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Results for “Autophagy”

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At least 163 records · Page 9Linked to original sources

PubMed2002

Molecular machinery required for autophagy and the cytoplasm to vacuole targeting (Cvt) pathway in S. cerevisiae.

Autophagy is a vacuolar trafficking pathway that targets subcellular constituents to the vacuole for degradation and recycling. In nutrient-rich conditions in yeast, a different vacuolar trafficking pathway, the cytoplasm to vacuole targeting (Cvt) pathway, transports the resident hydrolase aminopeptidase I to the vacuole, using many of the same molecular components as autophagy. The Cvt pathway is constitutive, whereas autophagy is induced by starvation. Recent studies have laid important groundwork for understanding the signaling mechanism that induces autophagy. Another key advance has been the identification of two novel conjugation systems that function in vesicle formation in both pathways. Finally, many autophagy- and Cvt-specific gene products, including those involved in lipid modification, vesicle expansion and cargo specificity, have been shown to localize to a novel perivacuolar membrane compartment. Additional analysis of this location will help in further dissecting the early events of vesicle formation and identifying the source of the sequestering membrane.

Autophagy↗

PubMed2002

Inhibition of autophagy in mitotic animal cells.

In nutrient-deprived cells autophagy recycles cytoplasmic constituents by engulfing and degrading them in membrane-bound autophagic vacuoles. The regulation of autophagic vacuole formation is poorly understood, but here we show this process is under strict cell-cycle control in cultured animal cells. We found strong inhibition of autophagic vacuole accumulation in nocodazole-arrested pseudo-prometaphase cells, and also in metaphase and anaphase cells generated on release from the nocodazole arrest. Autophagic vacuoles reappeared after closure of the nuclear envelope in telophase/G1. Treatment with phosphoinositide 3(PI3)-kinase inhibitors wortmannin, LY294002 and 3-methyladenine (known to inhibit the autophagic response in interphase cells) rescued autophagy in mitotic cells without inducing reassembly of vesiculated ER and Golgi compartments. The autophagy induced in mitotic cells was inhibited by amino acids, and the resulting autophagosomes contained proteins LC3 and Lamp1, known to be associated with autophagosomes in interphase cells. The mitotic inhibition of autophagy was not relieved by rapamycin treatment or in PDK1-/- embryonic stem cells, by microinjection of inhibitory antibodies against the class III PI3 kinase VPS34, or in cell lines lacking the p85 regulatory subunits of class IA PI3 kinases. Our results show that autophagy is under strict mitotic control and indicate a novel role for phosphoinositide 3-kinases or other wortmannin/LY294002-sensitive kinases in mitotic membrane traffic regulation.

Adenine↗

PubMed2006

Autophagy delays apoptotic death in breast cancer cells following DNA damage.

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.

Apoptosis↗

PubMed2004

Autophagy as a cell death and tumor suppressor mechanism.

Autophagy is characterized by sequestration of bulk cytoplasm and organelles in double or multimembrane autophagic vesicles, and their delivery to and subsequent degradation by the cell's own lysosomal system. Autophagy has multiple physiological functions in multicellular organisms, including protein degradation and organelle turnover. Genes and proteins that constitute the basic machinery of the autophagic process were first identified in the yeast system and some of their mammalian orthologues have been characterized as well. Increasing lines of evidence indicate that these molecular mechanisms may be recruited by an alternative, caspase-independent form of programmed cell death, named autophagic type II cell death. In some settings, autophagy and apoptosis seem to be interconnected positively or negatively, introducing the concept of 'molecular switches' between them. Additionally, mitochondria may be central organelles integrating the two types of cell death. Malignant transformation is frequently associated with suppression of autophagy. The recent implication of tumor suppressors like Beclin 1, DAP-kinase and PTEN in autophagic pathways indicates a causative role for autophagy deficiencies in cancer formation. Autophagic cell death induction by some anticancer agents underlines the potential utility of its induction as a new cancer treatment modality.

Animals↗

PubMed2006

Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival.

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.

Animals↗

PubMed2006

Autophagy is not a main contributor to the degradation of phospholipids in tobacco cells cultured under sucrose starvation conditions.

Net degradation of cellular components occurs in plant cells cultured under starvation conditions, and autophagy contributes to the degradation of intracellular proteins. In this study, we investigated the degradation of membrane phospholipids by autophagy in cultured tobacco (Nicotiana tabacum) cells. The amounts of total phospholipids and a major phospholipid, phosphatidylcholine (PC), decreased, whereas phosphorylcholine, a degradation product of PC, increased in response to deprivation of sucrose. The addition of glycerol to the culture medium inhibited both the degradation of phospholipids and the concomitant increase of phosphorylcholine. Glycerol, however, did not block autophagy, which was assessed by the accumulation of autolysosomes in the presence of a cysteine protease inhibitor. On the other hand, 3-methyladenine, an inhibitor of autophagy, did not affect the net degradation of PC. We labeled intracellular phospholipids by loading cells with a fluorochrome-labeled fatty acid and chased it under sucrose-free conditions. Glycerol slowed down the decrease in the amount of fluorochrome-labeled PC, suggesting that it inhibits the degradation process of PC. These results show that phospholipids are degraded by mechanisms different from autophagy in tobacco cells cultured under sucrose-free conditions.

Adenine↗

PubMed2005

Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein.

Autophagy is a process that is thought to occur in all eukaryotes in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon induction of autophagy, double membrane-bound structures called autophagosomes engulf portions of the cytoplasm and transfer them to the vacuole or lysosome for degradation. In this study, we have characterized two potential markers for autophagy in plants, the fluorescent dye monodansylcadaverine (MDC) and a green fluorescent protein (GFP)-AtATG8e fusion protein, and propose that they both label autophagosomes in Arabidopsis. Both markers label the same small, apparently membrane-bound structures found in cells under conditions that are known to induce autophagy such as starvation and senescence. They are usually seen in the cytoplasm, but occasionally can be observed within the vacuole, consistent with a function in the transfer of cytoplasmic material into the vacuole for degradation. MDC-staining and the GFP-AtATG8e fusion protein can now be used as very effective tools to complement biochemical and genetic approaches to the study of autophagy in plant systems.

Arabidopsis↗

PubMed2005

Autophagy is an immediate macrophage response to Legionella pneumophila.

After ingestion by macrophages, Legionella pneumophila enter spacious vacuoles that are quickly enveloped by endoplasmic reticulum (ER), then slowly transferred to lysosomes. Here we demonstrate that the macrophage autophagy machinery recognizes the pathogen phagosome as cargo for lysosome delivery. The autophagy conjugation enzyme Atg7 immediately translocated to phagosomes harbouring virulent Legionella. Subsequently, Atg8, a second autophagy enzyme, and monodansyl-cadaverine (MDC), a dye that accumulates in acidic autophagosomes, decorated the pathogen vacuoles. The autophagy machinery responded to 10-30 kDa species released into culture supernatants by Type IV secretion-competent Legionella, as judged by the macrophages' processing of Atg8 and formation of vacuoles that sequentially acquired Atg7, Atg8 and MDC. When compared with autophagosomes stimulated by rapamycin, Legionella vacuoles acquired Atg7, Atg8 and MDC more slowly, and Atg8 processing was also delayed. Moreover, compared with autophagosomes of Legionella-permissive naip5 mutant A/J macrophages, those of resistant C57BL/6 J macrophages matured quickly, preventing efficient Legionella replication. Accordingly, we discuss a model in which macrophages elevate autophagy as a barrier to infection, a decision influenced by regulatory interactions between Naip proteins and caspases.

Animals↗

PubMed2003

Autophagy genes are essential for dauer development and life-span extension in C. elegans.

Both dauer formation (a stage of developmental arrest) and adult life-span in Caenorhabditis elegans are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes. Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells. Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that bec-1, the C. elegans ortholog of the yeast and mammalian autophagy gene APG6/VPS30/beclin1, is essential for normal dauer morphogenesis and life-span extension. Dauer formation is associated with increased autophagy and also requires C. elegans orthologs of the yeast autophagy genes APG1, APG7, APG8, and AUT10. Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in C. elegans.

Animals↗

PubMed2024

Macrophage LRRK2 hyperactivity impairs autophagy and induces Paneth cell dysfunction.

LRRK2 polymorphisms (G2019S/N2081D) that increase susceptibility to Parkinson's disease and Crohn's disease (CD) lead to LRRK2 kinase hyperactivity and suppress autophagy. This connection suggests that LRRK2 kinase inhibition, a therapeutic strategy being explored for Parkinson's disease, may also benefit patients with CD. Paneth cell homeostasis is tightly regulated by autophagy, and their dysfunction is a precursor to gut inflammation in CD. Here, we found that patients with CD and mice carrying hyperactive LRRK2 polymorphisms developed Paneth cell dysfunction. We also found that LRRK2 kinase can be activated in the context of interactions between genes (genetic autophagy deficiency) and the environment (cigarette smoking). Unexpectedly, lamina propria immune cells were the main intestinal cell types that express LRRK2, instead of Paneth cells as previously suggested. We showed that LRRK2-mediated pro-inflammatory cytokine release from phagocytes impaired Paneth cell function, which was rescued by LRRK2 kinase inhibition through activation of autophagy. Together, these data suggest that LRRK2 kinase inhibitors maintain Paneth cell homeostasis by restoring autophagy and may represent a therapeutic strategy for CD.

Leucine-Rich Repeat Serine-Threonine Protein Kinas↗

PubMed2006

Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae.

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.

Aspergillus oryzae↗

PubMed2026

Crotonylome profiling identifies MLKL crotonylation in lupus nephritis associated with RAB1A-mTOR signalling and autophagy changes in tubular epithelial cells.

OBJECTIVE: To investigate whether MLKL crotonylation is associated with tubular autophagy-lysosome pathway homeostasis in lupus nephritis (LN) and to explore its relationship with RAB1A-mechanistic target of rapamycin (mTOR) signalling. METHODS: Crotonylome proteomics was performed in peripheral blood mononuclear cells from patients with LN, patients with systemic lupus erythematosus without nephritis and healthy controls. Renal biopsy tissues were evaluated for tubulointerstitial fibrosis and autophagy-lysosome pathway-related markers. Mechanistic studies were conducted in lipopolysaccharide-stimulated HK-2 cells. Autophagic flux was assessed using bafilomycin A1. The dependency of mTOR/autophagy changes on RAB1A was tested by siRNA-mediated knockdown. RESULTS: MLKL was identified as a differentially crotonylated protein in LN, with increased crotonylation at K95 and K219. Kidney tissues from patients with LN showed increased fibronectin and collagen III deposition compared with controls, whereas no significant difference was observed between class IV and class V LN. LC3 signal did not differ significantly between groups, whereas LAMP1 expression and LC3-LAMP1 co-localisation were reduced in LN. In HK-2 cells, crotonylation-deficient MLKL mutants were associated with increased LC3-II and reduced p62, whereas K219Q showed the opposite pattern. Autophagic flux assays using bafilomycin A1 showed that K219R-expressing cells had higher LC3-II levels than WT cells both before and after lysosomal inhibition, with comparable BafA1-induced LC3-II accumulation, consistent with increased autophagosome formation rather than impaired lysosomal degradation. HDAC1 knockdown increased MLKL crotonylation and was accompanied by mTOR activation. MLKL crotonylation enhanced RAB1A guanriphosphat osphate (GTP) binding without altering total RAB1A abundance. RAB1A knockdown in MLKL WT-expressing cells attenuated mTOR phosphorylation and partly reversed the autophagy-suppressive marker profile. Sodium crotonate induced an autophagy-suppressive marker profile that was partly reversed by rapamycin. CONCLUSION: MLKL crotonylation is associated with activation of the RAB1A-mTOR axis and altered tubular autophagy-lysosome pathway homeostasis in LN. These findings suggest that tubular injury-related changes in LN may not be fully reflected by glomerulus-based classification alone.

Humans↗

PubMed2006

Autophagy in cancer: good, bad, or both?

Autophagy has been recognized as an important cellular process for at least 50 years; however, it is only with the recent identification of key regulators of autophagy (Atg genes) that we have begun a mechanistic exploration of its importance in cancer. Recent studies suggest that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy. However, the role of autophagy in these processes is complicated and may, depending on the circumstances, have diametrically opposite consequences for the tumor. In this article, we discuss recent discoveries regarding autophagy in cancer.

Animals↗

PubMed2001

Autophagy and nuclear changes in FM3A breast tumor cells after epirubicin, medroxyprogesterone and tamoxifen treatment in vitro.

OBJECTIVE: Autophagy is a form of physiological programmed cell death which is observable after hormonal withdrawal. In this study, the FM3A murine breast tumor cell line was treated with epirubicin alone and with medroxyprogesterone acetate (MPA) or tamoxifen, to determine if structural and kinetic signs of autophagy may also occur in an enhanced manner during epirubicin sensitization via hormonal agents. METHODS: One-week soft agar colony growth, 96-hour values of plating and pulse thymidine labeling and electron microscopical examinations were performed following treatment with MPA and tamoxifen alone or with epirubicin. RESULTS: Tamoxifen induced signs of autophagy, which was enhanced when it was combined with MPA. Epirubicin also induced autophagy of secretory granules, which coalesced to form an intracytoplasmic lumen. Combining MPA with epirubicin enhanced the formation of apoptotic blebs and chromatin fragmentation. When epirubicin was combined with tamoxifen, peculiar nuclear structures were formed. CONCLUSIONS: Hormonal agents may modulate anthracycline activity towards specific patterns in eliciting cell death, via autophagy and/or as yet unknown nucleolus-specific interactions.

Antibiotics, Antineoplastic↗

PubMed1997

Inhibition and restimulation by insulin of cellular autophagy in distal tubular cells of the kidney in early diabetic rats.

Cellular autophagy in the cells of distal tubular segments (DT cells) of the kidney cortex in streptozotocin-diabetic rats was evaluated by quantitative electron microscopy. Five days after streptozotocin administration, volume and numerical densities of autophagic vacuoles (AVs) in DT cells were reduced by 56 and 57%, respectively. The correction of the diabetic state by insulin injection reversed the inhibition of cellular autophagy. Volume and numerical densities of AVs increased by 97 and 53%, respectively. Endogenous insulin replacement by islet transplantation showed the same effect on cellular autophagy. Volume and numerical densities of AVs increased by 82 and 34%, respectively, as compared with sham-operated diabetic animals. The data show that, during diabetic kidney hypertrophy, the cellular autophagy is inhibited in DT cells to the same extent as in proximal tubular cells, suggesting that DT cells contribute in a balanced manner to hypertrophic growth of the kidney cortex. Similarly, DT cells are involved in the catabolic reaction, i.e., stimulation of autophagy, after metabolic correction of the diabetic state by insulin.

Animals↗

PubMed2026

CRISPR screen identifies autophagy inhibition (GNS561) as a PARP inhibitor (AZD5305) combination strategy in small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is a deadly cancer with few treatment options and poor prognosis, creating a dire need for improving therapies. Poly (ADP-ribose) polymerase inhibitors (PARPi) have been tested as a treatment strategy, but patient response varies. We aimed to identify novel approaches to sensitize SCLC to PARPi through a genome-wide CRISPR dropout screen. METHODS: Genome-wide CRISPR dropout screening was conducted in two SCLC cell lines using the PARPi, olaparib, as the selection pressure. Stable shRNA-mediated knockdown cell lines were validated by Western blotting and tested for olaparib sensitivity by assaying for cell viability. Synergy between PARPi and autophagy inhibition was tested by treating SCLC cell lines and analyzing cell viability using SynergyFinder+. The therapeutic strategy combining AZD5305 (PARPi) and GNS561 (novel autophagy inhibitor) was tested in cell line-derived xenograft mouse models. RESULTS: CRISPR screening identified the loss of mTOR negative regulators as a mechanism of PARPi sensitivity in SCLC, and knockdown of TSC1 and TSC2 sensitized SCLC cell lines to olaparib. Therapeutic strategies combining PARPi and autophagy inhibition demonstrated synergy in SCLC cell lines, and combination therapy with AZD5305 and GNS561 was effective in cell line-derived xenograft mouse models. CONCLUSIONS: Autophagy inhibition downstream of the mTOR pathway is a mechanism of PARPi sensitivity in SCLC. This suggests that a therapeutic combination of autophagy inhibition and PARPi is a promising treatment strategy in SCLC, paving the way for the adoption of novel treatments in this disease context.

Autophagy↗

PubMed2005

The molecular machinery of autophagy: unanswered questions.

Autophagy is a process in which cytosol and organelles are sequestered within double-membrane vesicles that deliver the contents to the lysosome/vacuole for degradation and recycling of the resulting macromolecules. It plays an important role in the cellular response to stress, is involved in various developmental pathways and functions in tumor suppression, resistance to pathogens and extension of lifespan. Conversely, autophagy may be associated with certain myopathies and neurodegenerative conditions. Substantial progress has been made in identifying the proteins required for autophagy and in understanding its molecular basis; however, many questions remain. For example, Tor is one of the key regulatory proteins at the induction step that controls the function of a complex including Atg1 kinase, but the target of Atg1 is not known. Although autophagy is generally considered to be nonspecific, there are specific types of autophagy that utilize receptor and adaptor proteins such as Atg11; however, the means by which Atg11 connects the cargo with the sequestering vesicle, the autophagosome, is not understood. Formation of the autophagosome is a complex process and neither the mechanism of vesicle formation nor the donor membrane origin is known. The final breakdown of the sequestered cargo relies on well-characterized lysosomal/vacuolar proteases; the roles of lipases, by contrast, have not been elucidated, and we do not know how the integrity of the lysosome/vacuole membrane is maintained during degradation.

Autophagy↗

PubMed1997

Mutational analysis of Csc1/Vps4p: involvement of endosome in regulation of autophagy in yeast.

In the yeast Saccharomyces cerevisiae, autophagy, a bulk protein degradation in the vacuole, is induced in response to nutrient starvation. In a screen for mutations that result in induction of autophagy even in the presence of nutrients, we have isolated four mutants representing two csc complementation groups. These mutants induce autophagy of which activity is represented by activation of truncated alkaline phosphatase that is designed to be expressed in the cytosol. CSC1 was cloned by complementation of loss of viability phenotype of csc1-1 mutant and shown to be identical to END13/VPS4/GRD13. Though csc1-1 mutation is recessive, cells of delta csc1 do not induce autophagy in rich media, suggesting that csc1-1 allele is not a complete loss-of-function. Csc1p is a member of novel ATPase family named AAA protein including Sec18p/NSF, Cdc48p/p97, and Pas8p. Mutation site in csc1-1 is found in the SRH region that is highly conserved among AAA proteins. Cells of csc1-1 show sorting defect of CPY and the appearance of the class E compartment. These mutant phenotypes suggest the role of the protein that is involved in the traffic among the Golgi, endosome, and the vacuole in autophagy.

Adenosine Triphosphatases↗