bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒09‒29
sixteen papers selected by
Viktor Korolchuk, Newcastle University

  1. Nat Cell Biol. 2019 Sep 23.
      Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.
  2. Front Cell Dev Biol. 2019 ;7 179
      An imbalance between the production and clearance of macromolecules such as proteins, lipids and carbohydrates can lead to a category of diseases broadly known as macromolecule storage disorders. These include, but not limited to, neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease associated with accumulation of aggregation-prone proteins, Lafora and Pompe disease associated with glycogen accumulation, whilst lipid accumulation is characteristic to Niemann-Pick disease and Gaucher disease. One of the underlying factors contributing to the build-up of macromolecules in these storage disorders is the intracellular degradation pathway called autophagy. This process is the primary clearance route for unwanted macromolecules, either via bulk non-selective degradation, or selectively via aggrephagy, glycophagy and lipophagy. Since autophagy plays a vital role in maintaining cellular homeostasis, cell viability and human health, malfunction of this process could be detrimental. Indeed, defective autophagy has been reported in a number of macromolecule storage disorders where autophagy is impaired at distinct stages, such as at the level of autophagosome formation, autophagosome maturation or improper lysosomal degradation of the autophagic cargo. Of biomedical relevance, autophagy is regulated by multiple signaling pathways that are amenable to chemical perturbations by small molecules. Induction of autophagy has been shown to improve cell viability and exert beneficial effects in experimental models of various macromolecule storage disorders where the lysosomal functionality is not overtly compromised. In this review, we will discuss the role of autophagy in certain macromolecule storage disorders and highlight the potential therapeutic benefits of autophagy enhancers in these pathological conditions.
    Keywords:  autophagy; autophagy inducers; glycogen storage disorders; lipid storage disorders; macromolecule storage disorders; neurodegenerative disorders; proteinopathies; selective autophagy
  3. Exp Cell Res. 2019 Sep 21. pii: S0014-4827(19)30508-7. [Epub ahead of print] 111641
      Parkin is an ubiquitin ligase regulating mitochondrial quality control reactions, including the autophagic removal of depolarized mitochondria (mitophagy). Parkin-mediated protein ubiquitinations may be counteracted by deubiquitinating enzymes (DUBs). We conducted a high-content imaging screen of Parkin translocation to depolarized mitochondria after siRNA mediated silencing of each DUB in Parkin overexpressing HeLa cells. Knockdown of the ubiquitin-specific protease USP36 led to delayed Parkin translocation while only slightly disturbing the ubiquitination of mitochondrial proteins, but final autophagic elimination of mitochondria was severely disrupted. The localization of the nucleolar USP36 was not altered during mitophagy. However, the marker for transcriptional active chromatin, histone 2B Lys120 mono-ubiquitination was found reduced in USP36-silenced cells undergoing mitophagy. We observed a reduction of the mRNA and protein levels of Beclin-1 and its associated autophagy-related key regulator ATG14L in USP36 knockdown cells. Importantly, transfection of active ATG14L into USP36-silenced cells significantly restored Parkin-dependent mitophagy. We propose USP36 as regulator for the Parkin-dependent mitophagy at least in part via the Beclin-1-ATG14L pathway.
    Keywords:  Autophagy-related protein ATG14L; Beclin-1; Deubiquitinating enzyme; Mitophagy; Parkin; Ubiquitin-specific protease USP36
  4. Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 201904774. [Epub ahead of print]
      The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.
    Keywords:  AKT kinase; DEPDC5; GATOR1; Pim kinase; mTORC1
  5. EMBO Rep. 2019 Sep 23. e48150
      STK38 (also known as NDR1) is a Hippo pathway serine/threonine protein kinase with multifarious functions in normal and cancer cells. Using a context-dependent proximity-labeling assay, we identify more than 250 partners of STK38 and find that STK38 modulates its partnership depending on the cellular context by increasing its association with cytoplasmic proteins upon nutrient starvation-induced autophagy and with nuclear ones during ECM detachment. We show that STK38 shuttles between the nucleus and the cytoplasm and that its nuclear exit depends on both XPO1 (aka exportin-1, CRM1) and STK38 kinase activity. We further uncover that STK38 modulates XPO1 export activity by phosphorylating XPO1 on serine 1055, thus regulating its own nuclear exit. We expand our model to other cellular contexts by discovering that XPO1 phosphorylation by STK38 regulates also the nuclear exit of Beclin1 and YAP1, key regulator of autophagy and transcriptional effector, respectively. Collectively, our results reveal STK38 as an activator of XPO1, behaving as a gatekeeper of nuclear export. These observations establish a novel mechanism of XPO1-dependent cargo export regulation by phosphorylation of XPO1's C-terminal auto-inhibitory domain.
    Keywords:  APEX2; STK38; XPO1; YAP1; autophagy
  6. Cell Rep. 2019 Sep 24. pii: S2211-1247(19)31124-6. [Epub ahead of print]28(13): 3486-3496.e6
      The target of rapamycin complex 1 (TORC1) is a master regulator of cell homeostasis, which promotes anabolic reactions and synchronously inhibits catabolic processes such as autophagy-mediated protein degradation. Its prime autophagy target is Atg13, a subunit of the Atg1 kinase complex that acts as the gatekeeper of canonical autophagy. To study whether the activities of TORC1 and Atg1 are coupled through additional, more intricate control mechanisms than simply this linear pathway, we analyzed the epistatic relationship between TORC1 and Atg1 by using quantitative phosphoproteomics. Our in vivo data, combined with targeted in vitro TORC1 and Atg1 kinase assays, not only uncover numerous TORC1 and Atg1 effectors, but also suggest distinct bi-directional regulatory feedback loops and characterize Atg29 as a commonly regulated downstream target of both TORC1 and Atg1. Thus, an exquisitely multilayered regulatory network appears to coordinate TORC1 and Atg1 activities to robustly tune autophagy in response to nutritional cues.
    Keywords:  Atg29; autophagy; kinase; mass spectrometry; metabolism; phosphorylation; proteomics; signaling
  7. Cell Rep. 2019 Sep 24. pii: S2211-1247(19)31107-6. [Epub ahead of print]28(13): 3423-3434.e2
      Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase induce nucleophagy preferentially degrading only nucleolar components in budding yeast. Nucleolar proteins are relocated to sites proximal to the nucleus-vacuole junction (NVJ), where micronucleophagy occurs, whereas rDNA, which is embedded in the nucleolus under normal conditions, moves to NVJ-distal regions, causing rDNA dissociation from nucleolar proteins after TORC1 inactivation. This repositioning is mediated via chromosome linkage INM protein (CLIP)-cohibin complexes that tether rDNA to the inner nuclear membrane. Here, we show that TORC1 inactivation-induced rDNA condensation promotes the repositioning of rDNA and nucleolar proteins. Defects in condensin, Rpd3-Sin3 histone deacetylase (HDAC), and high-mobility group protein 1 (Hmo1), which are involved in TORC1 inactivation-induced rDNA condensation, compromised the repositioning and nucleophagic degradation of nucleolar proteins, although rDNA still escaped from nucleophagic degradation in these mutants. We propose a model in which rDNA condensation after TORC1 inactivation generates a motive force for the repositioning of rDNA and nucleolar proteins.
    Keywords:  Hmo1; Rpd3; TORC1; condensin; nucleolus; nucleophagy; rDNA; target of rapamycin complex 1
  8. Nat Commun. 2019 Sep 27. 10(1): 4399
      Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and Endoplasmic Reticulum-Mitochondria Contacts Sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality.
  9. Sci Rep. 2019 Sep 25. 9(1): 13896
      The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implications.
  10. Cell Rep. 2019 Sep 24. pii: S2211-1247(19)31097-6. [Epub ahead of print]28(13): 3329-3337.e5
      Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids (SILACs) and cultured cells, we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared with quiescent cell counterparts. Bioinformatic analysis reveals that 44 of these proteins participate in hemostasis, a process not previously linked with cellular senescence. We validated the expression of some of these SASP factors in cultured cells and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, selective removal of senescent cells using transgenic p16-3MR mice showed that clearing senescent cells attenuates the increased clotting caused by doxorubicin. Our study provides an in-depth, unbiased analysis of the SASP and unveils a function for cellular senescence in hemostasis.
    Keywords:  SASP; aging; cellular senescence; chemotherapy; clotting; coagulation; homeostasis; proteomics; secretion; thrombosis
  11. Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 201901759. [Epub ahead of print]
      Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.
    Keywords:  Saccharomyces cerevisiae; autophagy; mitophagy; phosphatase; protein phosphorylation
  12. J Biol Chem. 2019 Sep 22. pii: jbc.RA119.010036. [Epub ahead of print]
      The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation Akt is phosphorylated at Ser474, however how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here, we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 phosphorylation, without perturbing mTORC2 activity. This was achieved by utilising a chemical genetics approach, whereby ectopically expressed Ser474Ala Akt2 was engineered with a Trp80Ala mutation to confer resistance to the Akt inhibitor MK2206, and thus allow for its activation independently of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a and AS160) were reduced by approximately 50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model whereby Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.
    Keywords:  Akt PKB; Akt S474 phosphorylation; Akt W80A; GLUT4; MK2206; adipocyte; cell signaling; chemical genetics; glucose transport; insulin; mTOR complex (mTORC); phosphorylation; protein synthesis; serine/threonine protein kinase; substrate specificity
  13. Cells. 2019 Sep 12. pii: E1075. [Epub ahead of print]8(9):
      The mammalian target of rapamycin (mTOR), is a serine/threonine protein kinase and belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family. mTOR interacts with other subunits to form two distinct complexes, mTORC1 and mTORC2. mTORC1 coordinates cell growth and metabolism in response to environmental input, including growth factors, amino acid, energy and stress. mTORC2 mainly controls cell survival and migration through phosphorylating glucocorticoid-regulated kinase (SGK), protein kinase B (Akt), and protein kinase C (PKC) kinase families. The dysregulation of mTOR is involved in human diseases including cancer, cardiovascular diseases, neurodegenerative diseases, and epilepsy. Tissue damage caused by trauma, diseases or aging disrupt the tissue functions. Tissue regeneration after injuries is of significance for recovering the tissue homeostasis and functions. Mammals have very limited regenerative capacity in multiple tissues and organs, such as the heart and central nervous system (CNS). Thereby, understanding the mechanisms underlying tissue regeneration is crucial for tissue repair and regenerative medicine. mTOR is activated in multiple tissue injuries. In this review, we summarize the roles of mTOR signaling in tissue regeneration such as neurons, muscles, the liver and the intestine.
    Keywords:  intestine; liver; mTOR signaling; metabolism; muscle; neuron; tissue regeneration
  14. Eur J Pharmacol. 2019 Sep 19. pii: S0014-2999(19)30629-6. [Epub ahead of print] 172677
      We investigated the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), prosurvival kinase Akt, oxidative stress, and autophagy in the cytotoxicity of parkinsonian neurotoxin 1-methyl-4-phenyl piridinium (MPP+) towards SH-SY5Y human neuroblastoma cells. MPP + -mediated oxidative stress, mitochondrial depolarization, and apoptotic cell death were associated with rapid (within 2 h) activation of AMPK, its target Raptor, and prosurvival kinase Akt. Antioxidants N-acetylcysteine and butylated hydroxyanisole suppressed MPP + -induced cytotoxicity, AMPK, and Akt activation. A genetic or pharmacological inhibition of AMPK increased MPP + -triggered production of reactive oxygen species and cell death, and diminished Akt phosphorylation, while AMPK activation protected SH-SY5Y cells from MPP+. On the other hand, genetic or pharmacological inactivation of Akt stimulated MPP + -triggered oxidative stress and neurotoxicity, but did not affect AMPK activation. At later time-points (16-24 h), MPP + inhibited the main autophagy repressor mammalian target of rapamycin, which coincided with the increase in the levels of autophagy marker microtubule-associated protein 1 light-chain 3B. MPP + also increased the concentration of a selective autophagic target sequestosome-1/p62 and reduced the levels of lysosomal-associated membrane protein 1 and cytoplasmic acidification, suggesting that MPP + -induced autophagy was coupled with a decrease in autophagic flux. Nevertheless, further pharmacological inhibition of autophagy sensitized SH-SY5Y cells to MPP + -induced death. Antioxidants and AMPK knockdown reduced, whereas genetic inactivation of Akt potentiated neurotoxin-triggered autophagy. These results suggest that MPP + -induced oxidative stress stimulates AMPK, which protects SH-SY5Y cells through early activation of antioxidative Akt and late induction of cytoprotective autophagy.
    Keywords:  AMPK; Akt; Autophagy; MPP+; Neurons; Oxidative stress
  15. Proc Natl Acad Sci U S A. 2019 Sep 23. pii: 201903542. [Epub ahead of print]
      The contractile perivascular cells, pericytes (PC), are hijacked by glioblastoma (GB) to facilitate tumor progression. PC's protumorigenic function requires direct interaction with tumor cells and contributes to the establishment of immunotolerance to tumor growth. Cancer cells up-regulate their own chaperone-mediated autophagy (CMA), a process that delivers selective cytosolic proteins to lysosomes for degradation, with pro-oncogenic effects. However, the possible impact that cancer cells may have on CMA of surrounding host cells has not been explored. We analyzed the contribution of CMA to the GB-induced changes in PC biology. We have found that CMA is markedly up-regulated in PC in response to the oxidative burst that follows PC-GB cell interaction. Genetic manipulations to block the GB-induced up-regulation of CMA in PC allows them to maintain their proinflammatory function and to support the induction of effective antitumor T cell responses required for GB clearance. GB-induced up-regulation of CMA activity in PC is essential for their effective interaction with GB cells that help tumor growth. We show that CMA inhibition in PC promotes GB cell death and the release of high immunogenic levels of granulocyte-macrophage colony stimulating factor (GM-CSF), through deregulation of the expression of cell-to-cell interaction proteins and protein secretion. A GB mouse model grafted in vivo with CMA-defective PC shows reduced GB proliferation and effective immune response compared to mice grafted with control PC. Our findings identify abnormal up-regulation of CMA as a mechanism by which GB cells elicit the immunosuppressive function of PC and stabilize GB-PC interactions necessary for tumor cell survival.
    Keywords:  chaperone-mediated autophagy; glioblastoma; immunosuppressive function; pericytes; tumor
  16. Cell. 2019 Sep 13. pii: S0092-8674(19)30957-2. [Epub ahead of print]
      Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C.
    Keywords:  NCR1; NPC1; NPC2; Niemann-Pick type C proteins; X-ray crystallography; cryo-EM; lipid trafficking; sterol homeostasis; sterol membrane integration