Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival

Autophagy is a conserved mechanism responsible for the continuous clearance of unnecessary organelles or misfolded proteins in lysosomes. Three types of autophagy have been reported in the difference of substrate delivery to lysosome: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Among these types, CMA is a unique autophagy system that selectively degrades substrates detected by heat shock cognate protein 70 (HSC70). Recently, autophagic cell death has been reported to be involved in neuronal death following brain ischemia; however, the contribution of CMA to neuronal death/survival after ischemic stress has not been addressed. In the present study, we determined whether quantitative alterations in LAMP-2A, which is the key molecule in CMA, would modulate neuronal cell survival under hypoxic conditions. Incubation of Neuro2A cells in a hypoxic chamber (1% O(2), 5% CO(2)) increased the level of LAMP-2A and induced accumulation of LAMP-2A-positive lysosomes in the perinuclear area, which is a hallmark of CMA activation. The activation of CMA in response to hypoxia was also confirmed by the GAPDH-HaloTag CMA indicator system at the single cell level. Next, we asked whether CMA was involved in cell survival during hypoxia. Blocking LAMP-2A expression with siRNA increased the level of cleaved caspase-3 and the number of propidium iodide-positive cells after hypoxic stress regardless of whether macroautophagy could occur, whereas the administration of mycophenolic acid, a potent CMA activator, rescued hypoxia-mediated cell death. Finally, we asked whether CMA was activated in the neurons after middle cerebral artery occlusion in vivo. The expression of LAMP-2A was significantly increased in the ischemic hemisphere seven days after brain ischemia. These results indicate that CMA is activated during hypoxia and contributes to the survival of cells under these conditions.     

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.     

Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease

The presence of α-synuclein aggregates in the characteristic Lewy body pathology seen in idiopathic Parkinson''s disease (PD), together with α-synuclein gene mutations in familial PD, places α-synuclein at the center of PD pathogenesis. Decreased levels of the chaperone-mediated autophagy (CMA) proteins LAMP-2A and hsc70 in PD brain samples suggests compromised α-synuclein degradation by CMA may underpin the Lewy body pathology. Decreased CMA protein levels were not secondary to the various pathological changes associated with PD, including mitochondrial respiratory chain dysfunction, increased oxidative stress and proteasomal inhibition. However, decreased hsc70 and LAMP-2A protein levels in PD brains were associated with decreases in their respective mRNA levels. MicroRNA (miRNA) deregulation has been reported in PD brains and we have identified eight miRNAs predicted to regulate LAMP-2A or hsc70 expression that were reported to be increased in PD. Using a luciferase reporter assay in SH-SY5Y cells, four and three of these miRNAs significantly decreased luciferase activity expressed upstream of the lamp-2a and hsc70 3′UTR sequences respectively. We confirmed that transfection of these miRNAs also decreased endogenous LAMP-2A and hsc70 protein levels respectively and resulted in significant α-synuclein accumulation. The analysis of PD brains confirmed that six and two of these miRNAs were significantly increased in substantia nigra compacta and amygdala respectively. These data support the hypothesis that decreased CMA caused by miRNA-induced downregulation of CMA proteins plays an important role in the α-synuclein pathology associated with PD, and opens up a new avenue to investigate PD pathogenesis.     

The chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane

Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane.     

AMPK-dependent phosphorylation of lipid droplet protein PLIN2 triggers its degradation by CMA

Lipids stored in lipid droplets are hydrolyzed via either cytosolic lipases or a selective form of macroautophagy known as lipophagy. We recently demonstrated that chaperone-mediated autophagy (CMA) is required for the initiation of lipolysis by either of these independent lipolytic pathways. CMA selectively degrades the lipid droplet proteins perilipins (PLIN) 2 and 3 from the lipid droplet surface, thus, facilitating the recruitment of cytosolic lipases and autophagy effector proteins to the lipid droplets. PLIN2 phosphorylation was observed upon induction of lipolysis, but the phosphorylating kinase and the relation of this phosphorylation with CMA of PLIN2 remained unknown. Here, we report that phosphorylation of PLIN2 is dependent on AMPK and occurs after the interaction of PLIN2 with the CMA chaperone HSPA8/Hsc70. Our results highlight a role for posttranslational modifications in priming proteins to be amenable for degradation by CMA.     

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

LAMP2A 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.     

Structure of Transmembrane Domain of Lysosome-associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-mediated Autophagy

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.     

Chaperone-mediated autophagy prevents apoptosis by degrading BBC3/PUMA

Autophagy is a potentially inimical pathway and together with apoptosis, may be activated by similar stress stimuli that can lead to cell death. The molecular cues that dictate the cell fate choice between autophagy and apoptosis remain largely unknown. Here we report that the proapoptotic protein BBC3/PUMA (BCL2 binding component 3) is a bona fide substrate of chaperone-mediated autophagy (CMA). BBC3 associates with HSPA8/HSC70 (heat shock 70kDa protein 8), leading to its lysosome translocation and uptake. Inhibition of CMA results in stabilization of BBC3, which in turn sensitizes tumor cells to undergo apoptosis. We further demonstrate that upon TNF (tumor necrosis factor) treatment, IKBKB/IKKβ (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase β)-mediated BBC3 Ser10 phosphorylation is crucial for BBC3 stabilization via blocking its degradation by CMA. Mechanistically, Ser10 phosphorylation facilitates BBC3 translocation from the cytosol to mitochondria. BBC3 stabilization resulting from either Ser10 phosphorylation or CMA inhibition potentiates TNF-induced apoptotic cell death. Our findings thus reveal that the selective degradation of BBC3 underlies the prosurvival role of CMA and define a previously unappreciated proapoptotic role of IKBKB that acts through phosphorylation-mediated stabilization of BBC3, thereby promoting TNF-triggered apoptosis.     

Discovery of a novel type of autophagy targeting RNA

Regulated degradation of cellular components by lysosomes is essential to maintain biological homeostasis. In mammals, three forms of autophagy, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), have been identified. Here, we showed a novel type of autophagy, in which RNA is taken up directly into lysosomes for degradation. This pathway, which we term “RNautophagy,” is ATP-dependent, and unlike CMA, is independent of HSPA8/Hsc70. LAMP2C, a lysosomal membrane protein, serves as a receptor for this pathway. The cytosolic tail of LAMP2C specifically binds to almost all total RNA derived from mouse brain. The cytosolic sequence of LAMP2C and its affinity for RNA are evolutionarily conserved from nematodes to humans. Our findings shed light on the mechanisms underlying RNA homeostasis in higher eukaryotes.     

Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation

Chaperone-mediated autophagy (CMA), a selective form of protein lysosomal degradation, is maximally activated in stress situations to ensure maintenance of cellular homeostasis. CMA activity decreases with age and in several human chronic disorders, but in contrast, in most cancer cells, CMA is upregulated and required for tumor growth. However, the role of CMA in malignant transformation remains unknown. In this study, we demonstrate that CMA inhibition in fibroblasts augments the efficiency of MYC/c-Myc-driven cellular transformation. CMA blockage contributes to the increase of total and nuclear MYC, leading to enhancement of cell proliferation and colony formation. Impaired CMA functionality accentuates tumorigenesis-related metabolic changes observed upon MYC-transformation. Although not a direct CMA substrate, we have found that CMA regulates cellular MYC levels by controlling its proteasomal degradation. CMA promotes MYC ubiquitination and degradation by regulating the degradation of C330027C09Rik/KIAA1524/CIP2A (referred to hereafter as CIP2A), responsible for MYC stabilization. Ubiquitination and proteasomal degradation of MYC requires dephosphorylation at Ser62, and CIP2A inhibits the phosphatase responsible for this dephosphorylation. Failure to degrade CIP2A upon CMA blockage leads to increased levels of phosphorylated MYC (Ser62) and to stabilization of this oncogene. We demonstrate that this phosphorylation is essential for the CMA-mediated effect, since specific mutation of this site (Ser62 to Ala62) is enough to normalize MYC levels in CMA-incompetent cells. Altogether these data demonstrate that CMA mitigates MYC oncogenic activity by promoting its proteasomal degradation and reveal a novel tumor suppressive role for CMA in nontumorigenic cells.     

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.     

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats

Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis.     

Hispolon promotes MDM2 downregulation through chaperone-mediated autophagy

Amplification and overexpression of murine double minute (MDM2) has been observed in several human cancers. Some chemotherapeutic agents cause MDM2 ubiquitination and degradation in a proteasome-dependent system. In addition to the proteasome system, chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective misfolded protein degradation. Molecular chaperone heat shock cognate 70 protein (Hsc70) recognizes the misfolded proteins, which are then delivered to lysosome-associated membrane protein type 2A (LAMP2A) for lysosomal degradation. Our previous study reported that hispolon was able to induce cell apoptosis and downregulate MDM2 expression. In this study, our results showed that the proteasome inhibitor, MG132, could not inhibit hispolon-induced MDM2 downregulation. In contrast, both inhibition of lysosomes with NH₄Cl and inhibition of LAMP2A using siRNA partially attenuated hispolon-induced MDM2 downregulation. To determine whether Hsc70 recognizes MDM2 on amino acids 135-141, SMP14 antibody was used to compete with Hsc70 for interaction with MDM2. After Hsc70 knockdown, SMP14 antibody immunoprecipitated increased MDM2. We also found that hispolon induced increased association of Hsp70, Hsc70, Hsp90 and LAMP2A with MDM2. This association was inhibited in cells pretreated with geldanamycin (GA), an Hsp90 inhibitor. GA also attenuated hispolon-induced MDM2 downregulation. Meanwhile, inhibition of Hsc70 using siRNA attenuated hispolon-induced MDM2 downregulation. Our study provides the first example of the ability of hispolon to mediate MDM2 downregulation in lysosomes through the CMA pathway.     

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.     

Lysosome-associated protein 1 (LAMP-1) and Lysosome-associated protein 2 (LAMP-2) in a larger family carrier of Fabry disease

This study investigated the potential relationship between the expression levels of lysosome-associated membrane proteins (LAMP) 1 and 2 and responses to enzyme replacement therapy (ERT) in the members of a single family with Fabry disease (FD). LAMP levels were assessed by flow cytometry in leukocytes from 17 FD patients who received an eight-month course of ERT course and 101 healthy individuals. We found that phagocytic cells from the FD patients had higher expression levels of both LAMP-1 and LAMP-2, relative to the levels in phagocytes from the healthy controls (p = 0.001). Furthermore, the LAMP-1 and LAMP-2 levels in phagocytes from the FD carriers continuously decreased with ERT administration to reach levels similar to those in healthy controls. We suggest that LAMP-1 and LAMP-2 could be used as additional markers with which to assess ERT effectiveness in FD.