bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒10‒13
ten papers selected by
Viktor Korolchuk
Newcastle University


  1. FEBS Lett. 2019 Oct 11.
    Staiano L, Zappa F.
      Autophagy is widely considered as a housekeeping mechanism enabling cells to survive stress conditions and, in particular, nutrient deprivation. Autophagy begins with the formation of the phagophore that expands and closes around cytosolic material and damaged organelles destined for degradation. The execution of this complex machinery is guaranteed by the coordinated action of more than 40 ATG (Autophagy-related) proteins that control the entire process at different stages from the biogenesis of the autophagosome to cargo sequestration and fusion with lysosomes. Autophagosome biogenesis occurs at multiple intracellular sites, such as the endoplasmic reticulum (ER) and the plasma membrane (PM). Soon after the formation of the phagophore, the nascent autophagosome progressively grows in size and ultimately closes by recruiting intracellular membranes. In this review we focus on the contribution of three membrane sources - the ER, the ER-Golgi Intermediate Compartment (ERGIC) and the Golgi complex (GC) - to autophagosome biogenesis and expansion. We also highlight the interplay between the secretory pathway and autophagy in cells when nutrients are scarce.
    Keywords:  ATG2; ATG9; Autophagosome; Autophagosome-Lysosome fusion; Autophagy; Omegasome; Phagophore; Phosphoinositides; Tethers
    DOI:  https://doi.org/10.1002/1873-3468.13637
  2. EMBO Rep. 2019 Oct 10. e47728
    Koyano F, Yamano K, Kosako H, Kimura Y, Kimura M, Fujiki Y, Tanaka K, Matsuda N.
      Ubiquitylation of outer mitochondrial membrane (OMM) proteins is closely related to the onset of familial Parkinson's disease. Typically, a reduction in the mitochondrial membrane potential results in Parkin-mediated ubiquitylation of OMM proteins, which are then targeted for proteasomal and mitophagic degradation. The role of ubiquitylation of OMM proteins with non-degradative fates, however, remains poorly understood. In this study, we find that the mitochondrial E3 ubiquitin ligase MITOL/March5 translocates from depolarized mitochondria to peroxisomes following mitophagy stimulation. This unusual redistribution is mediated by peroxins (peroxisomal biogenesis factors) Pex3/16 and requires the E3 ligase activity of Parkin, which ubiquitylates K268 in the MITOL C-terminus, essential for p97/VCP-dependent mitochondrial extraction of MITOL. These findings imply that ubiquitylation directs peroxisomal translocation of MITOL upon mitophagy stimulation and reveal a novel role for ubiquitin as a sorting signal that allows certain specialized proteins to escape from damaged mitochondria.
    Keywords:   VCP ; March5; PINK1- and Parkin-mediated mitophagy; peroxin; ubiquitin
    DOI:  https://doi.org/10.15252/embr.201947728
  3. Science. 2019 Oct 11. 366(6462): 203-210
    Anandapadamanaban M, Masson GR, Perisic O, Berndt A, Kaufman J, Johnson CM, Santhanam B, Rogala KB, Sabatini DM, Williams RL.
      The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
    DOI:  https://doi.org/10.1126/science.aax3939
  4. Genetics. 2019 Oct;213(2): 329-360
    Blackwell TK, Sewell AK, Wu Z, Han M.
      The Target of Rapamycin (TOR or mTOR) is a serine/threonine kinase that regulates growth, development, and behaviors by modulating protein synthesis, autophagy, and multiple other cellular processes in response to changes in nutrients and other cues. Over recent years, TOR has been studied intensively in mammalian cell culture and genetic systems because of its importance in growth, metabolism, cancer, and aging. Through its advantages for unbiased, and high-throughput, genetic and in vivo studies, Caenorhabditis elegans has made major contributions to our understanding of TOR biology. Genetic analyses in the worm have revealed unexpected aspects of TOR functions and regulation, and have the potential to further expand our understanding of how growth and metabolic regulation influence development. In the aging field, C. elegans has played a leading role in revealing the promise of TOR inhibition as a strategy for extending life span, and identifying mechanisms that function upstream and downstream of TOR to influence aging. Here, we review the state of the TOR field in C. elegans, and focus on what we have learned about its functions in development, metabolism, and aging. We discuss knowledge gaps, including the potential pitfalls in translating findings back and forth across organisms, but also describe how TOR is important for C. elegans biology, and how C. elegans work has developed paradigms of great importance for the broader TOR field.
    Keywords:  Caenorhabditis elegans development; DAF-15; NPRL-2; NPRL-3; Nprl2; Nprl3; RAGA-1; RSKS-1; RagA; RagC; Raptor; Rheb; Rheb-1; Rictor; S6 kinase; TOR; TORC1; TORC2; WormBook; aging; growth regulation; metabolism; nutrient signaling; sphingolipid
    DOI:  https://doi.org/10.1534/genetics.119.302504
  5. Science. 2019 Oct 10. pii: eaay0166. [Epub ahead of print]
    Rogala KB, Gu X, Kedir JF, Abu-Remaileh M, Bianchi LF, Bottino AMS, Dueholm R, Niehaus A, Overwijn D, Fils AP, Zhou SX, Leary D, Laqtom NN, Brignole EJ, Sabatini DM.
      The mTORC1 protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag GTPase-Ragulator complex. Nutrients switch the heterodimeric Rag GTPases between four different nucleotide binding states, only one of which (RagA/B•GTP-RagC/D•GDP) permits mTORC1 association. We determined the structure of the supercomplex of Raptor with Rag-Ragulator to 3.2 Å resolution by cryo-electron microscopy. The Raptor α-solenoid directly detects the nucleotide state of RagA, while the Raptor "claw" threads between the GTPase domains to detect that of RagC. Mutations that disrupt Rag-Raptor binding inhibit mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we develop a model of active mTORC1 docked on the lysosome.
    DOI:  https://doi.org/10.1126/science.aay0166
  6. J Cell Biol. 2019 Oct 10. pii: jcb.201903018. [Epub ahead of print]
    Tiscione SA, Vivas O, Ginsburg KS, Bers DM, Ory DS, Santana LF, Dixon RE, Dickson EJ.
      Niemann-Pick type C1 (NPC1) protein is essential for the transport of externally derived cholesterol from lysosomes to other organelles. Deficiency of NPC1 underlies the progressive NPC1 neurodegenerative disorder. Currently, there are no curative therapies for this fatal disease. Given the Ca2+ hypothesis of neurodegeneration, which posits that altered Ca2+ dynamics contribute to neuropathology, we tested if disease mutations in NPC1 alter Ca2+ signaling and neuronal plasticity. We determine that NPC1 inhibition or disease mutations potentiate store-operated Ca2+ entry (SOCE) due to a presenilin 1 (PSEN1)-dependent reduction in ER Ca2+ levels alongside elevated expression of the molecular SOCE components ORAI1 and STIM1. Associated with this dysfunctional Ca2+ signaling is destabilization of neuronal dendritic spines. Knockdown of PSEN1 or inhibition of the SREBP pathway restores Ca2+ homeostasis, corrects differential protein expression, reduces cholesterol accumulation, and rescues spine density. These findings highlight lysosomes as a crucial signaling platform responsible for tuning ER Ca2+ signaling, SOCE, and synaptic architecture in health and disease.
    DOI:  https://doi.org/10.1083/jcb.201903018
  7. Cell Death Dis. 2019 Oct 10. 10(10): 771
    Loos F, Xie W, Sica V, Bravo-San Pedro JM, Souquère S, Pierron G, Lachkar S, Sauvat A, Petrazzuolo A, Jimenez AJ, Perez F, Maiuri MC, Kepp O, Kroemer G.
      The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.
    DOI:  https://doi.org/10.1038/s41419-019-2011-5
  8. Dev Cell. 2019 Oct 07. pii: S1534-5807(19)30770-1. [Epub ahead of print]51(1): 4-5
    Dillard C, Rusten TE.
      Cell competition eradicates weaker cells from an epithelium and is important for organ homeostasis. In this issue of Developmental Cell, Nagata et al. (2019) uncover that eradication of loser cells depends on autophagy-mediated cell death.
    DOI:  https://doi.org/10.1016/j.devcel.2019.09.014
  9. Proc Natl Acad Sci U S A. 2019 Oct 07. pii: 201911612. [Epub ahead of print]
    Young LN, Goerdeler F, Hurley JH.
      Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.
    Keywords:  autophagy; cryo-EM; lipid kinase
    DOI:  https://doi.org/10.1073/pnas.1911612116
  10. Hum Mol Genet. 2019 Oct 10. pii: ddz209. [Epub ahead of print]
    Bryan MR, O'Brien MT, Nordham KD, Rose DIR, Foshage AM, Joshi P, Nitin R, Uhouse MA, Di Pardo A, Zhang Z, Maglione V, Aschner M, Bowman AB.
      The molecular etiology linking the pathogenic mutations in the Huntingtin (Htt) gene with Huntington's Disease (HD) is unknown. Prior work suggests a role for Htt in neuronal autophagic function and mutant HTT protein disrupts autophagic cargo loading. Reductions in the bioavailability of the essential metal manganese (Mn) are seen in models of HD. Excess cellular Mn impacts autophagic function, but the target and molecular basis of these changes are unknown. Thus, we sought to determine if changes in cellular Mn status impact autophagic processes in a wild-type or mutant Htt dependent manner. We report that the HD genotype is associated with reduced Mn-induced autophagy, and that acute Mn exposure increases autophagosome induction/formation. To determine if a deficit in bioavailable Mn is mechanistically linked to the autophagy related HD cellular phenotypes, we examined autophagosomes by electron microscopy. We observed that a 24hr 100uM Mn restoration treatment protocol attenuated an established HD "cargo-recognition failure" in the STHdh HD model cells by increasing the percentage of filled autophagosomes. Mn restoration had no effect on HTT aggregate number, but a 72-hour co-treatment with CQ in GFP-72Q-expressing HEK293 cells increased the number of visible aggregates in a dose-dependent manner. As CQ prevents autophagic degradation this indicates that Mn restoration in HD cell models facilitates incorporation of aggregates into autophagosomes. Together, these findings suggest that defective Mn homeostasis in HD models is upstream of the impaired autophagic flux and provide proof-of-principle support for increasing bioavailable Mn in HD to restore autophagic function and promote aggregate clearance.
    DOI:  https://doi.org/10.1093/hmg/ddz209