Objective: Several clinical trials have suggested that autophagy inhibition is a promising approach for cancer therapy. However, the implications of autophagy in ameloblastoma (AB) remain undiscovered. This study investigated the dysregulated autophagy and its regulatory mechanisms in AB. Methods: The expression and distribution of autophagy-related proteins including B-cell lymphoma-2-interacting protein-1 (Beclin1), microtubule-associated protein 1 light chain 3 (LC3) II/I and lysosomal associated membrane protein 2 (LAMP2) were detected in AB and normal oral mucosa (NOM) tissues by immunohistochemistry and western blot analyses. Under transmission electron microscopy, the autophagy of AB was observed. LAMP2 was a potential target mRNA of miR-1-3p. Quantitative Real-time PCR (qRT-PCR) analysis was utilized for examining LAMP2 and miR-1-3p in AB tissues as well as AM-1 cells. The correlation between LAMP2 and miR-1-3p was analyzed in AB. After transfection with miR-1-3p mimic or inhibitor, LAMP2 expression, proliferation, migration, and invasion were separately detected in AM-1 cells. Rescue assays were finally presented. Results: Our results showed that Beclin1 was lowly expressed as well as LC3II/I and LAMP2 were highly expressed in AB. Autophagosomes were observed in AB. MiR-1-3p was lowly expressed in AB, which exhibited negative correlations to LAMP2 expression. MiR-1-3p up-regulation significantly lowered LAMP2 expression in AM-1 cells. Furthermore, miR-1-3p overexpression restrained proliferative, migrated, and invasive capacities of AM-1 cells, which were ameliorated by LAMP2 overexpression. Conclusion: Our findings demonstrated that miR-1-3p suppressed malignant phenotypes of AB through down-regulating LAMP2-mediated autophagy, which could become an underlying target for AB therapy.

The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds.

Metastatic gastric cancer (GC) has a poor prognosis, and elucidating the molecular mechanisms involved in metastasis may lead to the development of novel therapeutic modalities.

A cDNA encoding the chicken orthologue of dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208 was cloned by RT-PCR from RNA isolated from mature chicken bone marrow-derived dendritic cells (chBM-DCs). The cloned chicken DC-LAMP (chDC-LAMP) cDNA consists of 1281 nucleotides encoding an open reading frame of 426 amino acids (aa). Comparison of the deduced aa sequence of DC-LAMP with orthologous proteins from human and mouse revealed 27 and 24% identity, respectively. The predicted chDC-LAMP protein shares the characteristic features of LAMP family members. ChDC-LAMP mRNA, unlike its mammalian orthologues, was expressed in a wide range of tissues, at highest levels in the lung. Lymphoid tissues including thymus, spleen, bursa, ceacal tonsil and Meckel's diverticulum had high chDC-LAMP mRNA expression levels. ChDC-LAMP mRNA was expressed in all splenocyte subsets with the highest expression in Bu-1(+) B cells and KUL01(+) cells, which would include macrophages and DC. ChDC-LAMP mRNA was highly expressed in chBM-DC, whereas expression levels in chicken monocyte-derived macrophages (chMo-Mac) and the HD11 macrophage cell line were significantly lower. Following CD40L stimulation, chDC-LAMP mRNA expression levels were up-regulated in mature chBM-DC, chMo-Mac and HD11 cells whereas lipopolysaccharide (LPS) only up-regulated chDC-LAMP mRNA expression levels in chBM-DC. ChDC-LAMP is not solely expressed on chicken DC but can be used as a marker to differentiate between immature and mature DC.

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

Microtubule-associated protein 1 light chain 3 gamma (MAP1LC3C or LC3C) is a member of the microtubule-associated family of proteins that are essential in the formation of autophagosomes and lysosomal degradation of cargo. LC3C has tumor-suppressing activity, and its expression is dependent on kidney cancer tumor suppressors, such as von Hippel-Lindau protein and folliculin. Recently, we demonstrated that LC3C autophagy is regulated by noncanonical upstream regulatory complexes and targets for degradation postdivision midbody rings associated with cancer cell stemness. Here, we show that loss of LC3C leads to peripheral positioning of the lysosomes and lysosomal exocytosis (LE). This process is independent of the autophagic activity of LC3C. Analysis of isogenic cells with low and high LE shows substantial transcriptomic reprogramming with altered expression of zinc (Zn)-related genes and activity of polycomb repressor complex 2, accompanied by a robust decrease in intracellular Zn. In addition, metabolomic analysis revealed alterations in amino acid steady-state levels. Cells with augmented LE show increased tumor initiation properties and form aggressive tumors in xenograft models. Immunocytochemistry identified high levels of lysosomal-associated membrane protein 1 on the plasma membrane of cancer cells in human clear cell renal cell carcinoma and reduced levels of Zn, suggesting that LE occurs in clear cell renal cell carcinoma, potentially contributing to the loss of Zn. These data indicate that the reprogramming of lysosomal localization and Zn metabolism with implication for epigenetic remodeling in a subpopulation of tumor-propagating cancer cells is an important aspect of tumor-suppressing activity of LC3C.

Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia‑associated neuronal death in non‑human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK‑801 and the calcium chelator ethylene glycol‑bis (2‑aminoethylether)‑N, N, N', N'‑tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal‑associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α‑fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N‑Acetyl‑L‑cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity.

Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death.

Lysosomal membrane permeabilization (LMP) is observed under many pathological conditions, leading to cellular dysfunction and death. However, the mechanisms by which lysosomal membranes become leaky in vivo are not clear. Our data demonstrate that LMP occurs in neurons following controlled cortical impact induced (CCI) traumatic brain injury (TBI) in mice, leading to impaired macroautophagy (autophagy) and neuronal cell death. Comparison of LC-MS/MS lysosomal membrane lipid profiles from TBI and sham animals suggested a role for PLA2G4A/cPLA2 (phospholipase A2, group IVA [cytosolic, calcium-dependent]) in TBI-induced LMP. Activation of PLA2G4A caused LMP and inhibition of autophagy flux in cell lines and primary neurons. In vivo pharmacological inhibition of PLA2G4A attenuated TBI-induced LMP, as well as subsequent impairment of autophagy and neuronal loss, and was associated with improved neurological outcomes. Inhibition of PLA2G4A in vitro limited amyloid-β-induced LMP and inhibition of autophagy. Together, our data indicate that PLA2G4A -mediated lysosomal membrane damage is involved in neuronal cell death following CCI-induced TBI and potentially in other neurodegenerative disorders.Abbreviations: AACOCF3, arachidonyl trifluoromethyl ketone; ACTB/β-actin, actin, beta; AD, Alzheimer disease; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG12, autophagy related 12; BECN1, beclin 1, autophagy related; C1P, ceramide-1-phosphate; CCI, controlled cortical impact; CTSD, cathepsin D; CTSL, cathepsin L; GFP, green fluorescent protein; IF, immunofluorescence; LAMP1, lysosomal-associated membrane protein 1; LAMP2, lysosomal-associated membrane protein 2; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LMP, Lysosomal membrane permeabilization; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; MAP1LC3/LC3, microtuble-associated protein 1 light chain 3; NAGLU, alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); PC, diacyl glycerophosphatidylcholine; PE, diacyl glycerophosphatidylethanolamine; PE-O, plasmanyl glycerophosphatidylethanolamine; PE-P, plasmenyl glycerophosphatidylethanolamine; PLA2G4A/cPLA2, phospholipase A2, group IVA (cytosolic, calcium-dependent); RBFOX3, RNA binding protein, fox-1 homolog (C. elegans) 3; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUBA1/α-tubulin, tubulin, alpha; TBI, traumatic brain injury; TFEB, transcription factor EB; ULK1, unc-51 like kinase 1.

Lysosome plays important roles in cellular homeostasis, and its dysregulation contributes to tumor growth and survival. However, the understanding of regulation and the underlying mechanism of lysosome in cancer survival is incomplete. Here, we reveal a role for a histone acetylation-regulated long noncoding RNA termed lysosome cell death regulator (LCDR) in lung cancer cell survival, in which its knockdown promotes apoptosis. Mechanistically, LCDR binds to heterogenous nuclear ribonucleoprotein K (hnRNP K) to regulate the stability of the lysosomal-associated protein transmembrane 5 (LAPTM5) transcript that maintains the integrity of the lysosomal membrane. Knockdown of LCDR, hnRNP K, or LAPTM5 promotes lysosomal membrane permeabilization and lysosomal cell death, thus consequently resulting in apoptosis. LAPTM5 overexpression or cathepsin B inhibitor partially restores the effects of this axis on lysosomal cell death in vitro and in vivo. Similarly, targeting LCDR significantly decreased tumor growth of patient-derived xenografts of lung adenocarcinoma (LUAD) and had significant cell death using nanoparticles (NPs)-mediated systematic short interfering RNA delivery. Moreover, LCDR/hnRNP K/LAPTM5 are up-regulated in LUAD tissues, and coexpression of this axis shows the increased diagnostic value for LUAD. Collectively, we identified a long noncoding RNA that regulates lysosome function at the posttranscriptional level. These findings shed light on LCDR/hnRNP K/LAPTM5 as potential therapeutic targets, and targeting lysosome is a promising strategy in cancer treatment.

Purpose: Emerging evidence suggests that 27-Hydroxycholesterol (27-OHC) causes neurodegenerative diseases through the induction of cytotoxicity and cholesterol metabolism disorder. The objective of this study is to determine the impacts of 27-OHC on lysosomal membrane permeabilization (LMP) and pyroptosis in neurons in the development of neural degenerative diseases. Methods: In this study, SH-SY5Y cells and C6 cells were co-cultured in vitro to investigate the influence of 27-OHC on the function of lysosome, LMP and pyroptosis related factors in neuron. Lyso Tracker Red (LTR) was used to detect the changes of lysosome pH, volume and number. Acridine orange (AO) staining was also used to detect the LMP in neurons. Then the morphological changes of cells were observed by a scanning electron microscope (SEM). The content of lysosome function associated proteins [including Cathepsin B (CTSB), Cathepsin D (CTSD), lysosomal-associated membraneprotein-1 (LAMP-1), LAMP-2] and the pyroptosis associated proteins [including nod-like recepto P3 (NLRP3), gasdermin D (GSDMD), caspase-1 and interleukin (IL)-1β] were detected through Western blot. Results: Results showed higher levels of lysosome function associated proteins, such as CTSB (p < 0.05), CTSD (p < 0.05), LAMP-1 (p < 0.01), LAMP-2; p < 0.01) in 27-OHC treated group than that in the control group. AO staining and LTR staining showed that 27-OHC induced lysosome dysfunction with LMP. Content of pyroptosis related factor proteins, such as GSDMD (p < 0.01), NLRP3 (p < 0.001), caspase-1 (p < 0.01) and IL-1β (p < 0.01) were increased in 27-OHC treated neurons. Additionally, CTSB was leaked through LMP into the cytosol and induced pyroptosis. Results from the present study also suggested that the CTSB is involved in activation of pyroptosis. Conclusion: Our data indicate that 27-OHC contributes to the pathogenesis of cell death by inducing LMP and pyroptosis in neurons.

Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALR: autophagic lysosome reformation; ATG7: autophagy related 7; CTSB: cathepsin B; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HPCA: hippocalcin; i.c.v: intracerebroventricular; KEGG: kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; LSDs: lysosomal storage disorders; MAP2: microtubule-associated protein 2; MCAO: middle cerebral artery occlusion; mCTSB: mature CTSB; mCTSD: mature CTSD; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PBS: phosphate-buffered saline; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; proCTSD: pro-cathepsin D; RAPA: rapamycin; RNA-seq: RNA sequencing; RPS6KB/p70S6K: ribosomal protein S6 kinase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM: Structured Illumination Microscopy; SNAP25: synaptosomal-associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYT1: synaptotagmin I; TBST: tris-buffered saline Tween-20; TEM: transmission electron microscopy; TFEB: transcription factor EB; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; TUBB3: tubulin, beta 3 class III.

Autophagy and lysosomal activities play a key role in the cell by initiating and carrying out the degradation of misfolded proteins. Transcription factor EB (TFEB) functions as a master controller of lysosomal biogenesis and function during lysosomal stress, controlling most but, importantly, not all lysosomal genes. Here, we sought to better understand the regulation of lysosomal genes whose expression does not appear to be controlled by TFEB. Sixteen of these genes were screened for transactivation in response to diverse cellular insults. mRNA levels for lysosomal-associated membrane protein 3 (LAMP3), a gene that is highly up-regulated in many forms of cancer, including breast and cervical cancers, were significantly increased during the integrated stress response, which occurs in eukaryotic cells in response to accumulation of unfolded and misfolded proteins. Of note, results from siRNA-mediated knockdown of activating transcription factor 4 (ATF4) and overexpression of exogenous ATF4 cDNA indicated that ATF4 up-regulates LAMP3 mRNA levels. Finally, ChIP assays verified an ATF4-binding site in the LAMP3 gene promoter, and a dual-luciferase assay confirmed that this ATF4-binding site is indeed required for transcriptional up-regulation of LAMP3 These results reveal that ATF4 directly regulates LAMP3, representing the first identification of a gene for a lysosomal component whose expression is directly controlled by ATF4. This finding may provide a key link between stresses such as accumulation of unfolded proteins and modulation of autophagy, which removes them.

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth in vivo. Thus, the necessary impact of integral lysosomal function in cell rescue and death were illustrated.

Canine malignant mammary gland tumors present with a poor prognosis due to metastasis to other organs, such as lung and lymph node metastases. Unlike in human studies where obesity has been shown to increase the risk of breast cancer, this has not been well studied in veterinary science. In our preliminary study, we discovered that leptin downregulated cathepsin A, which is responsible for lysosomal-associated membrane protein 2a (LAMP2a) degradation. LAMP2a is a rate-limiting factor in chaperone-mediated autophagy and is highly active in malignant cancers. Therefore, in this study, alterations in metastatic capacity through cathepsin A by leptin, which are secreted at high levels in the blood of obese patients, were investigated. We used a canine inflammatory mammary gland adenocarcinoma (CHMp) cell line cultured with RPMI-1640 and 10% fetal bovine serum. The samples were then subjected to real-time polymerase chain reaction, Western blot, immunocytochemistry, and lysosome isolation to investigate and visualize the metastasis and chaperone-mediated autophagy-related proteins. Results showed that leptin downregulated cathepsin A expression at both transcript and protein levels, whereas LAMP2a, the rate-limiting factor of chaperone-mediated autophagy, was upregulated by inhibition of LAMP2a degradation. Furthermore, leptin promoted LAMP2a multimerization through the lysosomal mTORC2 (mTOR complex 2)/PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1)/AKT1 (Serine/threonine-protein kinase 1) pathway. These findings suggest that targeting leptin receptors can alleviate mammary gland cancer cell metastasis in dogs.

CREG1 is a small glycoprotein which has been proposed as a transcription repressor, a secretory ligand, a lysosomal, or a mitochondrial protein. This is largely because of lack of antibodies for immunolocalization validated through gain- and loss-of-function studies. In the present study, we demonstrate, using antibodies validated for immunofluorescence microscopy, that CREG1 is mainly localized to the endosomal-lysosomal compartment. Gain- and loss-of-function analyses reveal an important role for CREG1 in both macropinocytosis and clathrin-dependent endocytosis. CREG1 also promotes acidification of the endosomal-lysosomal compartment and increases lysosomal biogenesis. Functionally, overexpression of CREG1 enhances macroautophagy/autophagy and lysosome-mediated degradation, whereas knockdown or knockout of CREG1 has opposite effects. The function of CREG1 in lysosomal biogenesis is likely attributable to enhanced endocytic trafficking. Our results demonstrate that CREG1 is an endosomal-lysosomal protein implicated in endocytic trafficking and lysosomal biogenesis.Abbreviations: AIFM1/AIF: apoptosis inducing factor mitochondria associated 1; AO: acridine orange; ATP6V1H: ATPase H+ transporting V1 subunit H; CALR: calreticulin; CREG: cellular repressor of E1A stimulated genes; CTSC: cathepsin C; CTSD: cathepsin D; EBAG9/RCAS1: estrogen receptor binding site associated antigen 9; EIPA: 5-(N-ethyl-N-isopropyl)amiloride; ER: endoplasmic reticulum; GFP: green fluorescent protein; HEXA: hexosaminidase subunit alpha; IGF2R: insulin like growth factor 2 receptor; LAMP1: lysosomal associated membrane protein 1; M6PR: mannose-6-phosphate receptor, cation dependent; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; PDIA2: protein disulfide isomerase family A member 2; SQSTM1/p62: sequestosome 1; TF: transferrin; TFEB: transcription factor EB.

Mitochondria are key organelles for cellular metabolism, and regulate several processes including cell death and macroautophagy/autophagy. Here, we show that mitochondrial respiratory chain (RC) deficiency deactivates AMP-activated protein kinase (AMPK, a key regulator of energy homeostasis) signaling in tissue and in cultured cells. The deactivation of AMPK in RC-deficiency is due to increased expression of the AMPK-inhibiting protein FLCN (folliculin). AMPK is found to be necessary for basal lysosomal function, and AMPK deactivation in RC-deficiency inhibits lysosomal function by decreasing the activity of the lysosomal Ca2+ channel MCOLN1 (mucolipin 1). MCOLN1 is regulated by phosphoinositide kinase PIKFYVE and its product PtdIns(3,5)P2, which is also decreased in RC-deficiency. Notably, reactivation of AMPK, in a PIKFYVE-dependent manner, or of MCOLN1 in RC-deficient cells, restores lysosomal hydrolytic capacity. Building on these data and the literature, we propose that downregulation of the AMPK-PIKFYVE-PtdIns(3,5)P2-MCOLN1 pathway causes lysosomal Ca2+ accumulation and impaired lysosomal catabolism. Besides unveiling a novel role of AMPK in lysosomal function, this study points to the mechanism that links mitochondrial malfunction to impaired lysosomal catabolism, underscoring the importance of AMPK and the complexity of organelle cross-talk in the regulation of cellular homeostasis. Abbreviation: ΔΨm: mitochondrial transmembrane potential; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATP: adenosine triphosphate; ATP6V0A1: ATPase, H+ transporting, lysosomal, V0 subbunit A1; ATP6V1A: ATPase, H+ transporting, lysosomal, V0 subbunit A; BSA: bovine serum albumin; CCCP: carbonyl cyanide-m-chlorophenylhydrazone; CREB1: cAMP response element binding protein 1; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; EBSS: Earl's balanced salt solution; ER: endoplasmic reticulum; FBS: fetal bovine serum; FCCP: carbonyl cyanide-p-trifluoromethoxyphenolhydrazone; GFP: green fluorescent protein; GPN: glycyl-L-phenylalanine 2-naphthylamide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryonic fibroblast; MITF: melanocyte inducing transcription factor; ML1N*2-GFP: probe used to detect PtdIns(3,5)P2 based on the transmembrane domain of MCOLN1; MTORC1: mechanistic target of rapamycin kinase complex 1; NDUFS4: NADH:ubiquinone oxidoreductase subunit S4; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; pcDNA: plasmid cytomegalovirus promoter DNA; PCR: polymerase chain reaction; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; P/S: penicillin-streptomycin; PVDF: polyvinylidene fluoride; qPCR: quantitative real time polymerase chain reaction; RFP: red fluorescent protein; RNA: ribonucleic acid; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; TMRM: tetramethylrhodamine, methyl ester, perchlorate; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1; v-ATPase: vacuolar-type H+-translocating ATPase; WT: wild-type.

Coxsackievirus B3 (CVB3) is a prevalent etiological agent for viral myocarditis and neurological disorders, particularly in infants and young children. Virus-encoded proteinases have emerged as cytopathic factors that contribute to disease pathogenesis in part through targeting the cellular recycling machinery of autophagy. Although it is appreciated that CVB3 can usurp cellular macroautophagy/autophagy for pro-viral functions, the precise mechanisms by which viral proteinases disrupt autophagy remain incompletely understood. Here we identified TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, as a novel target of CVB3 proteinase 3 C. Time-course infections uncovered a significant loss of full-length TFEB and the emergence of a lower-molecular mass (~63 kDa) fragment. Cellular and in vitro cleavage assays revealed the involvement of viral proteinase 3 C in the proteolytic processing of TFEB, while site-directed mutagenesis identified the site of cleavage after glutamine 60. Assessment of TFEB transcriptional activity using a reporter construct discovered a loss of function of the cleavage fragment despite nuclear localization and retaining of the ability of DNA and protein binding. Furthermore, we showed that CVB3 infection was also able to trigger cleavage-independent nuclear translocation of TFEB that relied on the serine-threonine phosphatase PPP3/calcineurin. Finally, we demonstrated that both TFEB and TFEB [Δ60] serve roles in viral egress albeit through differing mechanisms. Collectively, this study reveals that CVB3 targets TFEB for proteolytic processing to disrupt host lysosomal function and enhance viral infection.Abbreviations:ACTB: actin beta; CLEAR: coordinated lysosomal enhancement and regulation; CVB3: coxsackievirus B3; DAPI: 4',6-diamidino-2-phenylindole; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LTR: LysoTracker Red; PPP3/calcineurin: protein phosphatase 3; PPP3CA: protein phosphatase 3 catalytic subunit A; p-TFEB: phospho-Ser211 TFEB; si-CON: scramble control siRNA; TFEB: transcription factor EB; TFEB [Δ60]: TFEB cleavage fragment that lacks the first 60 amino acids; VP1: viral capsid protein 1.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.