bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019–11–24
fourteen papers selected by
Viktor Korolchuk, Newcastle University
  1. Restriction of intracellular Salmonella replication by restoring TFEB-mediated xenophagy.
  2. Regulation of reticulophagy by the N-degron pathway.
  3. NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome.
  4. An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.
  5. Acetyltransferase GCN5 regulates autophagy and lysosome biogenesis by targeting TFEB.
  6. TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast.
  7. Targeting senescent cells in translational medicine.
  8. The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.
  9. Plasticity in salt bridge allows fusion-competent ubiquitylation of mitofusins and Cdc48 recognition.
  10. Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena.
  11. Excessive ER-phagy mediated by the autophagy receptor FAM134B results in ER stress, the unfolded protein response, and cell death in HeLa cells.
  12. Modulation of autophagy by RTN-1C: role in autophagosome biogenesis.
  13. Michael Lazarou: Building a body of research.
  14. Mitosis flips the switch on autophagy control.
  1. Autophagy. 2019 Nov 19. 1-14
    Restriction of intracellular Salmonella replication by restoring TFEB-mediated xenophagy.
    Veena Ammanathan, Piyush Mishra, Aravinda K Chavalmane, Sasikumar Muthusamy, Vidya Jadhav, Chandrashekaran Siddamadappa, Ravi Manjithaya.
      Macroautophagy/autophagy functions as a part of the innate immune system in clearing intracellular pathogens. Although this process is well known, the mechanisms that control antibacterial autophagy are not clear. In this study we show that during intracellular Salmonella typhimurium infection, the activity of TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, is suppressed by maintaining it in a phosphorylated state on the lysosomes. Furthermore, we have identified a novel, antibacterial small molecule autophagy (xenophagy) modulator, acacetin. The xenophagy effect exerted by acacetin occurs in an MTOR (mechanistic target of rapamycin kinase)-independent, TFEB-dependent manner. Acacetin treatment results in persistently maintaining active TFEB in the nucleus and also in TFEB mediated induction of functional lysosomes that target Salmonella-containing vacuoles (SCVs). The enhanced proteolytic activity due to deployment of lysosomes results in clamping down Salmonella replication in SCVs. Acacetin is effective as a xenophagy compound in an in vivo mouse model of infection and reduces intracellular Salmonella burden.Abbreviations: 3-MA: 3-methyladenine; BafA1: bafilomycin A1; CFU: colony-forming units; DQ-BSA: dye quenched-bovine serum albumin; EEA1: early endosome antigen 1; FITC: fluorescein isothiocyanate; FM 4-64: pyridinium,4-(6-[4-{diethylamino}phenyl]-1,3,5-hexatrienyl)-1-(3[triethylammonio] propyl)-dibromide; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; MAPILC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; RFP: red fluorescent protein; SCVs: Salmonella-containing vacuoles; SD: standard deviation; SDS: sodium dodecyl sulfate; SEM: standard mean error; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TFEB: transcription factor EB.
    Keywords:  Acacetin; MTOR-independent; Salmonella typhimurium; Salmonella-containing vacuoles; TFEB; autolysosomes; lysosomes; xenophagy
    DOI:  https://doi.org/10.1080/15548627.2019.1689770
  2. Autophagy. 2019 Nov 20.
    Regulation of reticulophagy by the N-degron pathway.
    Chang Hoon Ji, Hee Yeon Kim, Ah Jung Heo, Min Ju Lee, Daniel Youngjae Park, Dong Hyun Kim, Bo Yeon Kim, Yong Tae Kwon.
      Cellular homeostasis requires selective autophagic degradation of damaged or defective organelles, including the endoplasmic reticulum (ER). Previous studies have shown that specific ER transmembrane receptors recruit LC3 on autophagic membranes by using LC3-interacting domains. In this study, we showed that the N-degron pathway mediates ubiquitin (Ub)-dependent reticulophagy. During this 2-step process, the ER transmembrane E3 ligase TRIM13 undergoes auto-ubiquitination via lysine 63 (K63) linkage chains and acts as a ligand for the autophagic receptor SQSTM1/p62 (sequestosome 1). In parallel, ER-residing molecular chaperones, such as HSPA5/GRP78/BiP, are relocated to the cytosol and conjugated with the amino acid L-arginine (Arg) at the N-termini by ATE1 (arginyltransferase 1). The resulting N-terminal Arg (Nt-Arg) binds the ZZ domain of SQSTM1, inducing oligomerization of SQSTM1-TRIM13 complexes and facilitating recruitment of LC3 on phagophores to the sites of reticulophagy. We developed small molecule ligands to the SQSTM1 ZZ domain and demonstrate that these chemical mimics of Nt-Arg facilitate reticulophagy and autophagic protein quality control of misfolded aggregates in the ER.
    Keywords:  ER homeostasis; ER protein quality control; ER stress response; ER-phagy; N-degron pathway; N-terminal arginylation; SQSTM1/p62; TRIM13; alpha1-antitrypsin deficiency; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2019.1695402
  3. Nat Commun. 2019 Nov 21. 10(1): 5284
    NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome.
    Evandro F Fang, Yujun Hou, Sofie Lautrup, Martin Borch Jensen, Beimeng Yang, Tanima SenGupta, Domenica Caponio, Rojyar Khezri, Tyler G Demarest, Yahyah Aman, David Figueroa, Marya Morevati, Ho-Joon Lee, Hisaya Kato, Henok Kassahun, Jong-Hyuk Lee, Deborah Filippelli, Mustafa Nazir Okur, Aswin Mangerich, Deborah L Croteau, Yoshiro Maezawa, Costas A Lyssiotis, Jun Tao, Koutaro Yokote, Tor Erik Rusten, Mark P Mattson, Heinrich Jasper, Hilde Nilsen, Vilhelm A Bohr.
      Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.
    DOI:  https://doi.org/10.1038/s41467-019-13172-8
  4. Mol Cell. 2019 Nov 06. pii: S1097-2765(19)30794-4. [Epub ahead of print]
    An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.
    Richard I Odle, Simon A Walker, David Oxley, Andrew M Kidger, Kathryn Balmanno, Rebecca Gilley, Hanneke Okkenhaug, Oliver Florey, Nicholas T Ktistakis, Simon J Cook.
      Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.
    Keywords:  ATG13; ATG14; CDK1; RAPTOR; TFEB; ULK1; autophagy; mTOR; mitosis
    DOI:  https://doi.org/10.1016/j.molcel.2019.10.016
  5. EMBO Rep. 2019 Nov 21. e48335
    Acetyltransferase GCN5 regulates autophagy and lysosome biogenesis by targeting TFEB.
    Yusha Wang, Yewei Huang, Jiaqi Liu, Jinna Zhang, Mingming Xu, Zhiyuan You, Chao Peng, Zhefeng Gong, Wei Liu.
      Accumulating evidence highlights the role of histone acetyltransferase GCN5 in the regulation of cell metabolism in metazoans. Here, we report that GCN5 is a negative regulator of autophagy, a lysosome-dependent catabolic mechanism. In animal cells and Drosophila, GCN5 inhibits the biogenesis of autophagosomes and lysosomes by targeting TFEB, the master transcription factor for autophagy- and lysosome-related gene expression. We show that GCN5 is a specific TFEB acetyltransferase, and acetylation by GCN5 results in the decrease in TFEB transcriptional activity. Induction of autophagy inactivates GCN5, accompanied by reduced TFEB acetylation and increased lysosome formation. We further demonstrate that acetylation at K274 and K279 disrupts the dimerization of TFEB and the binding of TFEB to its target gene promoters. In a Tau-based neurodegenerative Drosophila model, deletion of dGcn5 improves the clearance of Tau protein aggregates and ameliorates the neurodegenerative phenotypes. Together, our results reveal GCN5 as a novel conserved TFEB regulator, and the regulatory mechanisms may be involved in autophagy- and lysosome-related physiological and pathological processes.
    Keywords:  GCN5; TFEB; acetylation; autophagy; lysosome
    DOI:  https://doi.org/10.15252/embr.201948335
  6. Biochem Biophys Res Commun. 2019 Nov 15. pii: S0006-291X(19)32184-9. [Epub ahead of print]
    TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast.
    Shamsul Morshed, Tasnuva Sharmin, Takashi Ushimaru.
      Microautophagy is promoted after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) kinase. Invagination of vacuolar membranes by endosomal sorting complex required for transport (ESCRT) is required for microautophagy. Vps27, a subunit of ESCRT-0, is recruited onto vacuolar membranes via dephosphorylation after TORC1 inactivation. Here, we showed that Hse1, another ESCRT-0 subunit, is also recruited onto vacuolar membranes after TORC1 inactivation, promoting formation of ESCRT-0 complex on vacuolar membranes. Hse1 recruitment was dependent on Vps27, whereas Vps27 recruitment was independent of Hse1. Not only Vps27 but also Hse1 was required for ESCRT-III recruitment onto vacuolar membranes and microautophagy induction after TORC1 inactivation. This study revealed that ESCRT-0 (Vps27-Hse1) complex formation on vacuolar membranes is important for microautophagy inactivation after TORC1 inactivation.
    Keywords:  ESCRT-0; Hse1; Microautophagy; TORC1; Vps27
    DOI:  https://doi.org/10.1016/j.bbrc.2019.11.064
  7. EMBO Mol Med. 2019 Nov 19. e10234
    Targeting senescent cells in translational medicine.
    Marta Paez-Ribes, Estela González-Gualda, Gary J Doherty, Daniel Muñoz-Espín.
      Organismal ageing is a complex process driving progressive impairment of functionality and regenerative potential of tissues. Cellular senescence is a state of stable cell cycle arrest occurring in response to damage and stress and is considered a hallmark of ageing. Senescent cells accumulate in multiple organs during ageing, contribute to tissue dysfunction and give rise to pathological manifestations. Senescence is therefore a defining feature of a variety of human age-related disorders, including cancer, and targeted elimination of these cells has recently emerged as a promising therapeutic approach to ameliorate tissue damage and promote repair and regeneration. In addition, in vivo identification of senescent cells has significant potential for early diagnosis of multiple pathologies. Here, we review existing senolytics, small molecules and drug delivery tools used in preclinical therapeutic strategies involving cellular senescence, as well as probes to trace senescent cells. We also review the clinical research landscape in senescence and discuss how identifying and targeting cellular senescence might positively affect pathological and ageing processes.
    Keywords:   SASP ; age-related disorders; cellular senescence; senolytic drugs; senoprobes
    DOI:  https://doi.org/10.15252/emmm.201810234
  8. Elife. 2019 Nov 21. pii: e50149. [Epub ahead of print]8
    The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.
    Heather P Harding, Adriana Ordonez, Felicity Allen, Leopold Parts, Alison J Inglis, Roger L Williams, David Ron.
      The eukaryotic translation initiation factor 2a (eIF2a) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2a kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2a phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.
    Keywords:  cell biology; genetics; genomics
    DOI:  https://doi.org/10.7554/eLife.50149
  9. Life Sci Alliance. 2019 Dec;pii: e201900491. [Epub ahead of print]2(6):
    Plasticity in salt bridge allows fusion-competent ubiquitylation of mitofusins and Cdc48 recognition.
    Vincent Anton, Ira Buntenbroich, Ramona Schuster, Felix Babatz, Tânia Simões, Selver Altin, Gaetano Calabrese, Jan Riemer, Astrid Schauss, Mafalda Escobar-Henriques.
      Mitofusins are dynamin-related GTPases that drive mitochondrial fusion by sequential events of oligomerization and GTP hydrolysis, followed by their ubiquitylation. Here, we show that fusion requires a trilateral salt bridge at a hinge point of the yeast mitofusin Fzo1, alternatingly forming before and after GTP hydrolysis. Mutations causative of Charcot-Marie-Tooth disease massively map to this hinge point site, underlining the disease relevance of the trilateral salt bridge. A triple charge swap rescues the activity of Fzo1, emphasizing the close coordination of the hinge residues with GTP hydrolysis. Subsequently, ubiquitylation of Fzo1 allows the AAA-ATPase ubiquitin-chaperone Cdc48 to resolve Fzo1 clusters, releasing the dynamin for the next fusion round. Furthermore, cross-complementation within the oligomer unexpectedly revealed ubiquitylated but fusion-incompetent Fzo1 intermediates. However, Cdc48 did not affect the ubiquitylated but fusion-incompetent variants, indicating that Fzo1 ubiquitylation is only controlled after membrane merging. Together, we present an integrated model on how mitochondrial outer membranes fuse, a critical process for their respiratory function but also putatively relevant for therapeutic interventions.
    DOI:  https://doi.org/10.26508/lsa.201900491
  10. Cell Death Differ. 2019 Nov 18.
    Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena.
    Viktória Kiss, András Jipa, Kata Varga, Szabolcs Takáts, Tamás Maruzs, Péter Lőrincz, Zsófia Simon-Vecsei, Szilárd Szikora, István Földi, Csaba Bajusz, Dávid Tóth, Péter Vilmos, Imre Gáspár, Paolo Ronchi, József Mihály, Gábor Juhász.
      Autophagy ensures the turnover of cytoplasm and requires the coordinated action of Atg proteins, some of which also have moonlighting functions in higher eukaryotes. Here we show that the transmembrane protein Atg9 is required for female fertility, and its loss leads to defects in actin cytoskeleton organization in the ovary and enhances filopodia formation in neurons in Drosophila. Atg9 localizes to the plasma membrane anchor points of actin cables and is also important for the integrity of the cortical actin network. Of note, such phenotypes are not seen in other Atg mutants, suggesting that these are independent of autophagy defects. Mechanistically, we identify the known actin regulators profilin and Ena/VASP as novel binding partners of Atg9 based on microscopy, biochemical, and genetic interactions. Accordingly, the localization of both profilin and Ena depends on Atg9. Taken together, our data identify a new and unexpected role for Atg9 in actin cytoskeleton regulation.
    DOI:  https://doi.org/10.1038/s41418-019-0452-0
  11. J Biol Chem. 2019 Nov 20. pii: jbc.RA119.008709. [Epub ahead of print]
    Excessive ER-phagy mediated by the autophagy receptor FAM134B results in ER stress, the unfolded protein response, and cell death in HeLa cells.
    Yangjie Liao, Bo Duan, Yufei Zhang, Xinmin Zhang, Bin Xia.
      Autophagy is typically a pro-survival cellular process that promotes the turnover of long-lived proteins and damaged organelles, but it can also induce cell death. We have previously reported that the small molecule Z36 induces autophagy along with autophagic cell death in HeLa cells. In this study, we analyzed differential gene expression in Z36-treated HeLa cells, and found that Z36-induced endoplasmic reticulum (ER)-phagy results in ER stress and the unfolded protein response (UPR). This result is in contrast to the common notion that autophagy is generally activated in response to ER stress and the UPR. We demonstrate that Z36 upregulates the expression levels of FAM134B, LC3, and Atg9, which together mediate excessive ER-phagy, characterized by forming increased numbers of autophagosomes with larger sizes. We noted that the excessive ER-phagy accelerates ER degradation and impairs ER homeostasis, and thereby triggers ER stress and the UPR, as well as ER-phagy-dependent cell death. Interestingly, overexpression of FAM134B alone in HeLa cells is sufficient to impair ER homeostasis and cause ER stress and cell death. These findings suggest a mechanism involving FAM134B activity for ER-phagy to promote cell death.
    Keywords:  Bcl-xL inhibitor; ER-phagy; FAM134B; Z36; autophagy; cell death; endoplasmic reticulum (ER); endoplasmic reticulum stress (ER stress); reticulophagy regulator 1 (RETREG1); unfolded protein response (UPR)
    DOI:  https://doi.org/10.1074/jbc.RA119.008709
  12. Cell Death Dis. 2019 Nov 18. 10(12): 868
    Modulation of autophagy by RTN-1C: role in autophagosome biogenesis.
    Manuela D' Eletto, Anna Risuglia, Serafina Oliverio, Bisan Mehdawy, Roberta Nardacci, Matteo Bordi, Federica Di Sano.
      The endoplasmic reticulum (ER) is a key organelle fundamental for the maintenance of cellular homeostasis and to determine the cell's fate under stress conditions. Among the known proteins that regulate ER structure and function there is Reticulon-1C (RTN-1C), a member of the reticulon family localized primarily on the ER membrane. We previously demonstrated that RTN-1C expression affects ER function and stress condition. ER is an essential site for the regulation of apoptotic pathways and it has also been recently recognized as an important component of autophagic signaling. Based on these evidences, we have investigated the impact of RTN-1C modulation on autophagy induction. Interestingly we found that reticulon overexpression is able to activate autophagic machinery and its silencing results in a significative inhibition of both basal and induced autophagic response. Using different experimental approaches we demonstrated that RTN-1C colocalizes with ATG16L and LC3II on the autophagosomes. Considering the key role of reticulon proteins in the control of ER membrane shaping and homeostasis, our data suggest the participation of RTN-1C in the autophagic vesicle biogenesis at the level of the ER compartment. Our data indicate a new mechanism by which this structural ER protein modulates cellular stress, that is at the basis of different autophagy-related pathologies.
    DOI:  https://doi.org/10.1038/s41419-019-2099-7
  13. J Cell Biol. 2019 Nov 19. pii: jcb.201911052. [Epub ahead of print]
    Michael Lazarou: Building a body of research.
    Marie Anne O'Donnell.
      Lazarou investigates the relationship between mitochondria and autophagy.
    DOI:  https://doi.org/10.1083/jcb.201911052
  14. Nat Rev Mol Cell Biol. 2019 Nov 22.
    Mitosis flips the switch on autophagy control.
    Joseph Willson.
      
    DOI:  https://doi.org/10.1038/s41580-019-0196-1
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