Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42345 (mTOR)
 26,049 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Autophagy is a vital response to nutrient starvation. Here, we screened a kinase-specific siRNA library using an autophagy assay in human embryonic kidney 293 cells that measures lipidation of the marker protein GFP-LC3 following amino acid starvation. This screen identified ULK1 in addition to other novel candidates that could be confirmed with multiple siRNAs. Knockdown of ULK1, but not the related kinase ULK2, inhibited the autophagic response. Also, ULK1 knockdown inhibited rapamycin-induced autophagy consistent with a role downstream of mTOR. Overexpression of ULK1 inhibited autophagy and this inhibition was independent of its kinase activity. Deletion of the PDZ domain-binding Val-Tyr-Ala motif at the ULK1 C terminus generated a more potent dominant-negative protein. Further deletions revealed that the minimal ULK1 dominant-negative region could be mapped to residues 1-351. Full-length ULK1 localized to cytoplasmic structures, some of which were GFP-LC3-positive, and this localization required the conserved C-terminal domain. In contrast, ULK1-(1-351) was diffuse in the cytoplasm. These experiments reveal at least two domains in ULK1 which likely function via unique sets of effectors to regulate autophagy.
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PMID:siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy. 1759 59

G(M1)-gangliosidosis is an autosomal recessive lysosomal lipid storage disorder, caused by mutations of the lysosomal beta-galactosidase (beta-gal) and results in the accumulation of G(M1). The underlying mechanisms of neurodegeneration are poorly understood. Here we demonstrate increased autophagy in beta-gal-deficient (beta-gal(-/-)) mouse brains as evidenced by elevation of LC3-II and beclin-1 levels. Activation of autophagy in the beta-gal(-/-) brain was found to be accompanied with enhanced Akt-mTOR and Erk signaling. In addition, the mitochondrial cytochrome c oxidase activity was significantly decreased in brains and cultured astrocytes from beta-gal(-/-) mouse. Mitochondria isolated from beta-gal(-/-) astrocytes were morphologically abnormal and had a decreased membrane potential. These cells were more sensitive to oxidative stress than wild type cells and this sensitivity was suppressed by ATP, an autophagy inhibitor 3-methyladenine and a pan-caspase inhibitor z-VAD-fmk. These results suggest activation of autophagy leading to mitochondrial dysfunction in the brain of G(M1)-gangliosidosis.
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PMID:Enhanced autophagy and mitochondrial aberrations in murine G(M1)-gangliosidosis. 1819 Jul 92

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 microM and 8 microM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A1, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing 3 mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.
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PMID:A method to measure cardiac autophagic flux in vivo. 1821 95

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.
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PMID:Autophagy plays a protective role during zVAD-induced necrotic cell death. 1825 89

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that remains the major infectious cause of birth defects, as well as being an important opportunistic pathogen. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved process responsible for the degradation of cytoplasmic macromolecules, and the elimination of damaged organelles via a lysosomal pathway. This process is also triggered in organisms by stressful conditions and by certain diseases. Previous observations have suggested that autophagy (also known as xenophagy in this case) may contribute to innate immunity against viral infections. Recent studies on HSV-1, another herpesvirus, have shown that HSV-1 is able to avoid this cellular defense by means of a viral protein, ICP34.5, which antagonizes the host autophagy response. However, it was not known whether HCMV was also able to counteract autophagy. Here, we show that HCMV infection drastically inhibits autophagosome formation in primary human fibroblasts. Autophagy was assessed by GFP-LC3 redistribution, LC3-II and p62 accumulation and electron microscopy. Inhibition of autophagy occurred early in the infection by a mechanism involving viral protein(s). Indeed, only infected cells expressing viral proteins displayed a striking decrease of autophagy; whereas bystander, non-infected cells displayed a level of autophagy similar to that of control cells. HCMV activated the mTOR signaling pathway, and rendered infected cells resistant to rapamycin-induced autophagy. Moreover, infected cells also became resistant to the stimulation of autophagy by lithium chloride, an mTOR-independent inducer of autophagy. These findings suggest that HCMV has developed efficient strategies for blocking the induction of autophagy during infection.
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PMID:Human cytomegalovirus controls a new autophagy-dependent cellular antiviral defense mechanism. 1834 Jan 11

In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.
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PMID:CCK-induced pancreatic growth is not limited by mitogenic capacity in mice. 1835 33

DNA mismatch repair (MMR) ensures the fidelity of DNA replication and is required for activation of cell cycle arrest and apoptosis in response to certain classes of DNA damage. We recently reported that MMR is also implicated in initiation of an autophagic response after MMR processing of 6-thioguanine (6-TG). It is now generally believed that autophagy is negatively controlled by mammalian target of rapamycin (mTOR) activity. To determine whether mTOR is involved in 6-TG-induced autophagy, we used rapamycin, a potential anticancer agent, to inhibit mTOR activity. Surprisingly, we find that rapamycin cotreatment inhibits 6-TG-induced autophagy in MMR-proficient human colorectal cancer HCT116 (MLH1(+)) and HT29 cells as measured by LC3 immunoblotting, GFP-LC3 relocalization, and acridine orange staining. Consistently, short interfering RNA silencing of the 70-kDa ribosomal S6 kinase 1 (S6K1), the downstream effector of mTOR, markedly reduces 6-TG-induced autophagy. Furthermore, we show that inhibition of mTOR by rapamycin induces the activation of Akt as shown by increased Akt phosphorylation at Ser(473) and the inhibition of 6-TG-induced apoptosis and cell death. Activated Akt is a well-known inhibitor of autophagy. In conclusion, our data indicate that mTOR-S6K1 positively regulates autophagy after MMR processing of 6-TG probably through its negative feedback inhibition of Akt.
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PMID:Mammalian target of rapamycin and S6 kinase 1 positively regulate 6-thioguanine-induced autophagy. 1838 46

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.
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PMID:Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer. 1842 12

The Bcl-2 family proteins are important regulators of type I programmed cell death apoptosis; however, their role in autophagic cell death (AuCD) or type II programmed cell death is still largely unknown. Here we report the cloning and characterization of a novel Bcl-2 homology domain 3 (BH3)-only protein, apolipoprotein L1 (apoL1), that, when overexpressed and accumulated intracellularly, induces AuCD in cells as characterized by the increasing formation of autophagic vacuoles and activating the translocation of LC3-II from the cytosol to the autophagic vacuoles. Wortmannin and 3-methyladenine, inhibitors of class III phosphatidylinostol 3-kinase and, subsequently, autophagy, blocked apoL1-induced AuCD. In addition, apoL1 failed to induce AuCD in autophagy-deficient ATG5(-/-) and ATG7(-/-) mouse embryonic fibroblast cells, suggesting that apoL1-induced cell death is indeed autophagy-dependent. Furthermore, a BH3 domain deletion construct of apoL1 failed to induce AuCD, demonstrating that apoL1 is a bona fide BH3-only pro-death protein. Moreover, we showed that apoL1 is inducible by p53 in p53-induced cell death and is a lipid-binding protein with high affinity for phosphatidic acid (PA) and cardiolipin (CL). Previously, it has been shown that PA directly interacted with mammalian target of rapamycin and positively regulated the ability of mammalian target of rapamycin to activate downstream effectors. In addition, CL has been shown to activate mitochondria-mediated apoptosis. Sequestering of PA and CL with apoL1 may alter the homeostasis between survival and death leading to AuCD. To our knowledge, this is the first BH3-only protein with lipid binding activity that, when overproduced intracellularly, induces AuCD.
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PMID:Apolipoprotein L1, a novel Bcl-2 homology domain 3-only lipid-binding protein, induces autophagic cell death. 1850 29

Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the neurodegeneration of Alzheimer's disease (AD). The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain, however, is unknown. In this study, we show that constitutive macroautophagy in primary cortical neurons is highly efficient, because newly formed autophagosomes are rapidly cleared by fusion with lysosomes, accounting for their scarcity in the healthy brain. Even after macroautophagy is strongly induced by suppressing mTOR (mammalian target of rapamycin) kinase activity with rapamycin or nutrient deprivation, active cathepsin-positive autolysosomes rather than LC3-II-positive autophagosomes predominate, implying efficient autophagosome clearance in healthy neurons. In contrast, selectively impeding late steps in macroautophagy by inhibiting cathepsin-mediated proteolysis within autolysosomes with cysteine- and aspartyl-protease inhibitors caused a marked accumulation of electron-dense double-membrane-limited AVs, containing cathepsin D and incompletely degraded LC3-II in perikarya and neurites. Similar structures accumulated in large numbers when fusion of autophagosomes with lysosomes was slowed by disrupting their transport on microtubules with vinblastine. Finally, we find that the autophagic vacuoles accumulating after protease inhibition or prolonged vinblastine treatment strongly resembled AVs that collect in dystrophic neurites in the AD brain and in an AD mouse model. We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone. Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway.
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PMID:Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease. 1859 67


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