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There are 39 results for Rapamycin (displaying 21 to 30).

Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane‐bound vesicles termed autophagosomes. The conserved Atg5–Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5–Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5–Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre‐autophagosomal structure but is essential for autophagy and cytoplasm‐to‐vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5–Atg12/Atg16 complex during autophagosome formation.

Localization of wild‐type Atg5 and Atg5 K160E, R171E to the PAS. (A) Yeast cells of the indicated genotype expressing Atg5–3 × mCherry or Atg5 K160E, R171E–3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope. Maximum projections of z‐stacks are shown. (B) Graph showing the percentage of mCherry‐positive cells that display Atg5–3 × mCherry dots. In total, 3 independent experiments were conducted and >400 cells were counted. (C) Graph showing the percentage of cells …

… expression constructs and Atg5–2 × Myc was immunoprecipitated with an anti‐Myc antibody. The precipitated protein was subjected to anti‐Atg5 and anti‐Atg12 western blotting. The substitution of R171 and K160 to glutamate caused the mutant protein to run higher. The same phenomenon was observed for the recombinant protein (Supplementary Figure 3). (D) Anti‐Atg12 western blot of cell fractions prepared from rapamycin‐treated yeast cells. The anti‐Pgk1 and anti‐Pex30 served as control for the cytosolic …

… showed reduced membrane binding in vivo we fractionated membranes from rapamycin‐treated yeast cells by separating the cytosol (S100) from the membrane fraction (P100) by ultracentrifugation. While the endogenous and the introduced wild‐type Atg5–Atg12 conjugates were present in the membrane fractions the Atg5 K160E, R171E mutant showed a severely reduced presence in the pellet fraction ( ; ). In addition, also when we expressed 9 × Myc‐tagged versions of wild type and K160E, R171E Atg5 we found …

Julia Romanov et al. The EMBO Journal November 2012

eIF4A inactivates TORC1 in response to amino acid starvation

Amino acids regulate TOR complex 1 ( TORC 1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC 1 recruitment to lysosomes where TORC 1 is activated by binding Rheb. How the absence of amino acids inactivates TORC 1 is less well understood. Amino acid starvation recruits the TSC 1/ TSC 2 complex to the vicinity of TORC 1 to inhibit Rheb; however, the upstream mechanisms regulating TSC 2 are not known. We identify here the eIF 4A‐containing eIF 4F translation initiation complex as an upstream regulator of TSC 2 in response to amino acid withdrawal in Drosophila . We find that TORC 1 and translation preinitiation complexes bind each other. Cells lacking eIF 4F components retain elevated TORC 1 activity upon amino acid removal. This effect is specific for eIF 4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC 1 inactivation upon amino acid removal.

S6K phosphorylation can be detected in cell lysates by dot‐blot analysis. Lysates of S2 cells treated with Schneider's medium containing (+) or lacking (−) amino acids for 30 min, spotted onto nitrocellulose membranes, and probed with the indicated antibodies. The elevated S6K phosphorylation observed upon eIF4A knockdown is caused by elevated TORC1 activity, since it can be abolished with rapamycin treatment. Kc167 cells were incubated for 5 days with negative control GFP or eIF4a dsRNA …

… ), or incubated with 10 μg/ml insulin for 60 min (lane 3), or 20 nM rapamycin for 30 min (lane 4), before lysis in CHAPS‐containing buffer. Representative of three biological replicates. (C′) Validation of the anti‐dRaptor antibody. Left panel: Kc167 cells were treated with the indicated dsRNAs for 5 days, prior to lysis. The band corresponding to the dRaptor protein, running at approximately 180 kDa, significantly decreases upon Raptor knockdown. Right panel: The Raptor band observed in a TOR …

… structures. Binding sites for other initiation factors are shown. BBinding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1‐independent fashion. Co‐immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for the indicated times. Representative of three biological replicates. CKnockdown of NAT1 using four independent, non‐overlapping dsRNAs leads to reduced TORC1 activity. Kc167 …

… ). In contrast, S2 cells with an eIF4A knockdown consistently retained a low but significantly elevated level of S6K phosphorylation upon the removal of all amino acids (Fig  A, lanes 3–4). The elevated S6K phosphorylation was abolished upon treatment with rapamycin (Fig  B), confirming that it is due to elevated TORC1 activity. This phenotype was also observed with two additional, non‐overlapping dsRNAs targeting eIF4A, thereby excluding possible off‐target effects (Fig  A, lanes 5–8 …

Foivos‐Filippos Tsokanos et al. The EMBO Journal May 2016

Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy

The majority of studies on autophagy, a cytoplasmic homeostatis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3‐kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3‐phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI‐1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.

… R336Q (E) transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 μm. Quantitation of p62 puncta per cell (K). Bars, s.e.m. (n=3 independent experiments, 30 cells per experiments). *P<0.05 (t‐test), ns: nonsignificant. (L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot. …

… , identifying the brakes in the system once it is set in motion by the upstream Tor‐dependent signalling systems has eluded a proper definition. And yet, the autophagic process must be tightly regulated to support cell survival when needed but also to avoid cell death and injury through excessive autophagy. A key signalling regulator of autophagy is the Akt/mTOR pathway. Inhibition of mTOR kinase by specific inhibitor, rapamycin or nutrient deprivation results in activation of autophagy ( ). Once …

Isabelle Vergne et al. The EMBO Journal August 2009

Species‐specific impact of the autophagy machinery on Chikungunya virus infection

Chikungunya virus (CHIKV) is a recently re‐emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52—but not its mouse orthologue—interacts with the non‐structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.

… labeled with p62 and capsid antibodies (F). Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I). Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells …

… ‐LC3‐B ( ). Stochastic optical reconstruction microscopy provided a high‐resolution image of p62 association with capsid ( online). Ultrastructural analysis revealed double‐membrane vesicles, containing and surrounded by nucleocapsids, immunolabelled for capsid and p62 ( online). Moreover, CHIKV induced autophagy in a mouse model for CHIKV ( ) [ ]. Autophagy induction with rapamycin in infected human cells significantly increased CHIKV replication (15 h: 1.6±0.2‐fold, P ⩽0.005; 24 h: 1.6±0.1‐fold …

Delphine Judith et al. EMBO Reports June 2013

Suppression of the HSF1‐mediated proteotoxic stress response by the metabolic stress sensor AMPK

Numerous extrinsic and intrinsic insults trigger the HSF 1‐mediated proteotoxic stress response ( PSR ), an ancient transcriptional program that is essential to proteostasis and survival under such conditions. In contrast to its well‐recognized mobilization by proteotoxic stress, little is known about how this powerful adaptive mechanism reacts to other stresses. Surprisingly, we discovered that metabolic stress suppresses the PSR . This suppression is largely mediated through the central metabolic sensor AMPK , which physically interacts with and phosphorylates HSF 1 at Ser121. Through AMPK activation, metabolic stress represses HSF 1, rendering cells vulnerable to proteotoxic stress. Conversely, proteotoxic stress inactivates AMPK and thereby interferes with the metabolic stress response. Importantly, metformin, a metabolic stressor and popular anti‐diabetic drug, inactivates HSF 1 and provokes proteotoxic stress within tumor cells, thereby impeding tumor growth. Thus, these findings uncover a novel interplay between the metabolic stress sensor AMPK and the proteotoxic stress sensor HSF 1 that profoundly impacts stress resistance, proteostasis, and malignant growth.

… cellular sensor of metabolic stress (El‐Mir et al , ). This activation is known to mediate numerous effects of metformin (Kahn et al , ). However, it has also been reported that metformin inhibits mTORC1 independently of AMPK (Kalender et al , ). We first asked whether mTORC1 inhibition by metformin mediates HSF1 suppression. To address this, we examined induction of Hsp genes in cells treated with rapamycin, a specific mTOR inhibitor. Successful inhibition of mTORC1 by rapamycin was evidenced …

Siyuan Dai et al. The EMBO Journal February 2015

Loss of autophagy in hypothalamic POMC neurons impairs lipolysis

Autophagy degrades cytoplasmic contents to achieve cellular homeostasis. We show that selective loss of autophagy in hypothalamic proopiomelanocortin (POMC) neurons decreases α‐melanocyte‐stimulating hormone (MSH) levels, promoting adiposity, impairing lipolysis and altering glucose homeostasis. Ageing reduces hypothalamic autophagy and α‐MSH levels, and aged‐mice phenocopy, the adiposity and lipolytic defect observed in POMC neuron autophagy‐null mice. Intraperitoneal isoproterenol restores lipolysis in both models, demonstrating normal adipocyte catecholamine responsiveness. We propose that an unconventional, autophagosome‐mediated form of secretion in POMC neurons controls energy balance by regulating α‐MSH production. Modulating hypothalamic autophagy might have implications for preventing obesity and metabolic syndrome of ageing.

… [ ]. A well‐known inhibitor of autophagy is the mammalian target of rapamycin (mTOR) [ ]. Hypothalamic mTOR regulates food intake [ ] suggesting that central mTOR might mediate these effects in part by modulating autophagy. In fact, autophagy in AgRP neurons has been shown to control food intake and energy balance [ ]. As autophagy declines with ageing [ ] it is plausible that decreased hypothalamic autophagy might contribute to the metabolic dysregulation observed with age. Recent studies in yeast …

Susmita Kaushik et al. EMBO Reports March 2012

TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC 1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB 11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB 1, as an interactor of TBC 1D14. TBC 1D14 binds to the TRAPP complex via an N‐terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy‐specific TRAPP subunit, forms part of a mammalian TRAPPIII‐like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC 1D14 and TRAPPIII regulate ATG 9 trafficking independently of ULK 1. We propose a model whereby TBC 1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG 9 required for initiation of autophagy.

… , the mammalian target of rapamycin complex 1 (mTORC1) is inactivated, which removes repression on the ULK (uncoordinated 51‐like kinase) complex, which consists of ULK1/2, ATG13, FIP200 and ATG101(Hara et al , ; Chan et al , ; Hosokawa et al , ; Mercer et al , ). The ULK1 complex then goes on to activate the autophagy‐specific phosphatidylinositol 3 kinase (PtdIns(3)K) complex, which includes ATG14, Beclin1, VPS34 and p150 and nucleates pools of phosphatidylinositol‐3‐phosphate (PtdIns(3)P …

Christopher A Lamb et al. The EMBO Journal February 2016

Structure of the Atg12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation

Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12–Atg5 conjugate functions as an E3‐like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12–Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.

… and by autophagy in response to starvation conditions or rapamycin treatment. In the vacuole, prApe1 is processed into a mature form (mApe1), which can be monitored by western blotting for Ape1. As shown in (low‐copy plasmid, bottom panels), autophagic activity was normal in these mutant cells, except for those expressing Atg12 F154R. These data, except for F154R mutant, indicate that the non‐covalent interaction between Atg12 and Atg5 is not necessary for the normal formation of the Atg8–PE conjugate …

Nobuo N Noda et al. EMBO Reports February 2013

PLIC proteins or ubiquilins regulate autophagy‐dependent cell survival during nutrient starvation

Ubiquilins (UBQLNs) are adaptor proteins thought to deliver ubiquitinated substrates to proteasomes. Here, we show a role for UBQLN in autophagy: enforced expression of UBQLN protects cells from starvation‐induced death, whereas depletion of UBQLN renders cells more susceptible. The UBQLN protective effect requires the autophagy‐related genes ATG5 and ATG7, two essential components of autophagy. The ubiquitin‐associated domain of UBQLN mediates both its association with autophagosomes and its protective effect against starvation. Depletion of UBQLN delays the delivery of autophagosomes to lysosomes. This study identifies a new role for UBQLN in regulating the maturation of autophagy, expanding the involvement of ubiquitin‐related proteins in this process.

… not covalently tag proteins, although they are thought to regulate the proteasomal degradation of ubiquitin conjugates ( ; ). UBQLNs also regulate the function of the thrombospondin receptor CD47 ( ) and G protein signalling ( ), and might also affect endocytosis through interaction with UIM‐containing proteins ( ; ); in addition, UBQLNs might interact with the kinase mammalian target of rapamycin (mTOR; ). Although mostly cytosolic, a fraction of UBQLN associates with cell membranes ( ), although …

Elsa‐Noah N'Diaye et al. EMBO Reports February 2009

Genome‐wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development

The mechanisms that coordinate the regulation of autophagy with developmental signaling during multicellular organism development remain largely unknown. Here, we show that impaired function of ribosomal protein RPL ‐43 causes an accumulation of SQST ‐1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we identify 139 genes that promote autophagy activity upon inactivation. Various signaling pathways, including Sma/Mab TGF ‐β signaling, lin‐35 /Rb signaling, the XBP ‐1‐mediated ER stress response, and the ATFS ‐1‐mediated mitochondrial stress response, regulate the expression of autophagy genes independently of the TFEB homolog HLH ‐30. Our study thus provides a framework for understanding the role of signaling pathways in regulating autophagy under physiological conditions.

… species . The mammalian target of rapamycin serine/threonine kinase complex 1 (mTORC1) and the Vps34 PI(3)K complex are major nodes for integrating various signaling pathways with autophagy regulation . Transcriptional regulation of autophagy genes confers another layer of regulation. The master positive regulator TFEB and the master repressor ZKSCAN3 transcriptionally regulate a network of genes involved in autophagosome and lysosome biogenesis . The forkhead transcription factor FoxO also …

Bin Guo et al. EMBO Reports June 2014
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