bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒07‒07
twelve papers selected by
Viktor Korolchuk
Newcastle University


  1. Autophagy. 2019 Jul 04. 1-3
    Abudu YP, Pankiv S, Mathai BJ, Lamark T, Johansen T, Simonsen A.
      Removal of damaged mitochondria is vital for cellular homeostasis especially in non-dividing cells, like neurons. Damaged mitochondria that cannot be repaired by the ubiquitin-proteasomal system are cleared by a form of selective autophagy known as mitophagy. Following damage, mitochondria become labelled with 'eat-me' signals that selectively determine their degradation. Recently, we identified the mitochondrial matrix proteins, NIPSNAP1 (nipsnap homolog 1) and NIPSNAP2 as 'eat-me' signals for damaged mitochondria. NIPSNAP1 and NIPSNAP2 accumulate on the mitochondrial outer membrane following mitochondrial depolarization, recruiting autophagy receptors and adaptors, as well as human Atg8 (autophagy-related 8)-family proteins to facilitate mitophagy. The NIPSNAPs allow a sustained recruitment of SQSTM1-like receptors (SLRs) to ensure efficient mitophagy. Zebrafish lacking Nipsnap1 show decreased mitophagy in the brain coupled with increased ROS production, loss of dopaminergic neurons and strongly reduced locomotion.
    Keywords:  Atg8; NIPSNAP1; NIPSNAP2; PINK1/Parkin; SLRs; mitophagy; ‘eat me’ signal
    DOI:  https://doi.org/10.1080/15548627.2019.1637642
  2. Autophagy. 2019 Jul 04. 1-14
    Tian X, Zheng P, Zhou C, Wang X, Ma H, Ma W, Zhou X, Teng J, Chen J.
      Autophagosome and lysosome fusion is an important macroautophagy/autophagy process for cargo degradation, and SNARE proteins, including STX17, SNAP29, VAMP7 and VAMP8, are key players in this process. However, the manner in which this process is precisely regulated is poorly understood. Here, we show that VAMP7B, a SNARE domain-disrupted isoform of R-SNARE protein VAMP7, competes with SNARE domain functional isoform VAMP7A to bind to STX17 and inhibits autophagosome-lysosome fusion. Moreover, we show that DIPK2A, a late endosome- and lysosome-localized protein, binds to VAMP7B, which inhibits the interaction of VAMP7B with STX17 and enhances the binding of STX17 to VAMP7A, thus enhancing autophagosome-lysosome fusion. Furthermore, DIPK2A participates in autophagic degradation of mitochondria proteins and alleviates apoptosis. Thus, we reveal a new aspect of autophagosome-lysosome fusion in which different isoforms of VAMP7 compete with STX17 and their regulation by DIPK2A. Abbreviations: DIPK2A: divergent protein kinase domain 2A; EEA1: early endosome antigen 1; GOLGA2: golgin A2; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MT-CO2: mitochondrially encoded cytochrome c oxidase II; PARP1: poly(ADP-ribose) polymerase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; REEP: receptor accessory protein; RTN4: reticulon 4; SNARE: SNAP receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TOMM20: translocase of outer mitochondrial membrane 20; VAMP7: vesicle associated membrane protein 7; VAMP8: vesicle associated membrane protein 8.
    Keywords:  Autophagosome-lysosome fusion; DIPK2A; STX17; VAMP7; autophagy
    DOI:  https://doi.org/10.1080/15548627.2019.1637199
  3. EMBO Rep. 2019 Jul;20(7): e46885
    Alsaadi RM, Losier TT, Tian W, Jackson A, Guo Z, Rubinsztein DC, Russell RC.
      Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn's disease (CD), and phosphorylation of CD-associated ATG16L1 T300A (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Phosphorylated ATG16L1 localizes to the site of internalized bacteria and stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy, indicating a role for ATG16L1 phosphorylation in the promotion of anti-bacterial autophagy. In contrast to wild-type ATG16L1, ULK1-mediated phosphorylation of caATG16L1 drives its destabilization in response to stress. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1 signalling to ATG16L1 is a double-edged sword, enhancing the function of the wild-type ATG16L1, but promoting degradation of caATG16L1.
    Keywords:  ATG16L1; Crohn's disease; ULK1; autophagy; caspase
    DOI:  https://doi.org/10.15252/embr.201846885
  4. Autophagy. 2019 Jul 01. 1-2
    Boyle KB, Ravenhill BJ, Randow F.
      The selective macroautophagy of prospective cargo necessitates activity of the autophagy machinery at cargo-determined locations. Whether phagophore membranes are recruited to, or are generated de novo at, the cargo is unknown. In our recent study we show that damaged Salmonella-containing vacuoles, marked by LGALS8/galectin-8, engage the cargo receptor CALCOCO2/NDP52 to recruit the autophagy-initiating ULK and TBK1 complexes and cause the formation of WIPI2-positive phagophore membranes. CALCOCO2 functions in the induction of autophagy by forming a trimer with RB1CC1/FIP200 and TBKBP1/SINTBAD-AZI2/NAP1, components of the ULK and TBK1 kinase complexes, respectively. Such recruitment of the upstream autophagy machinery to prospective cargo reveals how in complex eukaryotes detection of cargo-associated 'eat me' signals, induction of autophagy, and juxtaposition of cargo and phagophores are integrated.
    Keywords:  Autophagy; FIP200; NDP52; Salmonella; TBK1; ULK1; cargo receptor; eat-me signal; galectin-8
    DOI:  https://doi.org/10.1080/15548627.2019.1628548
  5. Science. 2019 Jul 05. 365(6448): 53-60
    Cui Y, Parashar S, Zahoor M, Needham PG, Mari M, Zhu M, Chen S, Ho HC, Reggiori F, Farhan H, Brodsky JL, Ferro-Novick S.
      The COPII-cargo adaptor complex Lst1-Sec23 selectively sorts proteins into vesicles that bud from the endoplasmic reticulum (ER) and traffic to the Golgi. Improperly folded proteins are prevented from exiting the ER and are degraded. ER-phagy is an autophagic degradation pathway that uses ER-resident receptors. Working in yeast, we found an unexpected role for Lst1-Sec23 in ER-phagy that was independent from its function in secretion. Up-regulation of the stress-inducible ER-phagy receptor Atg40 induced the association of Lst1-Sec23 with Atg40 at distinct ER domains to package ER into autophagosomes. Lst1-mediated ER-phagy played a vital role in maintaining cellular homeostasis by preventing the accumulation of an aggregation-prone protein in the ER. Lst1 function appears to be conserved because its mammalian homolog, SEC24C, was also required for ER-phagy.
    DOI:  https://doi.org/10.1126/science.aau9263
  6. J Mol Biol. 2019 Jun 28. pii: S0022-2836(19)30415-2. [Epub ahead of print]
    Sun D, Wu R, Li P, Yu L.
      The selective degradation of protein aggregates is called aggrephagy. Misfolded proteins are thought to form aggregates, which are then surrounded by selective autophagy receptors and targeted to autophagosomes for degradation. Recent studies of p62 bodies, PGL granules and stress granules indicate that proteins targeted for aggrephagy are not simple protein aggregates but rather form liquid-like protein condensates through liquid-liquid phase separation. The liquid-like properties of the condensates and hardening to a gel-like state may be crucial in the initiation of aggrephagy. Dysregulation of phase separation may cause human diseases. Here we review the potential roles of liquid-liquid phase separation in the process of aggrephagy.
    Keywords:  Aberrant stress granule; Aggrephagy; PGL granule; Phase separation; p62 body
    DOI:  https://doi.org/10.1016/j.jmb.2019.06.026
  7. Nat Commun. 2019 Jun 28. 10(1): 2865
    Cuéllar J, Ludlam WG, Tensmeyer NC, Aoba T, Dhavale M, Santiago C, Bueno-Carrasco MT, Mann MJ, Plimpton RL, Makaju A, Franklin S, Willardson BM, Valpuesta JM.
      The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, β-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar β-propeller proteins.
    DOI:  https://doi.org/10.1038/s41467-019-10781-1
  8. Cell. 2019 Jun 21. pii: S0092-8674(19)30627-0. [Epub ahead of print]
    Heckmann BL, Teubner BJW, Tummers B, Boada-Romero E, Harris L, Yang M, Guy CS, Zakharenko SS, Green DR.
      The expression of some proteins in the autophagy pathway declines with age, which may impact neurodegeneration in diseases, including Alzheimer's Disease. We have identified a novel non-canonical function of several autophagy proteins in the conjugation of LC3 to Rab5+, clathrin+ endosomes containing β-amyloid in a process of LC3-associated endocytosis (LANDO). We found that LANDO in microglia is a critical regulator of immune-mediated aggregate removal and microglial activation in a murine model of AD. Mice lacking LANDO but not canonical autophagy in the myeloid compartment or specifically in microglia have a robust increase in pro-inflammatory cytokine production in the hippocampus and increased levels of neurotoxic β-amyloid. This inflammation and β-amyloid deposition were associated with reactive microgliosis and tau hyperphosphorylation. LANDO-deficient AD mice displayed accelerated neurodegeneration, impaired neuronal signaling, and memory deficits. Our data support a protective role for LANDO in microglia in neurodegenerative pathologies resulting from β-amyloid deposition.
    Keywords:  Alzheimer’s Disease; LC3-associated endocytosis; LC3-associated phagocytosis; autophagy; microglia; neurodegeneration; neuroinflammation; receptor-mediated endocytosis; tau pathology; β-amyloid
    DOI:  https://doi.org/10.1016/j.cell.2019.05.056
  9. Biochem Biophys Res Commun. 2019 Jun 25. pii: S0006-291X(19)31267-7. [Epub ahead of print]
    Yazawa R, Nishida Y, Aoyama S, Tanida I, Miyatsuka T, Suzuki L, Himuro M, Haruna H, Takubo N, Shimizu T, Watada H.
      Autophagy is a mechanism of bulk protein degradation that plays an important role in regulating homeostasis in many organisms. Among several methods for evaluating its activity, a fluorescent reporter GFP-LC3-RFP-LC3ΔG, in which GFP-LC3 is cleaved by ATG4 following autophagic induction and degraded in lysosome, has been used for monitoring autophagic flux, which is the amount of lysosomal protein degradation. In this study, we modified this reporter by exchanging GFP for pHluorin, which is more sensitive to low pH, and RFP to mCherry, to construct pHluorin-LC3-mCherry reporter. Following starvation or mTOR inhibition, the increase of autophagic flux was detected by a decrease of the fluorescent ratio of pHluorin to mCherry; our reporter was also more sensitive to autophagy-inducing stimuli than the previous one. To establish monitoring cells for mouse genome-wide screening of regulators of autophagic flux based on CRISPR/Cas9 system, after evaluating knockout efficiency of clones of Cas9-expressing MEFs, we co-expressed our reporter and confirmed that autophagic flux was impaired in gRNA-mediated knockout of canonical autophagy genes. Finally, we performed genome-wide gRNA screening for genes inhibiting starvation-mediated autophagic flux and identified previously reported genes such as Atgs. Thus, we have successfully established a system for screening of genes regulating autophagic flux with our pHluorin-LC3-mCherry reporter in mice.
    Keywords:  Autophagic flux; Autophagy; CRISPR/Cas9; Next-generation sequencing; pHluorin
    DOI:  https://doi.org/10.1016/j.bbrc.2019.06.129
  10. Elife. 2019 Jul 04. pii: e45777. [Epub ahead of print]8
    Maeda S, Otomo C, Otomo T.
      An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid-transferring protein. ATG2A can extract lipids from membrane vesicles and unload them to other vesicles. Lipid transfer by ATG2A is more efficient between tethered vesicles than between untethered vesicles. The PI3P effectors WIPI4 and WIPI1 associate ATG2A stably to PI3P-containing vesicles, thereby facilitating ATG2A-mediated tethering and lipid transfer between PI3P-containing vesicles and PI3P-free vesicles. Based on these results, we propose that ATG2-mediated transfer of lipids from the ER to the phagophore enables phagophore expansion.
    Keywords:  biochemistry; cell biology; chemical biology; human
    DOI:  https://doi.org/10.7554/eLife.45777
  11. Cell Mol Life Sci. 2019 Jun 28.
    Bayne AN, Trempe JF.
      Parkinson's disease (PD) is a degenerative movement disorder resulting from the loss of specific neuron types in the midbrain. Early environmental and pathophysiological studies implicated mitochondrial damage and protein aggregation as the main causes of PD. These findings are now vindicated by the characterization of more than 20 genes implicated in rare familial forms of the disease. In particular, two proteins encoded by the Parkin and PINK1 genes, whose mutations cause early-onset autosomal recessive PD, function together in a mitochondrial quality control pathway. In this review, we will describe recent development in our understanding of their mechanisms of action, structure, and function. We explain how PINK1 acts as a mitochondrial damage sensor via the regulated proteolysis of its N-terminus and the phosphorylation of ubiquitin tethered to outer mitochondrial membrane proteins. In turn, phospho-ubiquitin recruits and activates Parkin via conformational changes that increase its ubiquitin ligase activity. We then describe how the formation of polyubiquitin chains on mitochondria triggers the recruitment of the autophagy machinery or the formation of mitochondria-derived vesicles. Finally, we discuss the evidence for the involvement of these mechanisms in physiological processes such as immunity and inflammation, as well as the links to other PD genes.
    Keywords:  Kinase; Mitochondria; PINK1; Parkin; Parkinson; Ubiquitin
    DOI:  https://doi.org/10.1007/s00018-019-03203-4
  12. Sci Rep. 2019 Jul 01. 9(1): 9455
    Arias-Fuenzalida J, Jarazo J, Walter J, Gomez-Giro G, Forster JI, Krueger R, Antony PMA, Schwamborn JC.
      Autophagic processes play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy can be affected at multiple and distinct steps of the pathway. Current analyses tools do not deliver the required detail for dissecting pathway intermediates. The development of new tools to analyze autophagic processes qualitatively and quantitatively in a more straightforward manner is required. Defining all autophagy pathway intermediates in a high-throughput manner is technologically challenging and has not been addressed yet. Here, we overcome those requirements and limitations by the developed of stable autophagy and mitophagy reporter-iPSC and the establishment of a novel high-throughput phenotyping platform utilizing automated high-content image analysis to assess autophagy and mitophagy pathway intermediates.
    DOI:  https://doi.org/10.1038/s41598-019-45917-2