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At least 271 records · Page 15Linked to original sources
PubMed2007

Methods for monitoring autophagy from yeast to human.

The increasing interest in autophagy in a wide range of organisms, accompanied by an ever-growing influx of researchers into this field, necessitates a good understanding of the methodologies available to monitor this process. In this review we discuss current approaches that can be used to follow the overall process of autophagy, as well as individual steps, from yeast to human. The majority of the review considers methods that apply to macroautophagy; however, we also consider alternative types of degradation including chaperone-mediated autophagy and microautophagy. This information is meant to provide a resource for newcomers as well as a stimulus for experienced researchers who may be prompted to develop additional assays to examine autophagy-related pathways.

Animals↗
PubMed2001

Beclin 1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function.

Beclin 1 encodes a Bcl-2-interacting coiled-coil protein with autophagy and tumor suppressor function and is monoallelically deleted in 40-75% of sporadic human breast and ovarian cancers. Beclin 1 contains a leucine-rich nuclear export signal motif raising the possibility that its autophagy and/or tumor suppressor function may require regulated, CRM1-dependent, nucleocytoplasmic transport. In this study, we show that wild-type Beclin 1 colocalizes with both intracytoplasmic organelles and nuclei in COS7 monkey kidney and MCF7 human breast carcinoma cells. Inhibition of CRM1-dependent nuclear export with leptomycin B or mutation of the nuclear export signal motif of Beclin 1 results in predominantly nuclear localization. Unlike wild-type Beclin 1, the nuclear export mutant of Beclin 1 fails to promote nutrient deprivation-induced autophagy and fails to inhibit in vitro clonigenicity and in vivo tumorigenicity of MCF7 cells. Thus, beclin 1 has a leptomycin B-sensitive leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function. These findings suggest that the CRM1 nuclear export pathway may be important in the functional regulation of autophagic growth control.

Amino Acid Sequence↗
PubMed2005

Cathepsins and BID are involved in the molecular switch between apoptosis and autophagy in breast cancer MCF-7 cells exposed to camptothecin.

The details of molecular switching points between apoptosis and autophagy in tumor cells have still not been fully elucidated. This study focused on the role of cathepsin B and its substrate, BID as molecular links between apoptosis and autophagy in human breast cancer MCF-7 cells exposed to camptothecin. Apoptosis occurred rapidly with a peak in 60 min after drug administration, whereas autophagy developed at a much slower rate with continuous progression during 24 h of cell exposure to the drug. CPT induced very rapid activation of cathepsin B. Inhibition of cathepsins by E64d prevented CPT-induced BAX and BID aggregation on mitochondria and reduced significantly reduced apoptotic cell number. The above effects were accompanied by an increase in autophagosome formation, measured by expression of MAP I LC3. BID knock down resulted in strong suppression of CPT-induced apoptosis and a shift of cell death towards autophagy, manifesting with an increase of Beclin 1 and MAP I LC3 cellular content.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Autophagy--a basic mechanism and a potential role for neurodegeneration.

Autophagy constitutes a fundamental survival strategy of cells; its disturbance contributes to the pathogenesis of cancer, liver and immune disease, pathogen infection, myopathies as well as neurodegenerative disorders such as Amyotrophic lateral sclerosis, Parkinson;s, Huntington;s and Alzheimer;s disease. The pathogenesis of neurodegenerative diseases also involves a gradual and progressive loss of neuronal cells. Cells may use different pathways for active self-destruction as reflected by different morphology: while in apoptosis (or "type I") nuclear fragmentation associated with cytoplasmic condensation but preservation of organelles is predominant, autophagic degradation of the cytoplasmic structures preceding nuclear collapse is a characteristic of a second type of programmed cell death (PCD). Linking autophagy to programmed cell death initiated a controversial discussion on how a suggested role of autophagy in cell suicide might meet with its established survival function. To some extent, the diverse morphologies can be associated with distinct biochemical and molecular events [caspase-dependent and -independent death programs, DAP-kinase activity, Ras-expression, induction of autophagy genes, fate of cytoskeleton, among others]. However, there is a broad overlap between cell death pathways. Conceivably, diverse PCD programs emerged during evolution, the conservation of which allows eukaryotic cells a flexible response to physiological or pathological demands.

Animals↗
PubMed1990

Non-selective autophagy.

Autophagy is the major process by which cells degrade their own cytoplasm. Autophagy begins with the sequestration of a portion of the cytoplasm by a membraneous organelle called a phagophore. The resulting vacuole (autophagosome) can fuse with an endocytic vacuole to form am amphisome, which subsequently fuses with a lysosome to have its mixed autophagic/endocytic content degraded by lysosomal enzymes. Autophagy is a non-selective bulk process as indicated by the fact that hepatocytic cytosol enzymes with widely different half-lives are sequestered at the same rate. Regulation of autophagy is exerted at the sequestration step by amino acids, purines, ATP-depleting metabolites, cyclic nucleotides, phosphorylation, and hormones like insulin, glucagon and alpha-adrenergic agonists.

Amino Acids↗
PubMed1978

[Autophagy in visual cell renewal processes (author's transl)].

The biological significance of autophagy in the metabolism of visual cells is discussed. In the process of autophagy, parts of the cytoplasm including cell organelles are surrounded by membranes. This formation is called an autophagic vacuole. Then, the contents of the vacuole are degraded by enzymes. Autophagy is a well known phenomenon occuring in various tissues. In visual cells, however, it was only recently first described by Remé and Young and Remé. Autophagy has been shown to occur in the visual cells of several normal species. The amount of autophagic activity follows a 24-hour rhythm, complementary to an overall protein synthesis. Thus, within a 24-hour period, one cycle of formation and degradation in visual cells is completed. Incubation of isolated retinas significantly increases the number of autophagic vacuoles in inner segments, indicating an adaptation of visual cells to changed metabolic conditions. It is assumed that changes in normal autophagic turnover may be an important factor in visual cell degenerative diseases, thus opening up a new field in retinal pathology.

Animals↗
PubMed2016

Variants in autophagy-related genes and clinical characteristics in melanoma: a population-based study.

Autophagy has been linked with melanoma risk and survival, but no polymorphisms in autophagy-related (ATG) genes have been investigated in relation to melanoma progression. We examined five single-nucleotide polymorphisms (SNPs) in three ATG genes (ATG5; ATG10; and ATG16L) with known or suspected impact on autophagic flux in an international population-based case-control study of melanoma. DNA from 911 melanoma patients was genotyped. An association was identified between (GG) (rs2241880) and earlier stage at diagnosis (OR 0.47; 95% Confidence Intervals (CI) = 0.27-0.81, P = 0.02) and a decrease in Breslow thickness (P = 0.03). The ATG16L heterozygous genotype (AG) (rs2241880) was associated with younger age at diagnosis (P = 0.02). Two SNPs in ATG5 were found to be associated with increased stage (rs2245214 CG, OR 1.47; 95% CI = 1.11-1.94, P = 0.03; rs510432 CC, OR 1.84; 95% CI = 1.12-3.02, P = 0.05). Finally, we identified inverse associations between ATG5 (GG rs2245214) and melanomas on the scalp or neck (OR 0.20, 95% CI = 0.05-0.86, P = 0.03); ATG10 (CC) (rs1864182) and brisk tumor infiltrating lymphocytes (TILs) (OR 0.42; 95% CI = 0.21-0.88, P = 0.02), and ATG5 (CC) (rs510432) with nonbrisk TILs (OR 0.55; 95% CI = 0.34-0.87, P = 0.01). Our data suggest that ATG SNPs might be differentially associated with specific host and tumor characteristics including age at diagnosis, TILs, and stage. These associations may be critical to understanding the role of autophagy in cancer, and further investigation will help characterize the contribution of these variants to melanoma progression.

Adult↗
PubMed2004

Autophagy in yeast: a TOR-mediated response to nutrient starvation.

TOR plays a key role in cell growth and cell-cycle progression, but in addition recent studies have shown that TOR is also involved in the regulation of a number of molecular processes associated with nutrient deprivation, such as autophagy. In budding yeast, TOR negatively regulates activation of Apg1 protein kinase, which is essential for the induction of autophagy. This review describes recent research in this field and the mechanism by which TOR mediates induction of autophagy.

Adaptor Proteins, Signal Transducing↗
PubMed2004

In vivo and in vitro reconstitution of Atg8 conjugation essential for autophagy.

In an analogous manner to protein ubiquitination, The C terminus of Atg8p, a yeast protein essential for autophagy, conjugates to a head group of phosphatidylethanolamine via an amide bond. Though physiological role of this reaction is assigned to membrane organization during autophagy, its molecular details are still unknown. Here, we show that Escherichia coli cells coexpressed Atg8p, Atg7p (E1), and Atg3p (E2) allowed to form conjugate of Atg8p with endogenous PE. Further, we established an in vitro Atg8p-PE reconstitution system using purified Atg8pG116, Atg7p, Atg3p, and PE-containing liposomes, demonstrating that the Atg7p and the Atg3p are minimal catalysts for Atg8p-PE conjugate reaction. Efficiency of this lipidation reaction depends on the state of the substrate, PE (phospholipid bilayer and its lipid composition). It is also suggested that the lipidation induces a conformational change in the N-terminal region of Atg8p. In vitro system developed here will provide a powerful system for further understanding the precise role of lipidation and interaction of two ubiquitin-like systems essential for autophagy.

Autophagy↗
PubMed2004

Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy.

Delivery of proteins and organelles to the vacuole by autophagy and the cytoplasm to vacuole targeting (Cvt) pathway involves novel rearrangements of membrane resulting in the formation of vesicles that fuse with the vacuole. The mechanism of vesicle formation and the origin of the membrane are complex issues still to be resolved. Atg18 and Atg21 are proteins essential to vesicle formation and together with Ygr223c form a novel family of phosphoinositide binding proteins that are associated with the vacuole and perivacuolar structures. Their localization requires the activity of Vps34, suggesting that phosphatidylinositol(3)phosphate may be essential for their function. The activity of Atg18 is vital for all forms of autophagy, whereas Atg21 is required for the Cvt pathway but not for nitrogen starvation-induced autophagy. The loss of Atg21 results in the absence of Atg8 from the pre-autophagosomal structure (PAS), which may be ascribed to a reduced rate of conjugation of Atg8 to phosphatidylethanolamine. A similar defect in localization of a second ubiquitin-like conjugate, Atg12-Atg5, suggests that Atg21 may be involved in the recruitment of membrane to the PAS.

Aminopeptidases↗
PubMed2000

Age-related decline in chaperone-mediated autophagy.

Intracellular protein degradation rates decrease with age in many tissues and organs. In cultured cells, chaperone-mediated autophagy, which is responsible for the selective degradation of cytosolic proteins in lysosomes, decreases with age. In this work we use lysosomes isolated from rat liver to analyze age-related changes in the levels and activities of the main components of chaperone-mediated autophagy. Lysosomes from "old" (22-month-old) rats show lower rates of chaperone-mediated autophagy, and both substrate binding to the lysosomal membrane and transport into lysosomes decline with age. A progressive age-related decrease in the levels of the lysosome-associated membrane protein type 2a that acts as a receptor for chaperone-mediated autophagy was responsible for decreased substrate binding in lysosomes from old rats as well as from late passage human fibroblasts. The cytosolic levels and activity of the 73-kDa heat-shock cognate protein required for substrate targeting to lysosomes were unchanged with age. The levels of lysosome-associated hsc73 were increased only in the oldest rats. This increase may be an attempt to compensate for reduced activity of the pathway with age.

Age Factors↗
PubMed2000

Selective degradation of annexins by chaperone-mediated autophagy.

Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. Analysis of the sequences of the different members of the annexin family revealed the presence of a pentapeptide biochemically related to KFERQ in some annexins but not in others. Such sequences have been proposed to be a targeting sequence for chaperone-mediated autophagy, a lysosomal pathway of protein degradation that is activated in confluent cells in response to removal of serum growth factors. We demonstrate that annexins II and VI, which contain KFERQ-like sequences, are degraded more rapidly in response to serum withdrawal, while annexins V and XI, without such sequences, are degraded at the same rate in the presence and absence of serum. Using isolated lysosomes, only the annexins containing KFERQ-like sequences are degraded by chaperone mediated-autophagy. Annexins V and XI could associate with lysosomes but did not enter the lysosomes and were not proteolytic substrates. Furthermore, four annexins containing KFERQ-like sequences, annexins I, II, IV, and VI, are enriched in lysosomes with high chaperone-mediated autophagy activity as expected for substrate proteins. These results provide striking evidence for the importance of KFERQ motifs in substrates of chaperone-mediated autophagy.

Amino Acid Motifs↗
PubMed2025

Inhibition of autophagy-lysosomal function exacerbates microglial and monocyte lipid metabolism reprograming and dysfunction after brain injury.

CNS has an overall higher level of lipids than all tissues except adipose and contains up to 25% of total body cholesterol. Recent data demonstrate a complex crosstalk between lipid metabolism and inflammation, suggesting potential contribution of the lipid-rich brain environment to neuroinflammation. While recent data support the importance of brain lipid environment to inflammatory changes observed in age related chronic neurodegenerative diseases, in vivo interactions between lipid environment, lipid metabolism and neuroinflammation in acute brain disease and injury remain poorly understood. Here we utilize a mouse model of traumatic brain injury (TBI) to demonstrate that acute neurotrauma leads to widespread lipid metabolism reprograming in all microglial and brain associated and infiltrating monocyte populations. Additionally, we identify unique microglial and monocyte populations with higher degree of lipid metabolism reprograming and pronounced accumulation of neutral storage lipids, including cholesteryl esters and triglycerides. These lipids accumulate not only in lipid droplets but also in the microglial and monocyte lysosomes and are associated with lysosomal dysfunction and inhibition of autophagy after TBI. Our data indicate that lipid accumulation in these cells is the result of altered lipid handling rather than lipid synthesis and is triggered by phagocytosis of lipid-rich myelin debris generated after TBI. Finally, we use mice with autophagy defects in microglia and monocytes to demonstrate that further inhibition of autophagy leads to more pronounced lipid metabolism reprograming and exacerbated cellular lipid accumulation. Our data suggest a pathological feedback loop, where lipid phagocytosis causes inhibition of autophagy-lysosomal function, which in turn exacerbates cellular lipid retention, reprograming and inflammation.

Journal Article↗
PubMed2001

Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p.

We here report the identification of AUT10 as a novel gene required for both the cytoplasm to vacuole targeting of proaminopeptidase I and starvation-induced autophagy. aut10Delta cells are impaired in maturation of proaminopeptidase I under starvation and non-starvation conditions. A lack of Aut10p causes a defect in autophagy prior to vacuolar uptake of autophagosomes. Homozygous aut10Delta diploids do not sporulate. Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10Delta cells and mature vacuolar proteinases are present. A biologically active Ha-tagged Aut10p, chromosomally expressed from its endogenous promoter, localizes in indirect immunofluorescence microscopy in the cytosol and on granulated structures, which appear clustered around the vacuolar membrane. This localization differs from known autophagy proteins.

Amino Acid Sequence↗
PubMed2006

Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis--a finding with potential importance for clinical anticancer therapies.

Antibiotics, Antineoplastic↗
PubMed1998

Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast, Saccharomyces cerevisiae.

Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy. Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apg14p has a coiled-coil motif at its N terminus region. We found that overproduction of Apg14p partially reversed the defect in autophagy induced by the apg6-1 mutation. The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole. However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-1 mutant, nor did the apg14 null mutation affect the CPY sorting pathway. Structural analysis of APG6 revealed that APG6 is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vps10p, to the late-Golgi from the endosome (Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J. Cell Biol. 137, 79-92). Subcellular fractionation indicated that Apg14p and Apg6p peripherally associated with a membrane structure(s). Apg14p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex. These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway. Apg14p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway.

Amino Acid Sequence↗
PubMed2005

Trs85 (Gsg1), a component of the TRAPP complexes, is required for the organization of the preautophagosomal structure during selective autophagy via the Cvt pathway.

Autophagosomes and Cvt vesicles are limited by two membrane layers. The biogenesis of these unconventional vesicles and the origin of their membranes are hardly understood. Here we identify in Saccharomyces cerevisiae Trs85, a nonessential component of the TRAPP complexes, to be required for the biogenesis of Cvt vesicles. The TRAPP complexes function in endoplasmic reticulum-to-Golgi and Golgi trafficking. Growing trs85delta cells show a defect in the organization of the preautophagosomal structure. Although proaminopeptidase I is normally recruited to the preautophagosomal structure, the recruitment of green fluorescent protein-Atg8 depends on Trs85. Autophagy proceeds in the absence of Trs85, albeit at a reduced rate. Our electron microscopic analysis demonstrated that the reduced autophagic rate of trs85delta cells does not result from a reduced size of the autophagosomes. Growing and starved cells lacking Trs85 did not show defects in vacuolar biogenesis; mature vacuolar proteinase B and carboxypeptidase Y were present. Also vacuolar acidification was normal in these cells. It is known that mutations impairing the integrity of the ER or Golgi block both autophagy and the Cvt pathway. But the phenotypes of trs85delta cells show striking differences to those seen in mutants with defects in the early secretory pathway. This suggests that Trs85 might play a direct role in the Cvt pathway and autophagy.

Autophagy↗
PubMed2003

Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy.

Macroautophagy is a catabolic membrane trafficking phenomenon that is observed in all eukaryotic cells in response to various stimuli, such as nitrogen starvation and challenge with specific hormones. In the yeast Saccharomyces cerevisiae, the induction of autophagy involves a direct signal transduction mechanism that affects membrane dynamics. In this system, the induction process modifies a constitutive trafficking pathway called the cytoplasm-to-vacuole targeting (Cvt) pathway, which transports the vacuolar hydrolase aminopeptidase I, from the formation of small Cvt vesicles to the formation of autophagosomes. Apg1 is one of the proteins required for the direct signal transduction cascade that modifies membrane dynamics. Although Apg1 is required for both the Cvt pathway and autophagy, we find that Apg1 kinase activity is required only for Cvt trafficking of aminopeptidase I but not for import via autophagy. In addition, the data support a novel role for Apg1 in nucleation of autophagosomes that is distinct from its catalytic kinase activity and imply a qualitative difference in the mechanism of autophagosome and Cvt vesicle formation.

Aminopeptidases↗