CMA activity increases in response to macroautophagic dysfunction in the early stages of HD, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to cellular failure and the onset of pathological manifestations.

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.