bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒09‒15
nine papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2019 Sep 12.
    Tang Z, Takahashi Y, Wang HG.
      The mechanism by which ATG2 (ATG2A and ATG2B in mammals) regulates autophagosome biogenesis remains largely unknown. In our recent study, we showed that ATG2A translocates to the mitochondria-associated ER membranes (MAM) to promote phagophore growth during nutrient starvation. Mechanistically, the mitochondrial translocase TOMM40 binds to a C-terminal domain of ATG2A, termed the MAM localization domain (MLD), and mediates its MAM translocation in a manner dependent on the TOMM receptor TOMM70. Moreover, ATG2A associates with ATG9A through its N-terminal domain and this interaction is required for phagophore expansion and efficient autophagic flux. These observations suggest that ATG2 operates a mechanism for phagophore expansion at the MAM through the TOMM40-TOMM70 complex and ATG9 during autophagy.
    Keywords:  ATG2; ATG9; TOM40; TOM70; mitochondria-associated ER membranes (MAM); phagophore expansion
  2. Autophagy. 2019 Sep 13. 1-2
    Yang Y, Klionsky DJ.
      UBQLNs (ubiquilins) are highly conserved proteins across species, characterized by interactions with proteasomes and ubiquitinated proteins via UBA and UBL domains, respectively. The role of UBQLNs as chaperone proteins of the ubiquitin-proteasome system (UPS) is well-defined; however, the connections between UBQLNs and autophagy remain unclear. A recent study published in Nature Cell Biology from Dr. Hugo J. Bellen's lab showed a novel role of UBQLNs in macroautophagy/autophagy regulation through v-ATPase-MTOR signaling using Drosophila and mammalian neuronal cells. Notably, the highlighted article also investigated the autophagy phenotype of a common amyotrophic lateral sclerosis (ALS)-associated mutation in the gene encoding UBQLN2, demonstrating the contribution of abnormal v-ATPase-MTOR-mediated autophagy in ALS pathogenesis.
    Keywords:  Lysosome; MTOR; macroautophagy; stress; v-ATPase
  3. Autophagy. 2019 Sep 12.
    Wu YW, Waldmann H.
      A forward chemical genetic approach led to identification of autogramins as novel autophagy inhibitors. Autogramins selectively target the cholesterol transfer protein GRAMD1A (GRAM domain containing 1A). Autogramins compete with cholesterol binding to the StART domain of GRAMD1A, thereby inhibiting its cholesterol transfer activity. GRAMD1A associates with phosphatidylinositol monophosphate via its GRAM domain. GRAMD1A accumulates at autophagosome initiation sites upon starvation. This protein is involved in cholesterol distribution in response to starvation and is required for autophagosome biogenesis. Therefore, we identify a novel function of GRAMD1A and a new role of cholesterol in macroautophagy/autophagy.
    Keywords:  GRAMD1A; autogramin; autophagosome formation; autophagy; autophagy inhibitor; chemical genetics; cholesterol; cholesterol binding protein; cholesterol transfer protein
  4. Autophagy. 2019 Sep 13. 1-17
    Andrejeva G, Gowan S, Lin G, Wong Te Fong AL, Shamsaei E, Parkes HG, Mui J, Raynaud FI, Asad Y, Vizcay-Barrena G, Nikitorowicz-Buniak J, Valenti M, Howell L, Fleck RA, Martin LA, Kirkin V, Leach MO, Chung YL.
      Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. Abbreviations: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.
    Keywords:  Autophagosome; CTP:phosphocholine cytidylyltransferase; autophagy; choline phospholipids; phosphatidylcholine; propargylcholine
  5. Dis Model Mech. 2019 Sep 13. pii: dmm.038596. [Epub ahead of print]
    Brown RA, Voit A, Srikanth MP, Thayer JA, Kingsbury TJ, Jacobson MA, Lipinski MM, Feldman RA, Awad O.
      Bi-allelic GBA1 mutations cause Gaucher's disease (GD), the most common lysosomal storage disorder (LSD). Neuronopathic manifestations in GD include neurodegeneration, which can be severe and rapidly progressive. GBA1 mutations are also the most frequent genetic risk factors for Parkinson's disease. Dysfunction of the autophagy-lysosomal pathway represents a key pathogenic event in GBA1-associated neurodegeneration. Using an induced-pluripotent stem cell (iPSC) model of GD, we previously demonstrated that lysosomal alterations in GD neurons are linked to dysfunction of the transcription factor EB (TFEB). TFEB controls the coordinated expression of autophagy and lysosomal genes and is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). To further investigate the mechanism of autophagy-lysosomal pathway dysfunction in neuronopathic GD, we examined mTORC1 kinase activity in GD iPSC-neuronal progenitors and differentiated neurons. We found that mTORC1 is hyperactive in GD cells as evidenced by increased phosphorylation of its downstream protein substrates. We also found that pharmacological inhibition of glucosylceramide synthase enzyme reversed mTORC1 hyperactivation, suggesting that increased mTORC1 activity is mediated by the abnormal accumulation of glycosphingolipids in the mutant cells. Treatment with the mTOR inhibitor Torin1 upregulated lysosomal biogenesis and enhanced autophagic clearance in GD neurons, confirming that lysosomal dysfunction is mediated by mTOR hyperactivation. Further analysis demonstrated that increased TFEB phosphorylation by mTORC1 results in decreased TFEB stability in GD cells. Our study uncovers a new mechanism contributing to autophagy-lysosomal pathway dysfunction in GD, and identifies the mTOR complex as a potential therapeutic target for treatment of GBA1-associated neurodegeneration.
    Keywords:  Gaucher's Disease; Lysosomes; TFEB; iPSC; mTOR
  6. J Biol Chem. 2019 Sep 12. pii: jbc.RA119.009110. [Epub ahead of print]
    Frendo-Cumbo S, Jaldin-Fincati JR, Coyaud E, Laurent EMN, Townsend L, Tan JMJ, Xavier RJ, Pillon NJ, Raught B, Wright DC, Brumell JH, Klip A.
      Connections between deficient autophagy and insulin resistance have emerged, however the mechanism through which reduced autophagy impairs insulin-signaling remains unknown. We examined mouse embryonic fibroblasts lacking Atg16l1 (ATG16L1 KO MEFs), an essential autophagy gene, and observed deficient insulin and insulin-like growth factor-1 signaling. ATG16L1 KO MEFs displayed reduced protein content of Insulin Receptor Substrate-1 (IRS1), pivotal to insulin signaling, while IRS1myc overexpression recovered downstream insulin signaling. Endogenous IRS1 protein content and insulin signaling were restored in ATG16L1 KO MEFs upon proteasome inhibition. Through proximity-dependent biotin identification (BioID) and co-immunoprecipitation, we found that kelch-like proteins KLHL9 and KLHL13, which together form an E3 ubiquitin (Ub) ligase complex with cullin 3 (CUL3), are novel IRS1 interactors. Expression of Klhl9 and Klhl13 was elevated in ATG16L1 KO MEFs and siRNA-mediated knockdown of Klhl9, Klhl13 or Cul3 recovered IRS1 expression. Moreover, Klhl13 and Cul3 knockdown increased insulin signaling. Notably, adipose tissue of high-fat fed mice displayed lower Atg16l1 mRNA expression and IRS1 protein content, and adipose tissue KLHL13 and CUL3 expression positively correlated to body mass index (BMI) in humans. We propose that ATG16L1 deficiency evokes insulin resistance through induction of Klhl9 and Klhl13, which, in complex with Cul3, promote proteasomal IRS1 degradation.
    Keywords:  Akt PKB; E3 ubiquitin ligase; Insulin signaling; Kelch-like gene family; autophagy; autophagy-related protein 16L1 (ATG16L1); cell signaling; insulin; insulin receptor substrate 1 (IRS-1)
  7. Nat Commun. 2019 Sep 13. 10(1): 4176
    Holdgaard SG, Cianfanelli V, Pupo E, Lambrughi M, Lubas M, Nielsen JC, Eibes S, Maiani E, Harder LM, Wesch N, Foged MM, Maeda K, Nazio F, de la Ballina LR, Dötsch V, Brech A, Frankel LB, Jäättelä M, Locatelli F, Barisic M, Andersen JS, Bekker-Jensen S, Lund AH, Rogov VV, Papaleo E, Lanzetti L, De Zio D, Cecconi F.
      The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.
  8. J Biol Chem. 2019 Sep 13. pii: jbc.RA118.007283. [Epub ahead of print]
    James C, Müller M, Goldberg MW, Lenz C, Urlaub H, Kehlenbach RH.
      VAPB (vesicle-associated membrane protein-associated protein B) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified APEX2 (enhanced ascorbate peroxidase 2)-approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and non-specific hits. Furthermore, we identified emerin, TMEM43 and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.
    Keywords:  APEX; RAPIDS; VAPB; biotin; emerin; endoplasmic reticulum (ER); nuclear envelope; nuclear pore; peroxidase; proteomic proximity mapping
  9. Gerontology. 2019 Sep 10. 1-9
    Princz A, Tavernarakis N.
      Posttranslational modifications are ubiquitous regulators of cellular processes. The regulatory role of SUMOylation, the attachment of a small ubiquitin-related modifier to a target protein, has been implicated in fundamental processes like cell division, DNA damage repair, mitochondrial homeostasis, and stress responses. Recently, it is gaining more attention in drug discovery as well. As life expectancy keeps rising, more individuals are at risk for developing age-associated diseases. This not only makes a person's life uncomfortable, but it also places an economic burden on society. Therefore, finding treatments for age-related diseases is an important issue. Understanding the basic mechanisms in the cell under normal and disease conditions is fundamental for drug discovery. There is an increasing number of reports showing that the ageing process could be influenced by SUMOylation. Similarly, SUMOylation is essential for proper neuronal function. In this review we summarize the latest results regarding the connection between SUMOylation and neurodegenerative diseases. We highlight the significance of specific SUMO target proteins and the importance of SUMO isoform specificity.
    Keywords:  Ageing; Alzheimer’s disease; Huntington’s disease; Neurodegeneration; Neuron; Parkinson’s disease; SUMO