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Increasing evidences have showed that autophagy played a significant role in oral squamous cell carcinoma (OSCC). Purpose of our study was to explore the prognostic value of autophagy‐related genes (ATGs) and screen autophagy‐related biomarkers for OSCC. RNA‐seq and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database following extracting ATG expression profiles. Then, differentially expressed analysis was performed in R software and a risk score model according to ATGs was established. Moreover, comprehensive bioinformatics analyses were used to screen autophagy‐related biomarkers which were later verified in OSCC tissues and cell lines. A total of 232 ATGs were extracted, and 37 genes were differentially expressed in OSCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that these genes were mainly located in autophagosome membrane and associated with autophagy. Furthermore, the risk score on basis of ATGs was identified as potential independent prognostic biomarker. Moreover, ATG12 and BID were identified as potential autophagy‐related biomarkers of OSCC. This study successfully constructed a risk model, and the risk score could predict the prognosis of OSCC patients accurately. Moreover, ATG12 and BID were identified as two potential independent prognostic autophagy‐related biomarkers and might provide new OSCC therapeutic targets.

Scavenger receptors are pattern recognition receptors that recognize both foreign and self-ligands, and initiate different mechanisms of cellular activation, often as co-receptors. The function of scavenger receptor CD36 in the immune system has mostly been studied in macrophages but it is also highly expressed by innate type B cells where its function is less explored. Here we report that CD36 is involved in macro-autophagy/autophagy in B cells, and in its absence, the humoral immune response is impaired. We found that CD36-deficient B cells exhibit a significantly reduced plasma cell formation, proliferation, mitochondrial mobilization and oxidative phosphorylation. These changes were accompanied by impaired initiation of autophagy, and we found that CD36 regulated autophagy and colocalized with autophagosome membrane protein MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). When we investigated T-cell-dependent immune responses, we found that mice with CD36 deficiency, specifically in B cells, exhibited attenuated germinal center responses, class switching, and antibody production as well as autophagosome formation. These findings establish a critical role for CD36 in B cell responses and may also contribute to our understanding of CD36-mediated autophagy in other cells as well as in B cell lymphomas that have been shown to express the receptor. Abbreviations: AICDA/AID: activation-induced cytidine deaminase; ATG5: autophagy related 5; ATP: adenosine triphosphate; BCR: B-cell receptor; CPG: unmethylated cytosine-guanosine; CQ: chloroquine; DC: dendritic cells; FOB: follicular B cells; GC: germinal center; Ig: immunoglobulin; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MZB: marginal zone B cells; NP-CGG: 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin; OCR: oxygen consumption rate; oxLDL: oxidized low-density lipoprotein; PC: plasma cells; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; SRBC: sheep red blood cells; Tfh: follicular helper T cells; TLR: toll-like receptor.

Macroautophagy/autophagy is an intracellular degradation process that delivers cytosolic materials and/or damaged organelles to lysosomes. De novo synthesis of the autophagosome membrane occurs within a phosphatidylinositol-3-phosphate-rich region of the endoplasmic reticulum, and subsequent expansion is critical for cargo encapsulation. This process is complex, especially in mammals, with many regulatory factors. In this study, by utilizing PRKN (parkin RBR E3 ubiquitin protein ligase)-mediated mitochondria autophagy (mitophagy)-inducing conditions in conjunction with chemical crosslinking and mass spectrometry, we identified human BCAS3 (BCAS3 microtubule associated cell migration factor) and C16orf70 (chromosome 16 open reading frame 70) as novel proteins that associate with the autophagosome formation site during both non-selective and selective autophagy. We demonstrate that BCAS3 and C16orf70 form a complex and that their association with the phagophore assembly site requires both proteins. In silico structural modeling, mutational analyses in cells and in vitro phosphoinositide-binding assays indicate that the WD40 repeat domain in human BCAS3 directly binds phosphatidylinositol-3-phosphate. Furthermore, overexpression of the BCAS3-C16orf70 complex affects the recruitment of several core autophagy proteins to the phagophore assembly site. This study demonstrates regulatory roles for human BCAS3 and C16orf70 in autophagic activity. Abbreviations: AO: antimycin A and oligomycin; Ash: assembly helper; ATG: autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; C16orf70: chromosome 16 open reading frame 70; DAPI: 4‘,6-diamidino-2-phenylindole; DKO: double knockout; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; fluoppi: fluorescent-based technology detecting protein-protein interactions; FIS1: fission, mitochondrial 1; FKBP: FKBP prolyl isomerase family member 1C; FRB: FKBP-rapamycin binding; hAG: humanized azami-green; IP: immunoprecipitation; IRES: internal ribosome entry site; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MS: mass spectrometry; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mtDNA: mitochondrial DNA; OPTN: optineurin; PFA: paraformaldehyde; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; PROPPIN: β-propellers that bind polyphosphoinositides; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like autophagy activating kinase 1; WDR45B/WIPI3: WD repeat domain 45B; WDR45/WIPI4: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting; WT: wild type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1

Pompe disease is a lysosomal storage disease caused by mutations within the GAA gene, which encodes acid α-glucosidase (GAA)—an enzyme necessary for lysosomal glycogen degradation. A lack of GAA results in an accumulation of glycogen in cardiac and skeletal muscle, as well as in motor neurons. The only FDA approved treatment for Pompe disease—an enzyme replacement therapy (ERT)—increases survival of patients, but has unmasked previously unrecognized clinical manifestations of Pompe disease. These clinical signs and symptoms include tracheo-bronchomalacia, vascular aneurysms, and gastro-intestinal discomfort. Together, these previously unrecognized pathologies indicate that GAA-deficiency impacts smooth muscle in addition to skeletal and cardiac muscle. Thus, we sought to characterize smooth muscle pathology in the airway, vascular, gastrointestinal, and genitourinary in the Gaa−/− mouse model. Increased levels of glycogen were present in smooth muscle cells of the aorta, trachea, esophagus, stomach, and bladder of Gaa−/− mice, compared to wild type mice. In addition, there was an increased abundance of both lysosome membrane protein (LAMP1) and autophagosome membrane protein (LC3) indicating vacuolar accumulation in several tissues. Taken together, we show that GAA deficiency results in subsequent pathology in smooth muscle cells, which may lead to life-threatening complications if not properly treated.

Cell senescence leads to a number of changes in the properties of mesenchymal stromal cells (MSCs). In particular, the number of damaged structures is increased producing negative effect on intracellular processes. Elimination of the damaged molecules and organelles occurs via autophagy that can be important in the context of aging. Cultivation under low oxygen level can be used as an approach for enhancement of MSC therapeutic properties and "slowing down" cell senescence. The goal of this work was to study some morphological and functional characteristics and expression of autophagy-associated genes during replicative senescence of MSCs under different oxygen concentration. The study revealed changes in the regulation of autophagy at the transcriptional level. Upregulation of the expression of autophagosome membrane growth genes ATG9A and ULK1, of the autophagosome maturation genes CTSD, CLN3, GAA, and GABARAPL1, of the autophagy regulation genes TP53, TGFB1, BCL2L1, FADD, and HTT was shown. These changes were accompanied by downregulation of IGF1 and TGM2 expression. Increase of the lysosomal compartment volume was observed in the senescent MSCs that also indicated increase of their degradation activity. The number of lysosomes was decreased following prolonged cultivation under low oxygen concentration (5%). The replicative senescence of MSCs under conditions of different oxygen levels led to the similar modifications in the expression of the autophagy-associated genes.

ATG12, a core autophagy protein, forms a conjugate with ATG5 to promote the formation of autophagosome membrane, and plays an important role in antiviral immunity. However, little is known about the function of ATG12 in fish. Here, we cloned the open reading frame (ORF) of large yellow croaker (Larimichthys crocea) ATG12 (LcATG12), which is 354 nucleotides long and encodes a protein of 117 amino acids. The deduced LcATG12 possesses a conserved APG12 domain (residues 31 to 117), and shares 91.45% identities with ATG12 in orange-spotted grouper (Epinephelus coioides). LcATG12 was constitutively expressed in all examined tissues, with the highest level in intestine. Its transcript was also detected in primary head kidney granulocytes (PKG), primary head kidney macrophages (PKM), primary head kidney lymphocytes (PKL), and large yellow croaker head kidney (LYCK) cell line, and was significantly up-regulated by poly(I:C). LcATG12 was regularly distributed in both cytoplasm and nucleus of LYCK and epithelioma papulosum cyprinid (EPC) cells. Overexpression of LcATG12 in EPC cells significantly inhibited the replication of spring viremia of carp virus (SVCV). Further studies reveled that LcATG12 could induce the occurrence of autophagy in LYCK cells. Furthermore, overexpression of LcATG12 in LYCK cells increased the expression levels of large yellow croaker type I interferons (IFNs, IFNc, IFNd, and IFNh), IFN regulatory factors (IRF3 and IRF7), and IFN-stimulated genes (PKR, Mx, and Viperin). All these data indicated that LcATG12 plays a role in the antiviral immunity possibly by inducing both autophagy and type I IFN response in large yellow croaker.

Autophagosomes are unique double-membrane organelles that enclose a portion of intracellular components for lysosome/vacuole delivery to maintain cellular homeostasis in eukaryotic cells. Genetic screening has revealed the requirement of autophagy-related proteins for autophagosome formation, although the origin of the autophagosome membrane remains elusive. The endomembrane system is a series of membranous organelles maintained by dynamic membrane flow between various compartments. In plants, there is accumulating evidence pointing to a link between autophagy and the endomembrane system, in particular between the endoplasmic reticulum and autophagosome. Here, we highlight and discuss about recent findings on plant autophagosome formation. We also look into the functional roles of endomembrane machineries in regard to the autophagy pathway in plants.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. Abbreviations AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5ʹ-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type

Age-related hearing loss (AHL) is typically caused by the irreversible death of hair cells (HCs). Autophagy is a constitutive pathway to strengthen cell survival under normal or stress condition. Our previous work suggested that impaired autophagy played an important role in the development of AHL in C57BL/6 mice, although the underlying mechanism of autophagy in AHL still needs to be investigated. SIRT1 as an important regulator involves in AHL and is also a regulator of autophagy. Thus, we hypothesized that the modulation between SIRT1 and autophagy contribute to HC death and the progressive hearing dysfunction in aging. In the auditory cell line HEI-OC1, SIRT1 modulated autophagosome induction because of SIRT1 deacetylating a core autophagy protein ATG9A. The deacetylation of ATG9A not only affects the autophagosome membrane formation but also acts as a sensor of endoplasmic reticulum (ER) stress inducing autophagy. Moreover, the silencing of SIRT1 facilitated cell death via autophagy inhibition, whereas SIRT1 and autophagy activation reversed the SIRT1 inhibition media cell death. Notably, resveratrol, the first natural agonist of SIRT1, altered the organ of Corti autophagy impairment of the 12-month-old C57BL/6 mice and delayed AHL. The activation of SIRT1 modulates the deacetylation status of ATG9A, which acts as a sensor of ER stress, providing a novel perspective in elucidating the link between ER stress and autophagy in aging. Because SIRT1 activation restores autophagy with reduced HC death and hearing loss, it could be used as a strategy to delay AHL.

During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER–autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.

Autophagy is an adaptive response for cell survival in which cytoplasmic components and organelles are degraded in bulk under normal and stress conditions. Trichomonas vaginalis is a parasite highly adaptable to stress conditions such as iron (IR) and glucose restriction (GR). Autophagy can be traced by detecting a key autophagy protein (Atg8) anchored to the autophagosome membrane by a lipid moiety. Our goal was to perform a morphological and cellular study of autophagy in T. vaginalis under GR, IR, and Rapamycin (Rapa) treatment using TvAtg8 as a putative autophagy marker. We cloned tvatg8a and tvatg8b and expressed and purified rTvAtg8a and rTvAtg8b to produce specific polyclonal antibodies. Autophagy vesicles were detected by indirect immunofluorescence assays and confirmed by ultrastructural analysis. The biogenesis of autophagosomes was detected, showing intact cytosolic cargo. TvAtg8 was detected as puncta signal with the anti-rTvAtg8b antibody that recognized soluble and lipid-associated TvAtg8b by Western blot assays in lysates from stress-inducing conditions. The TvAtg8b signal co-localized with the CytoID and lysotracker labeling (autolysosomes) that accumulated after E-64d treatment in GR parasites. Our data suggest that autophagy induced by starvation in T. vaginalis results in the formation of autophagosomes for which TvAtg8b could be a putative autophagy marker.

Intracellular deposits of pathological tau are the hallmark of a broad spectrum of neurodegenerative disorders collectively known as tauopathies, with Alzheimer's disease, a secondary tauopathy, being further characterized by extracellular amyloid plaques. A major obstacle in developing effective treatments for tauopathies is the presence of the blood-brain barrier, which restricts the access of therapeutic agents to the brain. An emerging technology to overcome this limitation is the application of low-intensity ultrasound which, together with intravenously injected microbubbles, transiently opens the blood-brain barrier, thereby facilitating the delivery of therapeutic agents into the brain. Interestingly, even in the absence of therapeutic agents, ultrasound has previously been shown to reduce amyloid plaques and improve cognitive functions in amyloid-depositing mice through microglial clearance. Ultrasound has also been shown to facilitate the delivery of antibody fragments against pathological tau in P301L tau transgenic mice; however, the effect of ultrasound alone has not been thoroughly investigated in a tauopathy mouse model. Methods: Here, we performed repeated scanning ultrasound treatments over a period of 15 weeks in K369I tau transgenic mice with an early-onset tau-related motor and memory phenotype. We used immunohistochemical and biochemical methods to analyze the effect of ultrasound on the mice and determine the underlying mechanism of action, together with an analysis of their motor and memory functions following repeated ultrasound treatments. Results: Repeated ultrasound treatments significantly reduced tau pathology in the absence of histological damage. Associated impaired motor functions showed improvement towards the end of the treatment regime, with memory functions showing a trend towards improvement. In assessing potential clearance mechanisms, we ruled out a role for ubiquitination of tau, a prerequisite for proteasomal clearance. However, the treatment regime induced the autophagy pathway in neurons as reflected by an increase in the autophagosome membrane marker LC3II and a reduction in the autophagic flux marker p62, along with a decrease of mTOR activity and an increase in beclin 1 levels. Moreover, there was a significant increase in the interaction of tau and p62 in the ultrasound-treated mice, suggesting removal of tau by autophagosomes. Conclusions: Our findings indicate that a neuronal protein aggregate clearance mechanism induced by ultrasound-mediated blood-brain barrier opening operates for tau, further supporting the potential of low-intensity ultrasound to treat neurodegenerative disorders.

Accompanying the precipitous age-related decline in human female fertility is an increase in the proportion of poor-quality oocytes within the ovary. The macroautophagy pathway, an essential protein degradation mechanism responsible for maintaining cell health, has not yet been thoroughly investigated in this phenomenon. The aim of this study was to characterize the macroautophagy pathway in an established mouse model of oocyte aging using in-depth image analysis-based methods and to determine mechanisms that account for the observed changes. Three autophagy pathway markers were selected for assessment of gene and protein expression in this model: Beclin 1; an initiator of autophagosome formation, Microtubule-associated protein 1 light chain 3B; a constituent of the autophagosome membrane, and lysosomal-associated membrane protein 1; a constituent of the lysosome membrane. Through quantitative image analysis of immunolabeled oocytes, this study revealed impairment of the macroautophagy pathway in the aged oocyte with an attenuation of both autophagosome and lysosome number. Additionally, an accumulation of amphisomes greater than 10 μm2 in area were observed in aging oocytes, and this accumulation was mimicked in oocytes treated with lysosomal inhibitor chloroquine. Overall, these findings implicate lysosomal dysfunction as a prominent mechanism by which these age-related changes may occur and highlight the importance of macroautophagy in maintaining mouse pre-ovulatory oocyte quality. This provides a basis for further investigation of dysfunctional autophagy in poor oocyte quality and for the development of therapeutic or preventative strategies to aid in the maintenance of pre-ovulatory oocyte health.

Mother-to-child transmission (MTCT) is the major cause of chronic infection of hepatitis B virus (HBV) in patients. However, whether and how HBV crosses the placenta to cause infection in utero remains unclear. In this study, we investigate the mechanism as to how the HBV virions pass through layers of the trophoblast. Our data demonstrate the exocytosis of virions from the trophoblast after exposure to HBV where the endocytosed HBV virions co-localized with an S100A10/AnxA2 complex and LC3, an autophagosome membrane marker. Knockdown of either AnxA2 or S100A10 in trophoblast cells led to a reduction of the amount of exo-virus in Transwell assay. Immunohistochemistry also showed a high expression of AnxA2 and S100A10 in the placental tissue samples of HBV-infected mothers with congenital HBV-positive infants (HBV+/+). We conclude that in HBV intrauterine infection and mother-to-child transmission, a proportion of HBV hijacks autophagic protein secretion pathway and translocate across the trophoblast via S100A10/AnxA2 complex and multivesicular body (MVB)-mediated exocytosis. Our study provides a potential target for the interference of the mechanisms of HBV intrauterine infection and mother-to-child transmission., Mother-to-child transmission is the major cause of chronic hepatitis B virus (HBV) infection. This study shows that an unconventional protein secretion pathway that depends on autophagy may be hijacked by HBV to complete the process of intracellular transport. In HBV-infected trophoblasts, AnxA2-S100A10-mediated exocytosis may result in HBV intrauterine transmission.

Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia. Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

All eukaryotes rely on the ubiquitin-proteasome system (UPS) and autophagy to control the abundance of key regulatory proteins and maintain a healthy intracellular environment. In the UPS, damaged or superfluous proteins are ubiquitinated and degraded in the proteasome, mediated by three types of ubiquitin enzymes: E1s (ubiquitin activating enzymes), E2s (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). Conversely, in autophagy, a vesicular autophagosome is formed that transfers damaged proteins and organelles to the vacuole, mediated by a series of ATGs (autophagy related genes). Despite the use of two completely different componential systems, the UPS and autophagy are closely interconnected and mutually regulated. During autophagy, ATG8 proteins, which are autophagosome markers, decorate the autophagosome membrane similarly to ubiquitination of damaged proteins. Ubiquitin is also involved in many selective autophagy processes and is thus a common factor of the UPS and autophagy. Additionally, the components of the UPS, such as the 26S proteasome, can be degraded via autophagy, and conversely, ATGs can be degraded by the UPS, indicating cross regulation between the two pathways. The UPS and autophagy cooperate and jointly regulate homeostasis of cellular components during plant development and stress response.

IntroductionThe lysosomal degradation pathway of autophagy is a highly conserved mechanism in eukaryotic cells and play an important role in cellular, tissue and organismal homeostasis by removing dysfunctional organelles, intracellular bacterial and aggregated proteins selectively (Zaffagnini et al. , 2016). Autophagy can be activated by cellular stress such as starvation, hypoxia, oxidative stress, protein aggregation, endoplasmic reticulum (ER) stress and others, which triggers the cell survival process and thus provides energy to the cells when energy is consumed (Levineet al. , 2019). Thus, autophagy is generally considered to be a cell survival process and play an important role in the maintenance of cellular homeostasis.At present, it is divided into three categories according to the occurrence process: Macroautophagy (also termed autophagy), Microautophagy, and Chaperone-mediated autophagy (CMA), the following refers to the first category unless otherwise specified (Wen et al. , 2016). It is generally accepted that autophagy is a more selective process than originally expected. Autophagy can be divided into different types, according to the different autophagy substrates(also called cargo), for example, aggrephagy for protein aggregates, lysophagy for damaged lysosomes, mitophagy for mitochondria and xenophagy for intracellular bacterial. Conservatively, most of these different types of selective autophagy pathways use a common mechanism.Up to now, there are 34 Autophagy-related (ATG) genes that is discovered in yeast and 15 of which are “core” ATG genes that are commonly necessary to the different pathways of autophagy (Nakatogawa et al. , 2009). The autophagosome biogenesis process can be divided into four steps, the first is the nucleation and expansion of the phagophore, the second is the formation of autophagosome, the third is the autophagosome and lysosome fuse to form autolysosome, and the last is degrades the cargos by enzymes in lysosomes.It is also generally conceded that the trigger of autophagy results in ATGs recruitment, Unc-51-like kinase 1 (ULK1) complex (consisting of ULK1/2, ATG13, FIP200 and ATG101), to the phagophore assembly site (PAS), a specific subcellular location where the nucleation of phagophore to be triggered. Then a cup-shaped structure isolation membrane is formed from the different source of membrane including mitochondria, endoplasmic reticulum. Meanwhile, ‘eat-me’ signals are necessary to cargo to be selectively recognized by several autophagy receptors that link the cargo to the autophagic membrane via their light chain 3 (LC3)-interacting region (LIR) by Atg8. The most primarily ‘eat-me’ signals is ubiquitin (Ub) chains. Atg8 is a ubiquitin-like protein, can be tightly bound to the autophagic membranes when it was cleaved at its C-termini by the ATG4 pro- teases to expose a C-terminal glycine producing the form I of the ATG8 molecule, and then conjugated to PE and being an PE-conjugated form II of ATG8 proteins. Yeast only owns the one kind of Atg8 protein, while mammals have at least seven if not more ATG8 proteins that can be fell into two subfamilies containing not less than three MAP1 light chain 3 (LC3A, B and C) and four gamma-aminobutyrate receptor-associated protein (GABARAP) and GABARAP-like proteins (ATG8L/GEC-1/GABARAPL1, GATE-16/GABARAPL2 and GABARAPL3) (Xin et al. , 2001; He et al. , 2003).Many proteins have been identified as autophagy receptors that can recognize the ‘eat-me’ signals of cargos, and the classic and wildly accepted autophagy receptors including p62, OPTN, TAX1BP1, NDP52 (Pohl et al. , 2019). Each of these receptors contains LC3 interacting regions (LIRs) that facilitate their interaction with LC3-like molecules on the autophagosome (Wildet al. , 2011; Katsuragi et al. , 2015; Tumbarello et al. , 2015).Furthermore, there is a clear etiological link between gene mutations that control autophagy and human diseases (especially neurodegenerative diseases, inflammatory diseases, and cancer) (Levine et al. , 2019). These diseases are largely untreatable and have no target for intervention that needed an interventional therapeutic target and an interventional target drug. Therefore, these genes that play an important role in autophagy have the opportunity to be the candidate therapeutic targets for these diseases. As we introduced above, the autophagy related genes mainly including Atgs and autophagy receptors. Among them, OPTN is well-documented to have a strong relationship with several disease, for example, neurodegenerative diseases, cancer, inflammatory diseases,etc . (Liu et al. , 2018; Weil et al. , 2018).OPTN is a conservative protein in many species, including human, macaques, rats, pigs, and bovine and is widely in liver, heart, brain, placenta, liver, skeletal muscle, kidney, pancreas, retina, optic nerve blood vessels and so on. OPTN was identified as a negative regulator of NF-κB that competitively binds ubiquitin with NEMO (NF-κB essential modulator) (Zhu et al. , 2007), and an autophagy receptor that connect the ubiquitinated autophagy substrate and LC3-positive autophagosome membrane (Wildet al. , 2011). What’s more, OPTN is also an autophagy inducer that induce autophagic process upon overexpression (Ying et al. , 2016). Breakthrough has been made in comprehending the molecular mechanisms of OPTN in autophagy since 2011. OPTN is an important autophagy adaptor, participate in almost every step of the autophagy process and not just as a autophagy receptor. But the existing review neither clearly clarify this point neither sort out the existing literature lucidly.Over the past decade of studies, OPTN has been involved in many biological process, and its role as autophagy receptor is a breakthrough finding which caught scientists attention in recent years. Contemporary, OPTN was closely associated with some incurable disease in human beings, such as ALS, glaucoma, Paget’s disease, Crohn’s disease, and recently cancer and diabetic nephropathy. However, the relationship between OPTN and these disease has not been well revealed and clarified. This review discusses the biological functions of OPTN in the process of autophagy and the perspective of autophagic mechanism that OPTN involved in several human diseases.The Protein Structure and Cellular Function of OPTNOptineurin(OPTN) was first identified by a yeast two-hybrid screening in order to find interacting proteins of Ad E3 14.7-kDa protein (E3-14.7K) that inhibit TNF-α functions (Li et al. , 1998), and named as FIP-2(14.7K-interacting protein). Then Klaus et al . using “Data base searching” found a NEMO-related protein that shows a strong homology to NEMO, which is the second time that OPTN come into the sight of scientists and name NRP (the first letter of each word in “NEMO-related protein”) (Schwamborn et al. , 2000). After then, more and more studies have sprung up like mushrooms that uncovering the enigma of OPTN in structure of protein, biological function and etiological link in disease.The human OPTN protein contains 577 amino acids and is a 74kD scaffold protein contains several structural domains that endow its multiple abilities. And the mouseOptn gene codes for a 584–amino acid protein (67 kD) which is 78% identical to human OPTN (Rezaie et al. , 2005). The public databases contain partial or complete sequences of OPTN homologues of macaques, rats, pigs, and bovine, all of which show a great extent of similarity to human OPTN, which means OPTN is a conserved gene that play an important role in vital activity. OPTN contains several structural domains and is consists of the structures including CC (coiled-coil) domain, LZ (leucine zipper) domain, LIR (LC3 interacting region) domain, UBAN (ubiquitin-binding domain (UBD) of ABIN proteins and NEMO) domain, ZF (zinc finger) domain. The LIR domain and UBD domain play an important role in autophagy and is the foundation of OPTN to function as an autophagy receptor. In detail, the LIR domain is the location that binding LC3II and the UBD domain is the place where attach the ubiquitinated cargos. The LC3II is an PE-conjugated form II of ATG8 proteins that can tightly bound to the autophagic membranes. So that, cargos are enclosed by autophagic membranes and turn into autophagosome and then degraded in autolysosome.Many biological function of OPTN was reported so far. Firstly, Tayebeh et al. using immunocytochemistry to found the intracellular localization of OPTN and observed the colocalization with the Golgi apparatus (Rezaieet al. , 2002). A deeper study bring this colocalization to light, OPTN plays a vital role in the maintenance of Golgi integrity (Parket al. , 2006).Secondly, OPTN shows strong homology with NEMO, and was identified as a negative regulator of NF-κB that competitively binds ubiquitin with NEMO, thus it may have role in NF-κB signaling regulation. Thirdly, Wild et al. fully proved the specific interactions between OPTN and LC3/GABARAP proteins by pull-down assays in MCF-7 cells, yeast two-hybrid transformations, and purified proteins in vitro (Wild et al. , 2011). The vital role of OPTN in autophagy has attracted greater attention in the last ten years.

Autophagy is an intracellular degradation process, which is highly conserved in eukaryotes. During this process, unwanted cytosolic constituents are sequestered and delivered into the vacuole/lysosome by a double-membrane organelle known as an autophagosome. The autophagosome initiates from a membrane sac named the phagophore, and after phagophore expansion and closure, the outer membrane fuses with the vacuole/lysosome to release the autophagic body into the vacuole. Membrane sources derived from the endomembrane system (e.g., Endoplasmic Reticulum, Golgi and endosome) have been implicated to contribute to autophagosome in different steps (initiation, expansion or maturation). Therefore, coordination between the autophagy-related (ATG) proteins and membrane tethers from the endomembrane system is required during autophagosome biogenesis. In this review, we will update recent findings with a focus on comparing the selected core ATG complexes and the endomembrane tethering machineries for shaping the autophagosome membrane in yeast, mammal, and plant systems.

Introduction: Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. Methods: 2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence, and Western blot. Autophagosomes were detected by electron microscopy and their maturation by real-time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. Results: We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. However, the binding activity of the cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosome synthesis, maturation, and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol and the nucleus, leading to disruption of the organelles, cellular digestion, and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN, and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. Discussion/Conclusion: Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.

Mutations in ubiquitin ligase Parkin are associated with Parkinson’s disease and defective mitophagy. Conceptually, Parkin-dependent mitophagy is classified into two phases; 1. Parkin recruits to and ubiquitinates mitochondrial proteins, 2. Formation of autophagosome membrane, sequestering mitochondria for degradation. Recently, endosomal machineries were reported to contribute to the later stage for membrane assembly. We report a role for endosomes in the events upstream of phase 1. We demonstrate that an endosomal ubiquitin ligase CARP2 associates with damaged mitochondria, and this association precedes that of Parkin. CARP2 interacts with Parkin, and stable recruitment of Parkin to damaged mitochondria was substantially reduced in CARP2 KO cells. Our study unravels a novel role of endosomes in modulating upstream pathways of Parkin-dependent mitophagy initiation.

Melia et al. review the molecular mechanisms and membrane modeling events underlying autophagosome biogenesis., Autophagosome biogenesis involves de novo formation of a membrane that elongates to sequester cytoplasmic cargo and closes to form a double-membrane vesicle (an autophagosome). This process has remained enigmatic since its initial discovery >50 yr ago, but our understanding of the mechanisms involved in autophagosome biogenesis has increased substantially during the last 20 yr. Several key questions do remain open, however, including, What determines the site of autophagosome nucleation? What is the origin and lipid composition of the autophagosome membrane? How is cargo sequestration regulated under nonselective and selective types of autophagy? This review provides key insight into the core molecular mechanisms underlying autophagosome biogenesis, with a specific emphasis on membrane modeling events, and highlights recent conceptual advances in the field.

The intestine is a main organ responsible for energy and nutrition absorption and digestion and is an important immune barrier in fish. In recent years, the intestinal health of fish has gained increasing attention in the aquaculture industry. The intestine is a direct target organ of starvation and is highly sensitive to nutritional changes. However, the adaptive responses of the fish intestine to starvation are not well studied. The present study aimed to reveal the effects of starvation on antioxidant systems, autophagy, and mitochondrial function in the intestine of Chinese perch. Therefore, Chinese perch (250 ± 22 g) were starved for 0, 7, and 14 days. Our results indicate that the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) were significantly increased in the intestine of Chinese perch after 7 days and 14 days of starvation, indicating that starvation could induce oxidative stress in the intestine. Meanwhile, the activities and mRNA expression of antioxidative enzymes, including superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalases (CAT), were upregulated in the intestine after the starvation treatment, which might be a compensatory response to oxidative stress. Starvation also increased the expression of nrf2 in the intestine, thereby resulting in the upregulation of antioxidative gene expression. However, the glutathione content and its generation in the intestine were decreased under starvation stress. The ultrastructural analysis of the intestine demonstrated that starvation induced autophagy in the intestine of Chinese perch. The RT–qPCR analysis showed that starvation induced the mRNA expression of genes involved in autophagosome membrane initiation, autophagosome membrane expansion, vesicle recycling, and cargo recruitment, including beclin1, ulk1, beclin1, lc3a, atg4, atg5, atg7, p62, and atg9. Mitochondria are the main ATP-generating organelles and target organelles for ROS. The present study indicates that starvation induced a decrease in the ATP content in the intestine of Chinese perch. In addition, starvation downregulated the expression of mitochondrial oxidative phosphorylation-related and mitochondrial ATP generation-related genes (cox1, atp5f1, atp5a1, and mt-cyb) in the intestine of Chinese perch, suggesting that starvation could suppress mitochondrial energy synthesis. Taken together, our data demonstrate that starvation can induce oxidative stress, disrupt antioxidant systems, and cause autophagy and mitochondrial dysfunction in the intestine of Chinese perch. These results provide insight into the mechanisms by which starvation disrupts intestinal homeostasis in fish.

Estrogen receptor α (ERα)low/- tumors are associated with breast cancer (BCa) endocrine resistance, where ERα low tumors show a poor prognosis and a molecular profile similar to triple negative BCa tumors. Interleukin-1 (IL-1) downregulates ERα accumulation in BCa cell lines, yet the cells can remain viable. In kind, IL-1 and ERα show inverse accumulation in BCa patient tumors and IL-1 is implicated in BCa progression. IL-1 represses the androgen receptor hormone receptor in prostate cancer cells concomitant with the upregulation of the prosurvival, autophagy-related protein, Sequestome-1 (p62/SQSTM1; hereinafter, p62); and given their similar etiology, we hypothesized that IL-1 also upregulates p62 in BCa cells concomitant with hormone receptor repression. To test our hypothesis, BCa cell lines were exposed to conditioned medium from IL-1-secreting bone marrow stromal cells (BMSCs), IL-1, or IL-1 receptor antagonist. Cells were analyzed for the accumulation of ERα, progesterone receptor (PR), p62, or the autophagosome membrane protein, microtubule-associated protein 1 light chain 3 (LC3), and for p62-LC3 interaction. We found that IL-1 is sufficient to mediate BMSC-induced ERα and PR repression, p62 and autophagy upregulation, and p62-LC3 interaction in ERα+ /PR+ BCa cell lines. However, IL-1 does not significantly elevate the high basal p62 accumulation or high basal autophagy in the ERα- /PR- BCa cell lines. Thus, our observations imply that IL-1 confers a prosurvival ERα- /PR- molecular phenotype in ERα+ /PR+ BCa cells that may be dependent on p62 function and autophagy and may underlie endocrine resistance.

Autophagy begins with the nucleation of phagophores, which then expand to give rise to the double-membrane autophagosomes. Autophagosomes ultimately fuse with lysosomes, where the cytosolic cargoes are degraded. Accumulation of autophagosomes is a hallmark of autophagy and neurodegenerative disorders including Alzheimer’s and Huntington’s disease. In recent years, the sources of autophagosome membrane have attracted a great deal of interests, even so, the membrane donors for autophagosomes are still under debate. In this review, we describe the probable sources of autophagosome membrane.

Within minutes of induction of autophagy by amino-acid starvation in mammalian cells, multiple autophagosomes form throughout the cell cytoplasm. During their formation, the autophagosomes sequester cytoplasmic material and deliver it to lysosomes for degradation. How these organelles can be so rapidly formed and how their formation is acutely regulated are major questions in the autophagy field. Protein and lipid trafficking from diverse cell compartments contribute membrane to, or regulate the formation of the autophagosome. In addition, recruitment of Atg8 (in yeast), and the ATG8-family members (in mammalian cells) to autophagosomes is required for efficient autophagy. Recently, it was discovered that the centrosome and centriolar satellites regulate autophagosome formation by delivery of an ATG8-family member, GABARAP, to the forming autophagosome membrane, the phagophore. We propose that GABARAP regulates phagophore expansion by activating the ULK complex, the amino-acid controlled initiator complex. This finding reveals a previously unknown link between the centrosome, centriolar satellites and autophagy.

The fusion of autophagosomes with lysosomes is an obligatory step in the self-eating process of autophagy. In this issue, Kumar et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201708039) identify a protein complex, the autophagosome recognition particle (ARP), that chaperones a key SNARE, syntaxin 17, to the autophagosome membrane. Intriguingly, this protein complex coordinates both delivery and membrane insertion as a prelude to fusion.

Neurodegenerative diseases, characterized by a progressive loss of brain function, affect the lives of millions of individuals worldwide. The complexity of the brain poses a challenge for scientists trying to map the biochemical and physiological pathways to identify areas of pathological errors. Brain samples of patients with neurodegenerative diseases have been shown to contain large amounts of misfolded and abnormally aggregated proteins, resulting in dysfunction in certain brain centers. Removal of these abnormal molecules is essential in maintaining protein homeostasis and overall neuronal health. Macroautophagy is a major route by which cells achieve this. Administration of certain autophagy-enhancing compounds has been shown to provide therapeutic effects for individuals with neurodegenerative conditions. SQSTM1/p62 is a scaffold protein closely involved in the macroautophagy process. p62 functions to anchor the ubiquitinated proteins to the autophagosome membrane, promoting degradation of unwanted molecules. Modulators targeting p62 to induce autophagy and promote its protective pathways for aggregate protein clearance have high potential in the treatment of these conditions. Additionally, causal relationships have been found between errors in regulation of SQSTM1/p62 and the development of a variety of neurodegenerative disorders, including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration. Furthermore, SQSTM1/p62 also serves as a signaling hub for multiple pathways associated with neurodegeneration, providing a potential therapeutic target in the treatment of neurodegenerative diseases. However, rational design of a p62-oriented autophagy modulator that can balance the negative and positive functions of multiple domains in p62 requires further efforts in the exploration of the protein structure and pathological basis.

An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid-transferring protein. ATG2A can extract lipids from membrane vesicles and unload them to other vesicles. Lipid transfer by ATG2A is more efficient between its tethered vesicles than between untethered vesicles. The PI3P effectors WIPI4 and WIPI1 associate ATG2A stably to PI3P-containing vesicles, thereby facilitating ATG2A-mediated tethering and lipid transfer between PI3P-containing vesicles and PI3P-free vesicles. Based on these results, we propose that ATG2-mediated transfer of lipids from the ER to the phagophore enables phagophore expansion.

CD133 is a pentaspan transmembrane protein that can serve as a biomarker for cancer stem cells, although its biochemical mechanism remains unclear. Here we report that CD133 expression enhances glioma cell tolerance of a nutrient-deprived microenvironment. Under starvation conditions, CD133-positive cells exhibited higher survival and decreased levels of apoptosis. These changes were dependent on activation of autophagy-associated gene signaling and were impaired by the autophagic inhibitor chloroquine. Furthermore, rapamycin up-regulated the level of autophagy and inversely reduced CD133 expression. Immunofluorescence confirmed that starvation promoted release of CD133 from the plasma membrane to the cytoplasm, with CD133 also partially co-localizing with LC3 upon starvation. Additionally, CD133 partially co-localized with Beclin1, Atg5, and lysosomes, indicating that CD133 directly participates in the autophagosome membrane fusion process and ultimately undergoes lysosomal degradation. Collectively, our results demonstrate that CD133 contributes to cell survival by regulating autophagy, and that targeting CD133-linked signaling and autophagy may be useful in improving anti-cancer treatments.

Glycoconjugates, glycans, carbohydrates, and sugars: these terms encompass a class of biomolecules that are diverse in both form and function ranging from free oligosaccharides, glycoproteins and proteoglycans, to glycolipids that make up a complex glycan-code that impacts normal physiology and disease. Recent data suggests that one mechanism by which glycoconjugates impact physiology is through the regulation of the process of autophagy. Autophagy is a degradative pathway necessary for differentiation, organism development, and the maintenance of cell and tissue homeostasis. In this review, we will highlight what is known about the regulation of autophagy by glycoconjugates focusing on signaling mechanisms from the extracellular surface and the regulatory roles of intracellular glycans. Glycan signaling from the extracellular matrix converges on “master” regulators of autophagy including AMPK and mTORC1, thus impacting their localization, activity and/or expression. Within the intracellular milieu, gangliosides are constituents of the autophagosome membrane, a subset of proteins composing the autophagic machinery are regulated by glycosylation, and oligosaccharide exposure in the cytosol triggers an autophagic response. The examples discussed provide some mechanistic insights into glycan regulation of autophagy and reveals areas for future investigation.

The normal function of pancreatic beta cells is vital to the control of blood glucose. Earlier research suggests that the traditional Chinese medicine Cortex Lycii Radicis may help protect pancreatic beta cells and improve insulin sensitivity. However, the specific effects and molecular mechanism of this herb have not been described. Thus, we investigated the role of Cortex Lycii Radicis in regulating the proliferation, apoptosis, and autophagy of INS-1 pancreatic beta cells. Our study revealed that Cortex Lycii Radicis extracts could promote INS-1 cell proliferation and inhibit cell apoptosis under high glucose conditions. We also evaluated the formation of autophagosomes and found that GFP-LC3 fusion protein was translocated to the autophagosome membrane. Autophagosomes increased in the group treated with Cortex Lycii Radicis compared with the no treatment control group, indicating that these extracts could promote the activation of autophagy in INS-1 cells. Our findings suggest a significant association of the Cortex Lycii Radicis extracts treatment with apoptosis and autophagy, which protects the function of pancreatic beta cells, providing evidence for the development of a new drug for diabetes treatment.

Selective autophagy is critical for regulating cellular homeostasis by mediating lysosomal turnover of a wide variety of substrates including proteins, aggregates, organelles, and pathogens via a growing class of molecules termed selective autophagy receptors. The molecular mechanisms of selective autophagy receptor action and regulation are complex. Selective autophagy receptors link their bound cargo to the autophagosomal membrane by interacting with lipidated ATG8 proteins (LC3/GABARAP) that are intimately associated with the autophagosome membrane. The cargo signals that selective autophagy receptors recognize are diverse but their recognition can be broadly grouped into two classes, ubiquitin-dependent cargo recognition versus ubiquitin-independent. The roles of post-translational modification of selective autophagy receptors in regulating these pathways in response to stimuli are an active area of research. Here we will review recent advances in the identification of selective autophagy receptors and their regulatory mechanisms. Given its importance in maintaining cellular homeostasis, disruption of autophagy can lead to disease including neurodegeneration and cancer. The role of autophagy in cancer is complex as autophagy can mediate promotion or inhibition of tumorigenesis. Here we will also review the importance of autophagy in cancer with a specific focus on the role of selective autophagy receptors.

The actin regulator JMY creates filament networks that move membranes during autophagy. Hu and Mullins find that JMY is normally inhibited by interaction with the STRAP protein, but upon starvation, JMY is recruited away from STRAP and activated by LC3., During autophagy, actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in nonstarved cells, cytoplasmic JMY colocalizes with STRAP, a regulator of JMY’s nuclear functions, on nonmotile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY’s de novo actin nucleation activity via a cryptic actin-binding sequence near JMY’s N terminus, and STRAP inhibits JMY’s ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3- and JMY-dependent actin network formation on membranes and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY’s actin assembly activities in trans during autophagy.

In a previous study by our group, we demonstrated that electroacupuncture (EA) activates the class I phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. There is considerable evidence that the downstream mammalian target of rapamycin complex 1 (mTORC1) plays an important role in autophagy following ischemic stroke. The aim of the present study was to determine whether EA exerts a neuroprotective effect through mTORC1-mediated autophagy following ischemia/reperfusion injury. Our results revealed that EA at the LI11 and ST36 acupoints attenuated motor dysfunction, improved neurological deficit outcomes and decreased the infarct volumes. The number of autophagosomes, autolysosomes and lysosomes was decreased following treatment with EA. Simultaneously, the levels of the autophagosome membrane maker, microtubule-associated protein 1 light chain 3 beta (LC3B)II/I, Unc-51-like kinase 1 (ULK1), autophagy related gene 13 Atg13) and Beclin1 (ser14) were decreased, whereas mTORC1 expression was increased in the peri-infarct cortex. These results suggest that EA protects against ischemic stroke through the inhibition of autophagosome formation and autophagy, which is mediated through the mTORC1-ULK complex-Beclin1 pathway.

Autophagy is a cytosolic degradative pathway, which through a series of complicated membrane rearrangements leads to the formation of a unique double membrane vesicle, the autophagosome. The use of fluorescent proteins has allowed visualizing the autophagosome formation in live cells and in real time, almost 40 years after electron microscopy studies observed these structures for the first time. In the last decade, live-cell imaging has been extensively used to study the dynamics of autophagosome formation in cultured mammalian cells. Hereby we will discuss how the live-cell imaging studies have tried to settle the debate about the origin of the autophagosome membrane and how they have described the way different autophagy proteins coordinate in space and time in order to drive autophagosome formation.

Selective autophagy and macroautophagy sequester specific organelles/substrates or bulk cytoplasm, respectively, inside autophagosomes as cargo for delivery to lysosomes. The mammalian ATG8 orthologues (MAP1LC3A/B/C and GABARAP/L1/L2) are ubiquitin (UB)-like proteins conjugated to the autophagosome membrane and are thought to facilitate cargo receptor recruitment, vesicle maturation, and lysosomal fusion. To elucidate the molecular functions of the ATG8 proteins, we engineered cells lacking genes for each subfamily as well as all six mammalian ATG8s. Loss of GABARAPs alone attenuates autophagic flux basally and in response to macroautophagic or selective autophagic stimuli, including parkin-dependent mitophagy, and cells lacking all ATG8 proteins accumulate cytoplasmic UB aggregates, which are resolved following ectopic expression of individual GABARAPs. Autophagosomes from cells lacking GABARAPs had reduced lysosomal content by quantitative proteomics, consistent with fusion defects, but accumulated regulators of late endosome (LE)/autophagosome maturation. Through interaction proteomics of proteins accumulating in GABARAP/L1/L2-deficient cells, we identified C18orf8/RMC1 as a new subunit of the CCZ1-MON1 RAB7 guanine exchange factor (GEF) that positively regulates RAB7 recruitment to LE/autophagosomes. This work defines unique roles for GABARAP and LC3 subfamilies in macroautophagy and selective autophagy and demonstrates how analysis of autophagic machinery in the absence of flux can identify new regulatory circuits.

SummarySelective autophagy comprises cytoplasm-to-lysosome trafficking routes that transport cargos using double-membrane vesicles (autophagosomes). Cargos are detected by receptor proteins, which typically also bind to lipid-conjugated LC3 proteins on autophagosome membranes. We dissected lysosomal delivery of four SQSTM1-like receptors by genome-wide CRISPR screening looking for novel autophagy-related (ATG) factors and trafficking routes. We uncovered new mammalian ATG factors including TMEM41B, an endoplasmic reticulum membrane protein required for autophagosome membrane expansion and/or closure. Furthermore, we found that certain receptors remain robustly targeted to the lysosome even in the absence of ATG7 or other LC3 conjugation factors. Lastly, we identified a unique genetic fingerprint behind receptor flux in ATG7KO cells, which includes factors implicated in nucleating autophagosome formation and vesicle trafficking factors. Our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

The two major objectives of macroautophagy are to sequester cargo away from the cytoplasm and deliver this material for breakdown in the lysosome. Sequestration is complete when the autophagosome membrane undergoes fission to produce separate inner and outer membranes, while delivery into the lysosome requires fusion of the outer autophagosome membrane with the lysosome membrane. Thus, the merging of membranes through fission and fusion underlies each of the pivotal events in macroautophagic clearance. How these merging events are controlled in the cell is poorly understood. Several recent studies however suggest that the two events may be temporally coordinated and rely upon members of the classic membrane fusion SNARE family as well as the autophagy-specific family of Atg8 proteins.

Autophagy is a ubiquitous eukaryotic cellular process, in which cells degrade their own cytoplasmic components by hydrolases within the lysosome. Among many processes, the proteolytic processing of autophagy mediator protein Atg8 is catalyzed by a cysteine protease Atg4, to form Atg8—phosphatidylethanolamine (PE) conjugation, one of the most important step for autophagosome formation. Deconjugation of existing Atg8-PE is also catalyzed by Atg4 facilitates the release of Atg8 from the autophagosome membrane to recycle autophagy progression. Lysosomal hydrolases, familiar as cathepsins, divided into diverse number of enzyme subtypes namely cysteine, serine and aspartic proteases contribute to autophagy by catalyzing the cleavage of peptide bonds of autophagic substrates and thus help disposing the autophagic flux, albeit with the help of many other factors. Even though the cathepsins are implicated in autophagic processes, cathepsin A shows contrasting effect by reducing the rate of chaperone-mediated autophagy through proteolytic processing of lysosome-associated membrane protein type 2a (Lamp2a). Moreover, other families of proteases, such as calpains and caspases, may cleave autophagy-related proteins, negating the execution of autophagic processes. In this review, the overall focus is on the functional role of proteases in autophagy mainly, lysosomal hydrolases known as cathepsins, the cysteine protease Atg4 in yeast and the four orthologs of yeast protease Atg4 in mammalian system termed as “autophagins.” The present review also highlights the fundamental processes of autophagy including dysregulation of Atg4 protease in diverse pathological conditions such as cancer, cardiac diseases, neurodegenerative and infectious diseases. The overall approach of this article has also been extended with a view to emphasize on therapeutic strategies by targeting dysregulation of Atg4 protease associated with various diseases.

Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolonged arginine depletion impaired mitochondrial oxidative phosphorylation function and depolarized mitochondrial membrane potential. Thus, reactive oxygen species (ROS) production significantly increased in both cytosolic and mitochondrial fractions, presumably leading to DNA damage accumulation. Addition of ROS scavenger N-acetyl cysteine or knockdown of ATG5 or BECLIN1 attenuated the chromatophagy phenotype. Our data uncover an atypical autophagy-related death pathway and suggest that mitochondrial damage is central to linking arginine starvation and chromatophagy in two distinct cellular compartments.

The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z' factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC(™)) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry-based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway.

Molecular Biology Autophagy serves an essential cellular-garbage disposal function. For example, the autophagy receptor p62 (or sequestosome-1) is needed for selective removal of intracellular pathogen material to the autophagosome for degradation. Notably, p62 must oligomerize to be able to bind to the autophagosome membrane. Horos et al. discovered that p62 also binds to small noncoding vault RNA 1-1, which inhibits autophagy by preventing p62 oligomerization. During starvation, vault RNA 1-1 expression decreases, and autophagy is reactivated. Demonstration of the direct regulation of protein function by an RNA raises the intriguing possibility of the existence of other physiological riboregulators, including Toll-like receptors or protein kinases important in viral infections. Cell 176 , 1054 (2019).

Several lines of evidence suggest that the mechanism underlying drug-induced neuronal apoptosis is initiated by the increased production of reactive oxygen species (ROS). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been shown to initiate an apoptotic cascade by increasing ROS in the dopaminergic neurons of the substantia nigra, leading to the morphological and physiological features associated with Parkinson's disease. Recently, it has been reported that autophagy, a type of programmed cell death independent of the apoptotic cascade, also plays a role in neuronal damage. Although autophagy is negatively regulated by the mammalian target of rapamycin receptor (mTOR), there is some evidence showing a novel function for the anti-apoptotic protein Bcl-2. Bcl-2 is proposed to play a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Nevertheless, it is unclear whether autophagy is also correlated with apoptotic signaling in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity. Therefore, we hypothesized that the MPP(+) toxicity generally associated with initiating the apoptotic signaling cascade also increases an autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell lines, we demonstrate that MPP(+) increases the expression of microtubule-associated protein light chain 3 (LC3-II), an autophagosome membrane marker and the mTOR signaling pathway, and Beclin 1 while decreasing the Bcl-2 levels. Moreover, these expressions correlate with a decreased binding ratio between Bcl-2 and Beclin 1, in effect limiting the regulation of the downstream autophagic markers, such as LC3-II. Our results indicate that MPP(+) can induce autophagy in SK-N-SH cells by decreasing the Bcl-2/Beclin 1 complex.

Healthy cells use autophagy as a general 'housekeeping' mechanism and to survive stress, including stress induced by nutrient deprivation. Autophagy is initiated at the isolation membrane (originally termed the phagophore), and the coordinated action of ATG (autophagy-related) proteins results in the expansion of this membrane to form the autophagosome. Although the biogenesis of the isolation membrane and the autophagosome is complex and incompletely understood, insight has been gained into the molecular processes involved in initiating the isolation membrane, the source from which this originates (for example, it was recently proposed that the isolation membrane forms from the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM)) and the role of ATG proteins and the vesicular trafficking machinery in autophagosome formation.

SummaryMechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems [1, 2]. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis [3, 4]. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation.

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12-ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1+ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.

Although autophagosome formation has attracted substantial attention, the origin and the source of the autophagosomal membrane remains unresolved. The present study was designed to investigate in vivo subcellular evidence for the biogenesis of autophagosomal membrane during spermiogenesis using transmission-electron microscopy (TEM), Western blots and immunohistochemistry in samples from the Chinese soft-shelled turtle. The testis expressed LC3-II protein, which was located within spermatids at different stages of differentiation and indicated active autophagy. TEM showed that numerous autophagosomes were developed inside spermatids. Many endoplasmic reticulum (ER) were transferred into a special “Chrysanthemum flower center” (CFC) in which several double-layer isolation membranes (IM) were formed and extended. The elongated IM always engulfed some cytoplasm and various structures. Narrow tubules connected the ends of multiple ER and the CFC. The CFC was more developed in spermatids with compact nuclei than in spermatids with granular nuclei. An IM could also be transformed from a single ER. Sometimes an IM extended from a trans-Golgi network and wrapped different structures. The plasma membrane of the spermatid invaginated to form vesicles that were distributed among various endosomes around the CFC during spermiogenesis. All this cellular evidence suggests that, in vivo, IM was developed mainly by CFC produced from ER within differentiating spermatids during spermiogenesis. Vesicles from Golgi complexes, plasma membranes and endosomes might also be the sources of the autophagosome membrane.

MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.