LAMP2A regulates cisplatin resistance in colorectal cancer through mediating autophagy.

BACKGROUND: Drug resistance is an important constraint on clinical outcomes in advanced cancers. LAMP2A is a limiting protein in molecular chaperone-mediated autophagy. This study was aimed to explore LAMP2A function in cisplatin (cis-diamminedichloroplatinum, DDP) resistance colorectal cancer (CRC) to seek new ideas for CRC clinical treatment. METHODS: In this study, LAMP2A expression was analyzed by molecular experimental techniques,such as qRT-PCR and western blot. Then, LAMP2A in cells was interfered by cell transfection experiments. Subsequently, the function of LAMP2A on proliferation, migration, invasion, DDP sensitivity, and autophagy of CRC/DDP cells were further investigated by a series of experiments, such as CCK-8, transwell, and western blot. RESULTS: We revealed that LAMP2A was clearly augmented in DDP-resistant CRC and was related to poor patient prognosis. Functionally, LAMP2A insertion remarkably CRC/DDP proliferation, migration, invasion ability and DDP resistance by strengthen autophagy. In contrast, LAMP2A knockdown limited the proliferation, migration, and invasion while heightened cellular sensitivity to DDP by restraining autophagy in CRC/DDP cells. Furthermore, LAMP2A silencing was able to curb tumor formation and enhance sensitivity to DDP in vivo. CONCLUSION: In summary, LAMP2A boosted malignant progression and DDP resistance in CRC/DDP cells through mediating autophagy. Clarifying LAMP2A function in DDP resistance is promising to seek cancer therapies biomarkers targeting LAMP2A activity.

Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

[Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells]. / åˆ†å­ä¼´ä¾£ä»‹å¯¼çš„è‡ªå™¬å¯¹èƒ†çº¢ç´ è¯±å¯¼çš„å°é¼ å°èƒ¶è´¨ç»†èƒžæŸä¼¤çš„å½±å“.

OBJECTIVES: To investigate the effect of chaperone-mediated autophagy (CMA) on the damage of mouse microglial BV2 cells induce by unconjugated bilirubin (UCB). METHODS: The BV2 cell experiments were divided into two parts. (1) For the CMA activation experiment: control group (treated with an equal volume of dimethyl sulfoxide), QX77 group (treated with 20 µmol/L QX77 for 24 hours), UCB group (treated with 40 µmol/L UCB for 24 hours), and UCB+QX77 group (treated with both 20 µmol/L QX77 and 40 µmol/L UCB for 24 hours). (2) For the cell transfection experiment: LAMP2A silencing control group (treated with an equal volume of dimethyl sulfoxide), LAMP2A silencing control+UCB group (treated with 40 µmol/L UCB for 24 hours), LAMP2A silencing group (treated with an equal volume of dimethyl sulfoxide), and LAMP2A silencing+UCB group (treated with 40 µmol/L UCB for 24 hours). The cell viability was assessed using the modified MTT method. The expression levels of p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by Western blot. The relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative polymerase chain reaction. Levels of IL-6 and TNF-α in the cell culture supernatant were measured using ELISA. The co-localization of heat shock cognate protein 70 with p65 and NLRP3 was detected by immunofluorescence. RESULTS: Compared to the UCB group, the cell viability in the UCB+QX77 group increased, and the expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as the mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α decreased (P<0.05). Compared to the control group, there was co-localization of heat shock cognate protein 70 with p65 and NLRP3 in both the UCB and UCB+QX77 groups. After silencing the LAMP2A gene, compared to the LAMP2A silencing control+UCB group, the LAMP2A silencing+UCB group showed increased expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as increased mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α (P<0.05). CONCLUSIONS: CMA is inhibited in UCB-induced BV2 cell damage, and activating CMA may reduce p65 and NLRP3 protein levels, suppress inflammatory responses, and counteract bilirubin neurotoxicity.

ALKBH1 promotes HIF-1α-mediated glycolysis by inhibiting N-glycosylation of LAMP2A.

ALKBH1 is a typical demethylase of nucleic acids, which is correlated with multiple types of biological processes and human diseases. Recent studies are focused on the demethylation of ALKBH1, but little is known about its non-demethylase function. Here, we demonstrate that ALKBH1 regulates the glycolysis process through HIF-1α signaling in a demethylase-independent manner. We observed that depletion of ALKBH1 inhibits glycolysis flux and extracellular acidification, which is attributable to reduced HIF-1α protein levels, and it can be rescued by reintroducing HIF-1α. Mechanistically, ALKBH1 knockdown enhances chaperone-mediated autophagy (CMA)-mediated HIF-1α degradation by facilitating the interaction between HIF-1α and LAMP2A. Furthermore, we identify that ALKBH1 competitively binds to the OST48, resulting in compromised structural integrity of oligosaccharyltransferase (OST) complex and subsequent defective N-glycosylation of LAMPs, particularly LAMP2A. Abnormal glycosylation of LAMP2A disrupts lysosomal homeostasis and hinders the efficient degradation of HIF-1α through CMA. Moreover, NGI-1, a small-molecule inhibitor that selectively targets the OST complex, could inhibit the glycosylation of LAMPs caused by ALKBH1 silencing, leading to impaired CMA activity and disruption of lysosomal homeostasis. In conclusion, we have revealed a non-demethylation role of ALKBH1 in regulating N-glycosylation of LAMPs by interacting with OST subunits and CMA-mediated degradation of HIF-1α.

TRPML1 contributes to antimony-induced nephrotoxicity by initiating ferroptosis via chaperone-mediated autophagy.

Evidence suggests that ferroptosis participates in kidney injury. However, the role of ferroptosis in antimony (Sb) induced nephrotoxicity and the mechanism are unknown. Here, we demonstrated that Sb induced injury in renal tubular epithelial cells (RTECs) and ferroptosis. Inhibition of ferroptosis reduced RTECs injury. Besides, elimination of reactive oxygen species (ROS) alleviated ferroptosis and RTECs injury. Moreover, exposure to Sb not only increased the co-localization of glutathione peroxidase 4 (GPX4) and LAMP1, but also decreased the levels of MEF2D and LRRK2, while increased the levels of HSC70, HSP90, and LAMP2a. These findings suggest that Sb activates chaperone-mediated autophagy (CMA), enhances lysosomal transport and subsequent degradation of GPX4, ultimately leads to ferroptosis. Additionally, up-regulation of lysosomal cationic channel, TRPML1, mitigated RTECs injury and ferroptosis. Mechanistically, up-regulation of TRPML1 mitigated the changes in CMA-associated proteins induced by Sb, diminished the binding of HSC70, HSP90, and TRPML1 with LAMP2a. Furthermore, NAC restored the decreased TRPML1 level caused by Sb. In summary, deficiency of TRPML1, secondary to increased ROS induced by Sb, facilitates the CMA-dependent degradation of GPX4, thereby leading to ferroptosis and RTECs injury. These findings provide insights into the mechanism underlying Sb-induced nephrotoxicity and propose TRPML1 as a promising therapeutic target.

LAMP2A, LAMP2B and LAMP2C: similar structures, divergent roles.

LAMP2 (lysosomal associated membrane protein 2) is one of the major protein components of the lysosomal membrane. There currently exist three LAMP2 isoforms, LAMP2A, LAMP2B and LAMP2C, and they vary in distribution and function. LAMP2A serves as a receptor and channel for transporting cytosolic proteins in a process called chaperone-mediated autophagy (CMA). LAMP2B is required for autophagosome-lysosome fusion in cardiomyocytes and is one of the components of exosome membranes. LAMP2C is primarily implicated in a novel type of autophagy in which nucleic acids are taken up into lysosomes for degradation. In this review, the current evidence for the function of each LAMP2 isoform in various pathophysiological processes and human diseases, as well as their possible mechanisms, are comprehensively summarized. We discuss the evolutionary patterns of the three isoforms in vertebrates and provide technical guidance on investigating these isoforms. We are also concerned with the newly arising questions in this particular research area that remain unanswered. Advances in the functions of the three LAMP2 isoforms will uncover new links between lysosomal dysfunction, autophagy and human diseases.Abbreviation: ACSL4: acyl-CoA synthetase long-chain family member 4; AD: Alzheimer disease; Ag: antigens; APP: amyloid beta precursor protein; ATG14: autophagy related 14; AVSF: autophagic vacuoles with unique sarcolemmal features; BBC3/PUMA: BCL2 binding component 3; CCD: C-terminal coiled coil domain; CMA: chaperone-mediated autophagy; CVDs: cardiovascular diseases; DDIT4/REDD1: DNA damage inducible transcript 4; ECs: endothelial cells; ER: endoplasmic reticulum; ESCs: embryonic stem cells; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/ß-glucocerebrosidase: glucosylceramidase beta; GSCs: glioblastoma stem cells; HCC: hepatocellular carcinoma; HD: Huntington disease; HSCs: hematopoietic stem cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IL3: interleukin 3; IR: ischemia-reperfusion; LAMP2: lysosomal associated membrane protein 2; LDs: lipid droplets; LRRK2: leucine rich repeat kinase 2; MA: macroautophagy; MHC: major histocompatibility complex; MST1: macrophage stimulating 1; NAFLD: nonalcoholic fatty liver disease; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NLRP3: NLR family pyrin domain containing 3; PARK7: Parkinsonism associated deglycase; PD: Parkinson disease; PEA15/PED: proliferation and apoptosis adaptor protein 15; PKM/PKM2: pyruvate kinase M1/2; RA: rheumatoid arthritis; RARA: retinoic acid receptor alpha; RCAN1: regulator of calcineurin 1; RCC: renal cell carcinoma; RDA: RNautophagy and DNautophagy; RNAi: RNA interference; RND3: Rho Family GTPase 3; SG-NOS3/eNOS: deleterious glutathionylated NOS3; SLE: systemic lupus erythematosus; TAMs: tumor-associated macrophages; TME: tumor microenvironment; UCHL1: ubiquitin C-terminal hydrolase L1; VAMP8: vesicle associated membrane protein 8.

LAMP2A, and other chaperone-mediated autophagy related proteins, do not decline with age in genetically heterogeneous UM-HET3 mice.

Chaperone-mediated autophagy (CMA) selectively degrades proteins that are crucial for glycolysis, fatty acid metabolism, and the progression of several age-associated diseases. Several previous studies, each of which evaluated males of a single inbred mouse or rat strain, have reported that CMA declines with age in many tissues, attributed to an age-related loss of LAMP2A, the primary and indispensable component of the CMA translocation complex. This has led to a paradigm in the field of CMA research, stating that the age-associated decline in LAMP2A in turn decreases CMA, contributing to the pathogenesis of late-life disease. We assessed LAMP2A levels and CMA substrate uptake in both sexes of the genetically heterogeneous UM-HET3 mouse stock, which is the current global standard for the evaluation of anti-aging interventions. We found no evidence for age-related changes in LAMP2A levels, CMA substrate uptake, or whole liver levels of CMA degradation targets, despite identifying sex differences in CMA.

Benzo(a)pyrene regulates chaperone-mediated autophagy via heat shock protein 90.

AIMS: Some studies have shown that the Benzo(a)pyrene (BaP) exposure induced oxidative damage, DNA damage and autophagy, but the molecular mechanism is not clear. Heat shock protein 90 (HSP90) is regarded as an important target in cancer therapy and a key factor in autophagy. Therefore, this study aims to clarify the new mechanism of BaP regulating CMA through HSP90. MAIN METHODS: C57BL mice were fed with BaP at a dose of 25.3 mg/kg. A549 cells were treated with different concerntrations of BaP, and MTT assay was used to observe the effect of BaP on the proliferation of A549 cells. DNA damage was detected by alkaline comet assay. Focus experiment for detection of γ-H2AX by immunofluorescence. The mRNA expression of HSP90, HSC70 and Lamp-2a was detected by qPCR. The protein expressions of HSP90, HSC70 and Lamp-2a were detected by Western blot. Next, we knocked down HSP90 expression by the HSP90 Inhibitor, NVP-AUY 922, exposed or HSP90α shRNA lentivirus transduction in A549 cells. KEY FINDINGS: In these studies, we first found that heat shock protein 90 (HSP90), heat shock cognate 70 (HSC70) and lysosomal-associated membrane protein type 2 receptor (Lamp-2a) expressions of C57BL mice lung tissue and A549 cells exposed to BaP were significant increase, as well as BaP induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by comet assay and γ-H2AX foci analysis in A549 cells. Our results demonstrated BaP induced CMA and caused DNA damage. Next, we knocked down HSP90 expression by the HSP90 Inhibitor, NVP-AUY 922, exposed or HSP90α shRNA lentivirus transduction in A549 cells. HSC70 and Lamp-2a expressions of these cells exposed to BaP were not significant increase, which showed that BaP inducted CMA was mediated by HSP90. Further, HSP90α shRNA prevented BaP induced of BaP which suggested BaP regulated CMA and caused DNA damage by HSP90. Our results elucidated a new mechanism of BaP regulated CMA through HSP90. SIGNIFICANCE: BaP regulated CMA through HSP90. HSP90 is involved in the regulation of gene instability induced by DNA damage by BaP, which promotes CMA. Our study also revealed that BaP regulates CMA through HSP90. This study fills the gap of the effect of BaP on autophagy and its mechanism, which will lead to a more comprehensive understanding of the action mechanism of BaP.

Knockout validation of LAMP2A antibodies for immunostaining in human cancer cells.

LAMP2A is the rate-limiting factor of chaperone-mediated autophagy (CMA), a unique selective protein degradative pathway. To date LAMP2A antibodies are not knockout (KO)-validated in human cells. We have recently generated human isoform-specific LAMP2A KO cells, and here we assessed the specificity of select commercial LAMP2A antibodies on wild-type and LAMP2A KO human cancer cells. While all tested antibodies were suitable for immunoblotting, the anti-LAMP2A antibody (ab18528) is likely to exhibit an off-target reactivity in immunostaining approaches using human cancer cells, and alternative antibodies, which seem more appropriate, are available.

Thyroid hormone upregulates LAMP2 expression and lysosome activity.

Thyroid hormone (T3)-induced autophagy and its biological significance have been extensively investigated in recent years. However, limited studies to date have focused on the important role of lysosomes in autophagy. In this study, we explored the effects of T3 on lysosomal protein expression and trafficking in detail. Our findings showed that T3 activates rapid lysosomal turnover and expression of numerous lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, in a thyroid hormone receptor-dependent manner. In a murine model, LAMP2 protein was specifically induced in mice with hyperthyroidism. T3-promoted microtubule assembly was significantly disrupted by vinblastine, resulting in accumulation of the lipid droplet marker PLIN2. In the presence of the lysosomal autophagy inhibitors bafilomycin A1, chloroquine and ammonium chloride, we observed substantial accumulation of LAMP2 but not LAMP1 protein. T3 further enhanced the protein levels of ectopically expressed LAMP1 and LAMP2. Upon knockdown of LAMP2, cavities of lysosomes and lipid droplets accumulated in the presence of T3, although the changes in LAMP1 and PLIN2 expression were less pronounced. More specifically, the protective effect of T3 against ER stress-induced death was abolished by knockdown of LAMP2. Our collective results indicate that T3 not only promotes lysosomal gene expression but also LAMP protein stability and microtubule assembly, leading to enhancement of lysosomal activity in digesting any additional autophagosomal burden.

Chaperone-mediated autophagy promotes breast cancer angiogenesis via regulation of aerobic glycolysis.

Evidence shows that chaperone-mediated autophagy (CMA) is involved in cancer cell pathogenesis and progression. However, the potential role of CMA in breast cancer angiogenesis remains unknown. We first manipulated CMA activity by knockdown and overexpressing of lysosome-associated membrane protein type 2A (LAMP2A) in MDA-MB-231, MDA-MB-436, T47D and MCF7 cells. We found that the tube formation, migration and proliferation abilities of human umbilical vein endothelial cells (HUVECs) were inhibited after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A knockdown. While the above changes were promoted after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A overexpression. Moreover, we found that CMA could promote VEGFA expression in breast cancer cells and in xenograft model through upregulating lactate production. Finally, we found that lactate regulation in breast cancer cells is hexokinase 2 (HK2) dependent, and knockdown of HK2 can significantly reduce the ability of CMA-mediated tube formation capacity of HUVECs. Collectively, these results indicate that CMA could promote breast cancer angiogenesis via regulation of HK2-dependent aerobic glycolysis, which may serve as an attractive target for breast cancer therapies.

A rare and fatal cause of hypertrophic cardiomyopathy: Danon disease.

Danon disease is a rare and fatal disease caused by a mutation in the lysosome-associated membrane protein 2 gene. Impaired intracellular autophagy causes lysosomal vacuoles to accumulate mainly in myocardial and skeletal muscle cells, leading to hypertrophic cardiomyopathy, skeletal myopathy, and varying degrees of intellectual disability. Two distinct childhood presentations of Danon disease are described in this report.

Experimental diabetes exacerbates autophagic flux impairment during myocardial I/R injury through calpain-mediated cleavage of Atg5/LAMP2.

To explore the role of autophagic flux in the increased susceptibility of the experimental diabetic heart to ischaemia-reperfusion (I/R) injury, we established STZ-induced diabetic mice and performed I/R. In vitro, neonatal mouse cardiomyocytes were subjected to high glucose and hypoxia/reoxygenation challenge to mimic diabetic I/R injury. We found that experimental diabetes aggravated I/R-induced injury than compared with nondiabetic mice. Autophagic flux was impaired in I/R hearts, and the impairment was exacerbated in diabetic mice subjected to I/R with defective autophagosome formation and clearance. Calpains, calcium-dependent thiol proteases, were upregulated and highly activated after I/R of diabetes, while calpain inhibition attenuated cardiac function and cell death and partially restored autophagic flux. The expression levels of Atg5 and LAMP2, two crucial autophagy-related proteins, were significantly degraded in diabetic I/R hearts, alterations that were associated with calpain activation and could be reversed by calpain inhibition. Co-overexpression of Atg5 and LAMP2 reduced myocardial injury and normalized autophagic flux. In conclusion, experimental diabetes exacerbates autophagic flux impairment of cardiomyocytes under I/R stress, resulting in worse I/R-induced injury. Calpain activation and cleavage of Atg5 and LAMP2 at least partially account for the deterioration of autophagic flux impairment.

LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development.

Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells.

Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.

Immunohistochemical Detection of the Chaperone-Mediated Autophagy Markers LAMP2A and HSPA8 in Formalin-Fixed and Paraffin-Embedded Tissues.

Autophagy is crucial for maintaining cellular homeostasis and its deregulation is involved in disease development, including cancer. The key players of chaperone-mediated autophagy (CMA), a particular selective subtype of autophagy, are HSPA8 and LAMP2A. Both proteins can be immunohistochemically detected in formalin-fixed paraffin-embedded (FFPE) tissue. LAMP2A is frequently overexpressed in a variety of cancers where it likely supports cancer cell survival and resistance to anti-cancer therapies in a context-dependent manner. Here we present the immunohistochemical staining protocol of antibodies against LAMP2A and HSPA8, using an automated staining system, suitable for routine diagnostics. Additionally, we also suggest a staining evaluation method.

Germline FOXJ2 overexpression causes male infertility via aberrant autophagy activation by LAMP2A upregulation.

Spermatogenesis is a complex biological process that produces haploid spermatozoa and requires precise regulation by many tissue-specific factors. In this study, we explored the role and mechanism of Fork head box J2 (FOXJ2, which is highly expressed in spermatocytes) in the regulation of spermatogenesis using a germline-specific conditional Foxj2 knock-in mouse model (Stra8-Cre; Foxj2 tg/tg mouse). Foxj2 overexpression in mouse testes led to spermatogenesis failure, which started at the initiation of meiosis, and resulted in male infertility. Lysosomes and autophagy-related genes were upregulated in Stra8-cre; Foxj2 tg/tg mouse testes and the number of autolysosomes in the spermatocytes in Stra8-cre; Foxj2 tg/tg mice was increased. Chromatin immunoprecipitation-PCR and Dual-luciferase reporter assays showed that Lamp2 (encoding lysosome-associated membrane protein-2) was a target of FOXJ2. Foxj2 overexpression increased the expression levels of Lamp2a and Hsc70 (70-kDa cytoplasmic heat shock protein) in the Stra8-cre; Foxj2 tg/tg mouse testes. Our results suggested that Foxj2 overexpression in the germ cells of mouse testes affects chaperone-mediated autophagy by upregulating LAMP2A, leading to spermatogenesis failure at the initiation of meiosis, thus resulting in male infertility. Our findings provide a new insight into the function of FOXJ2 in spermatogenesis and the significance of autophagy regulation in spermatogenesis.

Chaperone-mediated autophagy protects cardiomyocytes against hypoxic-cell death.

Chaperone-mediated autophagy (CMA) is a chaperone-dependent process of selective cytosolic protein turnover that targets specific proteins to lysosomes for degradation. Enhancing protein degradation mechanisms has been shown to be beneficial in multiple models of cardiac disease, including myocardial infarction (MI) and ischemia-reperfusion (I/R) injury. However, the causal role of CMA in cardiomyocyte injury and death is largely unknown. Hypoxia is an important contributor to both MI and I/R damage, which are major, precedent causes of heart failure. Upregulating CMA was hypothesized to protect against hypoxia-induced cardiomyocyte death. Lysosome-associated membrane protein 2a (Lamp2a) overexpression and knockdown were used to causally study CMA's role in hypoxically stressed cardiomyocytes. LAMP2a protein levels were used as both a primary indicator and driver of CMA function. Hypoxic stress was stimulated by CoCl2 treatment, which increased LAMP2a protein levels (+1.4-fold) and induced cardiomyocyte apoptosis (+3.2-4.0-fold). Lamp2a siRNA knockdown (-3.2-fold) of control cardiomyocytes increased apoptosis (+1.8-fold) suggesting that loss of CMA is detrimental for cardiomyocyte survival. However, there was neither an additive nor a synergistic effect on cell death when Lamp2a-silenced cells were treated with CoCl2. Conversely, Lamp2a overexpression (+3.0-fold) successfully reduced hypoxia-induced apoptosis by ∼50%. LAMP2a was also significantly increased (+1.7-fold) in ischemic heart failure patient samples, similar to hypoxically stressed cardiomyocytes. The failing ischemic hearts may have had insufficient CMA activation. To our knowledge, this study for the first time establishes a protective role for CMA (via Lamp2a overexpression) against hypoxia-induced cardiomyocyte loss and reveals the intriguing possibility that CMA activation may offer a cardioprotective treatment for ischemic heart disease.

LAMP2A regulates the loading of proteins into exosomes.

Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.