Chaperone-Mediated Autophagy Promotes Beclin1 Degradation in Persistently Infected Hepatitis C Virus Cell Culture.
ABSTRACT: Liver cirrhosis is an independent risk factor for hepatocellular carcinoma (HCC). The mechanisms that contribute to HCC development in the cirrhotic microenvironment are unknown. We found that HCC grown in the highly stressed cirrhotic microenvironment undergoes autophagy switching from a protective state characterized by high macroautophagy with low chaperone-mediated autophagy (CMA) to an HCC-promoting state characterized by low macroautophagy with high CMA. This study examined how the stress response executes oncogenic cell programming through autophagy switching using hepatitis C virus cell culture. Protein kinase R-like endoplasmic reticulum kinase expression increased to high levels in hepatitis C virus culture. Protein kinase R-like endoplasmic reticulum kinase-dependent activation of nuclear factor erythroid 2-related factor (Nrf2) led to increased transcription of the cytoprotective genes: heat shock cognate 70 kDa protein and lysosome-associated membrane protein 2A (LAMP2A) and precipitated the induction of CMA. CMA selectively targeted beclin1 degradation, leading to accumulation of the autophagy flux protein p62 due to impaired autophagosome-endosome fusion. This impaired autophagosome-endosome fusion due to beclin1 degradation inhibited endocytosis and degradation of epidermal growth factor receptor. Silencing Nrf2 and LAMP2A reduced cell viability, suggesting that the stress response activates CMA as a compensatory mechanism of cell survival. We report a novel mechanism through which stress response triggers oncogenic Nrf2 signaling that promotes autophagy switching to favor cell survival.
Project description:Macroautophagy and chaperone-mediated autophagy (CMA) represent two major lysosomal degradation processes and often compensate for one another to facilitate cell survival. The aim of this study was to determine whether these autophagy pathways could compensate for one another to promote HCC cell survival in the cirrhotic liver. Analysis of normal liver tissue showed no expression of glypican-3 or p62 proteins, suggesting that macroautophagy is the major contributor to autophagic flux under non-pathological conditions. Of 46 cirrhotic livers with HCC examined, 39 (84%) of HCCs showed increased expression of p62, and 36 (78%) showed increased expression of glypican-3, while adjacent non-tumorous hepatocytes were negative for expression of p62 and glypican-3, similar to normal liver tissue. These results suggest that macroautophagy flux is impaired in HCC. Furthermore, more than 95% of HCCs showed altered expression of LAMP-2A compared to the surrounding non-tumorous cirrhotic liver, consistent with induction of CMA in HCC. Elevated expression of glucose-regulated protein 78 (GRP78) and heat shock cognate protein (Hsc70) were detected in 100% of HCC and adjacent non-tumorous cirrhotic livers, suggesting that unresolved ER-stress is associated with HCC risk in liver cirrhosis. Interestingly, inhibition of lysosomal degradation using hydroxychloroquine (HCQ) induced expression of the tumor suppressor p53, promoted apoptosis, and inhibited HCC growth, whereas activation of autophagy using an mTOR inhibitor (Torin1) promoted HCC growth. Results of this study suggest that induction of CMA compensates for the impairment of macroautophagy to promote HCC survival in the cirrhotic liver.
Project description:Recent data have shown that the expression of lysosome-associated membrane protein type 2?A (LAMP2A), the key protein in the chaperone-mediated autophagy (CMA) pathway, is elevated in breast tumor tissues. However, the exact effects and mechanisms of CMA during breast cancer metastasis remain largely unknown. In this study, we found that the LAMP2A protein level was significantly elevated in human breast cancer tissues, particularly in metastatic carcinoma. The increased LAMP2A level was also positively correlated with the histologic grade of ductal breast cancer. High LAMP2A levels also predicted shorter overall survival of breast cancer patients. Downregulation of CMA activity by LAMP2A knockdown significantly inhibited the growth and metastasis of both MDA-MB-231 and MDA-MB-468 breast cancer cells in vivo and in vitro, while upregulation of CMA activity by LAMP2A overexpression had the opposite effect. Mechanistically, we found that elevated CMA activity mediated increased growth and metastasis of human breast cancer cells by downregulating the activity of autophagy-related gene 5 (ATG5)-dependent macroautophagy. Collectively, these results indicate that the anti-macroautophagic property is a key feature of CMA-mediated tumorigenesis and metastasis and may, in some contexts, serve as an attractive target for breast cancer therapies.
Project description:The autophagy-lysosome pathway plays a fundamental role in the clearance of aggregated proteins and protection against cellular stress and neurodegenerative conditions. Alterations in autophagy processes, including macroautophagy and chaperone-mediated autophagy (CMA), have been described in Parkinson disease (PD). CMA is a selective autophagic process that depends on LAMP2A (lysosomal-associated membrane protein 2A), a mammal and bird-specific membrane glycoprotein that translocates cytosolic proteins containing a KFERQ-like peptide motif across the lysosomal membrane. Drosophila reportedly lack CMA and use endosomal microautophagy (eMI) as an alternative selective autophagic process. Here we report that neuronal expression of human LAMP2A protected Drosophila against starvation and oxidative stress, and delayed locomotor decline in aging flies without extending their lifespan. LAMP2A also prevented the progressive locomotor and oxidative defects induced by neuronal expression of PD-associated human SNCA (synuclein alpha) with alanine-to-proline mutation at position 30 (SNCA<sup>A30P</sup>). Using KFERQ-tagged fluorescent biosensors, we observed that LAMP2A expression stimulated selective autophagy in the adult brain and not in the larval fat body, but did not increase this process under starvation conditions. Noteworthy, we found that neurally expressed LAMP2A markedly upregulated levels of Drosophila Atg5, a key macroautophagy initiation protein, and that it increased the density of Atg8a/LC3-positive puncta, which reflects the formation of autophagosomes. Furthermore, LAMP2A efficiently prevented accumulation of the autophagy defect marker Ref(2)P/p62 in the adult brain under acute oxidative stress. These results indicate that LAMP2A can potentiate autophagic flux in the Drosophila brain, leading to enhanced stress resistance and neuroprotection.<h4>Abbreviations</h4>Act5C: actin 5C; a.E.: after eclosion; Atg5: autophagy-related 5; Atg8a/LC3: autophagy-related 8a; CMA: chaperone-mediated autophagy; DHE: dihydroethidium; elav: embryonic lethal abnormal vision; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; GABARAP: GABA typeA receptor-associated protein; Hsc70-4: heat shock protein cognate 4; HSPA8/Hsc70: heat shock protein family A (Hsp70) member 8; LAMP2: lysosomal associated membrane protein 2; MDA: malondialdehyde; PA-mCherry: photoactivable mCherry; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PD: Parkinson disease; Ref(2)P/p62: refractory to sigma P; ROS: reactive oxygen species; RpL32/rp49: ribosomal protein L32; RT-PCR: reverse transcription polymerase chain reaction; SING: startle-induced negative geotaxis; SNCA/?-synuclein: synuclein alpha; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; UAS: upstream activating sequence.
Project description:Chaperone-mediated autophagy (CMA) is a selective degradative process for cytosolic proteins that contributes to the maintenance of proteostasis. The signaling mechanisms that control CMA are not fully understood but might involve response to stress conditions including oxidative stress. Considering the role of CMA in redoxtasis and proteostasis, we sought to determine if the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2) has an impact on CMA modulation. In this work, we identified and validated 2 NFE2L2 binding sequences in the LAMP2 gene and demonstrated in several human and mouse cell types that NFE2L2 deficiency and overexpression was linked to reduced and increased LAMP2A levels, respectively. Accordingly, lysosomal LAMP2A levels were drastically reduced in nfe2l2-knockout hepatocytes, which also displayed a marked decrease in CMA activity. Oxidant challenge with paraquat or hydrogen peroxide, or pharmacological activation of NFE2L2 with sulforaphane or dimethyl fumarate also increased LAMP2A levels and CMA activity. Overall, our study identifies for the first time basal and inducible regulation of LAMP2A, and consequently CMA activity, by NFE2L2.<h4>Abbreviations</h4>ACTB: actin, beta, ARE: antioxidant response element; ATG5: autophagy related 5; BACH1: BTB domain and CNC homolog 1; ChIP: chromatin immunoprecipitation; CMA: chaperone-mediated autophagy; DHE: dihydroethidium; DMF: dimethyl fumarate; ENCODE: Encyclopedia of DNA elements at the University of California, Santa Cruz; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA: glucosylceramidase beta; GFP: green fluorescent protein; HMOX1: heme oxygenase 1; H<sub>2</sub>O<sub>2</sub>: hydrogen peroxide; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KEAP1: kelch like ECH associated protein 1; LAMP2A: lysosomal associated membrane protein 2A; LAMP2B: lysosomal associated membrane protein 2B; LAMP2C: lysosomal associated membrane protein 2C; LAMP1: lysosomal associated membrane protein 1; MAFF: MAF bZIP transcription factor F; MAFK: MAF bZIP transcription factor K; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PQ: paraquat; PI: protease inhibitors; qRT-PCR: quantitative real-time polymerase chain reaction; RNASE: ribonuclease A family member; SFN: sulforaphane; SQSTM1/p62: sequestosome 1; TBP: TATA-box binding protein.
Project description:Alpha-synuclein (ASYN) is crucial in Parkinson disease (PD) pathogenesis. Increased levels of wild type (WT) ASYN expression are sufficient to cause PD in humans. The manner of post-transcriptional regulation of ASYN levels is controversial. Previously, we had shown that WT ASYN can be degraded by chaperone-mediated autophagy (CMA) in isolated liver lysosomes. Whether this occurs in a cellular and, in particular, in a neuronal cell context is unclear. Using a mutant ASYN form that lacks the CMA recognition motif and RNA interference against the rate-limiting step in the CMA pathway, Lamp2a, we show here that CMA is indeed involved in WT ASYN degradation in PC12 and SH-SY5Y cells, and in primary cortical and midbrain neurons. However, the extent of involvement varies between cell types, potentially because of differences in compensatory mechanisms. CMA inhibition leads to an accumulation of soluble high molecular weight and detergent-insoluble species of ASYN, suggesting that CMA dysfunction may play a role in the generation of such aberrant species in PD. ASYN and Lamp2a are developmentally regulated in parallel in cortical neuron cultures and in vivo in the central nervous system, and they physically interact as indicated by co-immunoprecipitation. In contrast to previous reports, inhibition of macroautophagy, but not the proteasome, also leads to WT ASYN accumulation, suggesting that this lysosomal pathway is also involved in normal ASYN turnover. These results indicate that CMA and macroautophagy are important pathways for WT ASYN degradation in neurons and underline the importance of CMA as degradation machinery in the nervous system.
Project description:Cystinosis is a lysosomal storage disorder caused by defects in CTNS, the gene that encodes the lysosomal cystine transporter cystinosin. Patients with nephropathic cystinosis are characterized by endocrine defects, defective proximal tubule cell (PTC) function, the development of Fanconi syndrome and, eventually, end-stage renal disease. Kidney disease is developed despite the use of cysteamine, a drug that decreases lysosomal cystine overload but fails to correct overload-independent defects. Chaperone-mediated autophagy (CMA), a selective form of autophagy, is defective in cystinotic mouse fibroblasts, and treatment with cysteamine is unable to correct CMA defects in vivo, but whether the vesicular trafficking mechanisms that lead to defective CMA in cystinosis are manifested in human PTCs is not currently known and whether PTC-specific mechanisms are corrected upon CMA upregulation remains to be elucidated. Here, using CRISPR-Cas9 technology, we develop a new human PTC line with defective cystinosin expression (CTNS-KO PTCs). We show that the expression and localization of the CMA receptor, LAMP2A, is defective in CTNS-KO PTCs. The expression of the lipidated form of LC3B, a marker for another form of autophagy (macroautophagy), is decreased in CTNS-KO PTCs indicating decreased autophagosome numbers under basal conditions. However, the autophagic flux is functional, as measured by induction by starvation or by blockage using the v-ATPase inhibitor bafilomycin A, and by degradation of the macroautophagy substrate SQSTM1 under starvation and proteasome-inhibited conditions. Previous studies showed that LAMP2A accumulates in Rab11-positive vesicles in cystinotic cells. Here, we show defective Rab11 expression, localization and trafficking in CTNS-KO PTCs as determined by confocal microscopy, immunoblotting and TIRFM. We also show that both Rab11 expression and trafficking in cystinotic PTCs are rescued by the upregulation of CMA using small-molecule CMA activators. Cystinotic PTCs are characterized by PTC de-differentiation accompanied by loss of the endocytic receptor megalin, and megalin recycling is regulated by Rab11. Here we show that megalin plasma membrane localization is defective in CTNS-KO PTCs and its expression is rescued by treatment with CMA activators. Altogether, our data support that CMA upregulation has the potential to improve PTC function in cystinosis.
Project description:Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of selective soluble proteins. Lysosome-associated membrane protein type 2a (LAMP2A) is the key receptor protein of CMA; downregulation of LAMP2A leads to CMA blockade. Although CMA activation has been involved in cancer growth, CMA status and functions in non-small cell lung cancer (NSCLC) by focusing on the roles in regulating chemosensitivity remain to be clarified. In this study, we found that LAMP2A expression is elevated in NSCLC cell lines and patient's tumors, conferring poor survival and platinum resistance in NSCLC patients. LAMP2A knockdown in NSCLC cells suppressed cell proliferation and colony formation and increased the sensitivity to chemotherapeutic drugs in vitro. Furthermore, we found that intrinsic apoptosis signaling is the mechanism of cell death involved with CMA blockade. Remarkably, LAMP2A knockdown repressed tumorigenicity and sensitized the tumors to cisplatin treatment in NSCLC-bearing mice. Our discoveries suggest that LAMP2A is involved in the regulation of cancer malignant phenotypes and represents a promising new target against chemoresistant NSCLC.
Project description:Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags.
Project description:Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/?-synuclein accumulation in brain. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. <i>LRRK2</i> (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2<sup>R1441G</sup> knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition 'KFERQ'-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. However, such difference was not observed after the 'KFERQ'-motif was mutated. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. LAMP2A-knockdown reduced total lysosomal activity and clearance of 'KFERQ'-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. AR7 also attenuated the progressive accumulation of both intracellular and extracellular SNCA oligomers in prolonged cultures of mutant cortical neurons (DIV21), indicating that oligomer accumulation can be suppressed by CMA activation. Activation of autophagic pathways to reduce aged-related accumulation of pathogenic SNCA oligomers is a viable disease-modifying therapeutic strategy for PD.<b>Abbreviations:</b> 3-MA: 3-methyladenine; AR7: 7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; CMA: chaperone-mediated autophagy; CQ: chloroquine; CSF: cerebrospinal fluid; DDM: n-dodecyl ?-D-maltoside; DIV: days in vitro; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GWAS: genome-wide association studies; HSPA8/HSC70: heat shock protein 8; KFERQ: CMA recognition pentapeptide; KI: knockin; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LDH: lactate dehydrogenase; LRRK2: leucine-rich repeat kinase 2; MEF: mouse embryonic fibroblast; NDUFS4: NADH:ubiquinone oxidoreductase core subunit S4; NE: novel epitope; PD: Parkinson disease; RARA/RAR?: retinoic acid receptor, alpha; SNCA: synuclein, alpha; TUBB3/TUJ1: tubulin, beta 3 class III; WT: wild-type.
Project description:The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Here, we show CMA substrate accumulation in cystinotic kidney proximal tubule cells. We also found mislocalization of the CMA lysosomal receptor LAMP2A and impaired substrate translocation into the lysosome caused by defective CMA in cystinosis. The impaired LAMP2A trafficking and localization were rescued either by the expression of wild-type cystinosin or by the disease-associated point mutant CTNS-K280R, which has no cystine transporter activity. Defective LAMP2A trafficking in cystinosis was found to associate with decreased expression of the small GTPase Rab11 and the Rab7 effector RILP. Defective Rab11 trafficking in cystinosis was rescued by treatment with small-molecule CMA activators. RILP expression was restored by up-regulation of the transcription factor EB (TFEB), which was down-regulated in cystinosis. Although LAMP2A expression is independent of TFEB, TFEB up-regulation corrected lysosome distribution and lysosomal LAMP2A localization in Ctns-/- cells but not Rab11 defects. The up-regulation of Rab11, Rab7, or RILP, but not its truncated form RILP-C33, rescued LAMP2A-defective trafficking in cystinosis, whereas dominant-negative Rab11 or Rab7 impaired LAMP2A trafficking. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.