bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒06‒21
28 papers selected by
Viktor Korolchuk
Newcastle University

  1. Nat Commun. 2020 Jun 19. 11(1): 3148
    Son SM, Park SJ, Stamatakou E, Vicinanza M, Menzies FM, Rubinsztein DC.
      Macroautophagy ("autophagy") is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism.
  2. J Cell Biol. 2020 Sep 07. pii: e201912144. [Epub ahead of print]219(9):
    Yamano K, Kikuchi R, Kojima W, Hayashida R, Koyano F, Kawawaki J, Shoda T, Demizu Y, Naito M, Tanaka K, Matsuda N.
      Damaged mitochondria are selectively eliminated in a process called mitophagy. Parkin and PINK1, proteins mutated in Parkinson's disease, amplify ubiquitin signals on damaged mitochondria with the subsequent activation of autophagic machinery. Autophagy adaptors are thought to link ubiquitinated mitochondria and autophagy through ATG8 protein binding. Here, we establish methods for inducing mitophagy by mitochondria-targeted ubiquitin chains and chemical-induced mitochondrial ubiquitination. Using these tools, we reveal that the ubiquitin signal is sufficient for mitophagy and that PINK1 and Parkin are unnecessary for autophagy activation per se. Furthermore, using phase-separated fluorescent foci, we show that the critical autophagy adaptor OPTN forms a complex with ATG9A vesicles. Disruption of OPTN-ATG9A interactions does not induce mitophagy. Therefore, in addition to binding ATG8 proteins, the critical autophagy adaptors also bind the autophagy core units that contribute to the formation of multivalent interactions in the de novo synthesis of autophagosomal membranes near ubiquitinated mitochondria.
  3. Autophagy. 2020 Jun 19.
    Mesquita A, Glenn J, Jenny A.
      As one of the major, highly conserved catabolic pathways, autophagy delivers cytosolic components to lysosomes for degradation. It is essential for development, cellular homeostasis, and coping with stress. Reduced autophagy increases susceptibility to protein aggregation diseases and leads to phenotypes associated with aging. Of the three major forms of autophagy, macroautophagy (MA) can degrade organelles or aggregated proteins, and chaperone-mediated autophagy is specific for soluble proteins containing KFERQ-related targeting motifs. During endosomal microautophagy (eMI), cytoplasmic proteins are engulfed into late endosomes in an ESCRT machinery-dependent manner. eMI can be KFERQ-specific or occur in bulk and be induced by prolonged starvation. Its physiological regulation and function, however, are not understood. Here, we show that eMI in the Drosophila fat body, akin to the mammalian liver, is induced upon oxidative or genotoxic stress in an ESCRT and partially Hsc70-4-dependent manner. Interestingly, eMI activation is selective, as ER stress fails to elicit a response. Intriguingly, we find that reducing MA leads to a compensatory enhancement of eMI, suggesting a tight interplay between these degradative processes. Furthermore, we show that mutations in DNA damage response genes are sufficient to trigger eMI and that the response to oxidative stress is under the control of MAPK/JNK signaling. Our data suggest that, controlled by various signaling pathways, eMI allows an organ to react and adapt to specific types of stress and is thus likely critical to prevent disease.
    Keywords:  DNA damage; ER stress; ROS; autophagy; microautophagy; oxidative stress; proteostasis
  4. Curr Top Med Chem. 2020 Jun 18.
    Kabir MT, Uddin MS, Abdeen A, Ashraf GM, Perveen A, Hafeez A, Bin-Jumah MN, Abdel-Daim MM.
      Several proteolytic systems including ubiquitin (Ub)-proteasome system (UPS), chaperone-mediated autophagy (CMA), and macroautophagy are used by the mammalian cells to remove misfolded proteins (MPs). UPS mediates degradation of most of the MPs, where Ub-conjugated substrates are deubiquitinated, unfolded, and pass through the proteasome's narrow chamber, and eventually break into smaller peptides. It has been observed that the substrates that show a specific degradation signal, the KFERQ sequence motif, can be delivered to and go through CMA-mediated degradation in lysosomes. Macroautophagy can help in the degradation of substrates that are prone to aggregation and resistant to both the CMA and UPS. In the aforesaid case, cargoes are separated into autophagosomes before lysosomal hydrolase-mediated degradation. Even though the majority of the aggregated and MPs in the human proteome can be removed via cellular protein quality control (PQC), some mutant and native proteins tend to aggregate into β-sheet-rich oligomers that exhibit resistance to all identified proteolytic processes and can, therefore, grow into extracellular plaques or inclusion bodies. Indeed, the buildup of protease-resistant aggregated and MPs is a usual process underlying various protein misfolding disorders, including neurodegenerative diseases (NDs) for example Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and prion diseases. In this article, we have focused on the contribution of PQC in the degradation of pathogenic proteins in NDs.
    Keywords:  Protein misfolding; amyloid β; chaperone mediated autophagy; macroautophagy; neurodegeneration; tau.; ubiquitin-proteasome system
  5. Int J Mol Sci. 2020 Jun 12. pii: E4196. [Epub ahead of print]21(12):
    Csizmadia T, Lőw P.
      Deubiquitinating enzymes (DUBs) have an essential role in several cell biological processes via removing the various ubiquitin patterns as posttranslational modification forms from the target proteins. These enzymes also contribute to the normal cytoplasmic ubiquitin pool during the recycling of this molecule. Autophagy, a summary name of the lysosome dependent self-degradative processes, is necessary for maintaining normal cellular homeostatic equilibrium. Numerous forms of autophagy are known depending on how the cellular self-material is delivered into the lysosomal lumen. In this review we focus on the colorful role of DUBs in autophagic processes and discuss the mechanistic contribution of these molecules to normal cellular homeostasis via the possible regulation forms of autophagic mechanisms.
    Keywords:  DUB; cargo degradation; lysosome; ubiquitin; vesicle fusion
  6. Cell Stress. 2020 May 14. 4(6): 147-150
    Chen Z, Berquez M, Luciani A.
      Dysregulation of the mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of intermediary metabolism caused by the deficiency of methylmalonyl-CoA mutase (MMUT) - a mitochondrial enzyme that mediates the degradation of certain amino acids and lipids. The loss of MMUT activity triggers an accumulation of toxic endogenous metabolites causing severe organ dysfunctions and life-threatening complications. How MMUT deficiency instigates mitochondrial distress and tissue damage remains poorly understood. Using cell and animal-based models, we recently discovered that MMUT deficiency disables the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, impeding their delivery and subsequent dismantling by macroautophagy/autophagy-lysosome degradation systems (Luciani et al. Nat Commun. 11(1):970). This promotes an accumulation of damaged and/or dysfunctional mitochondria that spark epithelial distress and tissue damage. Using a systems biology approach based on drug-disease network perturbation modeling, we predicted targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient-derived kidney cells and ameliorates disease-relevant phenotypes in mmut-deficient zebrafish. These results unveil a link between primary MMUT deficiency, defective mitophagy, and cell distress, offering promising therapeutic avenues for MMA and other mitochondria-related diseases.
    Keywords:  cell damage; inherited metabolic disorders; kidney tubule; metabolism; mitochondria; mitophagy; organelle quality control; oxidative stress
  7. Autophagy. 2020 Jun 19. 1-10
    Gibson JF, Prajsnar TK, Hill CJ, Tooke AK, Serba JJ, Tonge RD, Foster SJ, Grierson AJ, Ingham PW, Renshaw SA, Johnston SA.
      Macroautophagy/autophagy functions to degrade cellular components and intracellular pathogens. Autophagy receptors, including SQSTM1/p62, target intracellular pathogens. Staphylococcus aureus is a significant pathogen of humans, especially in immunocompromise. S. aureus may use neutrophils as a proliferative niche, but their intracellular fate following phagocytosis has not been analyzed in vivo. In vitro, SQSTM1 can colocalize with intracellular Staphylococcus aureus, but whether SQSTM1 is beneficial or detrimental in host defense against S. aureus in vivo is unknown. Here we determine the fate and location of S. aureus within neutrophils throughout zebrafish infection. We show Lc3 and Sqstm1 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil and that Lc3 marking of bacterial phagosomes within neutrophils may precede bacterial degradation. Finally, we show Sqstm1 is important for controlling cytosolic bacteria, demonstrating for the first time a key role of Sqstm1 in autophagic control of S. aureus in neutrophils.ABBREVIATIONS: AR: autophagy receptor; CFU: colony-forming unit; CHT: caudal hematopoietic tissue; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; LWT: london wild-type: lyz: lysozyme; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification; UBD: ubiquitin binding domain.
    Keywords:   staphylococcus aureus ; Autophagy; bacterial infection; host-pathogen interactions; neutrophil; sqstm1/p62; xenophagy; zebrafish
  8. Sci Rep. 2020 Jun 15. 10(1): 9643
    Oliva Trejo JA, Tanida I, Suzuki C, Kakuta S, Tada N, Uchiyama Y.
      We generated a new transgenic mouse model that expresses a pHluorin-mKate2 fluorescent protein fused with human LC3B (PK-LC3 mice) for monitoring autophagy activity in neurons of the central nervous system. Histological analysis revealed fluorescent puncta in neurons of the cerebral cortex, hippocampus, cerebellar Purkinje cells, and anterior spinal regions. Using CLEM analysis, we confirmed that PK-LC3-positive puncta in the perikarya of Purkinje cells correspond to autophagic structures. To validate the usability of PK-LC3 mice, we quantified PK-LC3 puncta in Purkinje cells of mice kept in normal feeding conditions and of mice starved for 24 hours. Our results showed a significant increase in autophagosome number and in individual puncta areal size following starvation. To confirm these results, we used morphometry at the electron microscopic level to analyze the volume densities of autophagosomes and lysosomes/autolysosomes in Purkinje cells of PK-LC3 mice. The results revealed that the volume densities of autophagic structures increase significantly after starvation. Together, our data show that PK-LC3 mice are suitable for monitoring autophagy flux in Purkinje cells of the cerebellum, and potentially other areas in the central nervous system.
  9. Cell. 2020 Jun 11. pii: S0092-8674(20)30615-2. [Epub ahead of print]
    Qian H, Wu X, Du X, Yao X, Zhao X, Lee J, Yang H, Yan N.
      Lysosomal cholesterol egress requires two proteins, NPC1 and NPC2, whose defects are responsible for Niemann-Pick disease type C (NPC). Here, we present systematic structural characterizations that reveal the molecular basis for low-pH-dependent cholesterol delivery from NPC2 to the transmembrane (TM) domain of NPC1. At pH 8.0, similar structures of NPC1 were obtained in nanodiscs and in detergent at resolutions of 3.6 Å and 3.0 Å, respectively. A tunnel connecting the N-terminal domain (NTD) and the transmembrane sterol-sensing domain (SSD) was unveiled. At pH 5.5, the NTD exhibits two conformations, suggesting the motion for cholesterol delivery to the tunnel. A putative cholesterol molecule is found at the membrane boundary of the tunnel, and TM2 moves toward formation of a surface pocket on the SSD. Finally, the structure of the NPC1-NPC2 complex at 4.0 Å resolution was obtained at pH 5.5, elucidating the molecular basis for cholesterol handoff from NPC2 to NPC1(NTD).
    Keywords:  NPC1; NPC2; Niemann-Pick disease type C; cholesterol transport; cryo-EM; lysosomal cholesterol egress; structural biology
  10. Sci Rep. 2020 Jun 19. 10(1): 9972
    Yasueda A, Kayama H, Murohashi M, Nishimura J, Wakame K, Komatsu KI, Ogino T, Miyoshi N, Takahashi H, Uemura M, Matsuda C, Kitagawa T, Takeda K, Ito T, Doki Y, Eguchi H, Shimizu S, Mizushima T.
      Disturbed activation of autophagy is implicated in the pathogenesis of inflammatory bowel disease. Accordingly, several autophagy-related genes have been identified as Crohn's disease susceptibility genes. We screened the autophagy activators from a library including 3,922 natural extracts using a high-throughput assay system. The extracts identified as autophagy activators were administered to mice with 2% dextran sodium sulfate (DSS). Among the autophagy inducers, Sanguisorba officinalis L. (SO) suppressed DSS-induced colitis. To identify the mechanism by which SO ameliorates colitis, epithelial cell and innate myeloid cells-specific Atg7-deficient mice (Villin-cre; Atg7f/f and LysM-cre; Atg7f/f mice, respectively) were analyzed. SO-mediated inhibition of colitis was observed in Villin-cre; Atg7f/f mice. However, SO and a mixture of its components including catechin acid, ellagic acid, gallic acid, and ziyuglycoside II (Mix4) did not suppressed colitis in LysM-cre; Atg7f/f mice. In large intestinal macrophages (Mφ) of Atg7f/f mice, SO and Mix4 upregulated the expression of marker genes of anti-inflammatory Mφ including Arg1, Cd206, and Relma. However, these alterations were not induced in LysM-cre; Atg7f/f mice. These findings indicate that SO and its active components ameliorate DSS-induced colitis by providing intestinal Mφ with anti-inflammatory profiles via promotion of Atg7-dependent autophagy.
  11. Nat Commun. 2020 Jun 19. 11(1): 3106
    Park Y, Park J, Hwang HJ, Kim B, Jeong K, Chang J, Lee JB, Kim YK.
      Nonsense-mediated mRNA decay (NMD) typifies an mRNA surveillance pathway. Because NMD necessitates a translation event to recognize a premature termination codon on mRNAs, truncated misfolded polypeptides (NMD-polypeptides) could potentially be generated from NMD substrates as byproducts. Here, we show that when the ubiquitin-proteasome system is overwhelmed, various misfolded polypeptides including NMD-polypeptides accumulate in the aggresome: a perinuclear nonmembranous compartment eventually cleared by autophagy. Hyperphosphorylation of the key NMD factor UPF1 is required for selective targeting of the misfolded polypeptide aggregates toward the aggresome via the CTIF-eEF1A1-DCTN1 complex: the aggresome-targeting cellular machinery. Visualization at a single-particle level reveals that UPF1 increases the frequency and fidelity of movement of CTIF aggregates toward the aggresome. Furthermore, the apoptosis induced by proteotoxic stresses is suppressed by UPF1 hyperphosphorylation. Altogether, our data provide evidence that UPF1 functions in the regulation of a protein surveillance as well as an mRNA quality control.
  12. Autophagy. 2020 Jun 16. 1-3
    Lei Y, Klionsky DJ.
      A key feature of macroautophagy (hereafter autophagy) is the formation of the phagophore, a double-membrane compartment sequestering cargos and finally maturing into a vesicle termed an autophagosome; however, where these membranes originate from is not clear. In a previous study, researchers from the Rubinsztein lab proposed a model in which the autophagosome can evolve from the RAB11A-positive recycling endosome. In their recent paper, they determine that DNM2 (dynamin 2) functions in scission of the recycling endosome, and the release of the autophagosome precursor. These findings explain how the centronuclear myopathy (CNM) mutation in DNM2 results in the accumulation of immature autophagic structures.
    Keywords:  Autophagosome; RAB11; centronuclear myopathy; dynamin 2; recycling endosome; scission
  13. Cell Death Discov. 2020 ;6 45
    Obergasteiger J, Frapporti G, Lamonaca G, Pizzi S, Picard A, Lavdas AA, Pischedda F, Piccoli G, Hilfiker S, Lobbestael E, Baekelandt V, Hicks AA, Corti C, Pramstaller PP, Volta M.
      The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.
    Keywords:  Cellular neuroscience; Macroautophagy
  14. Cell Chem Biol. 2020 Jun 18. pii: S2451-9456(20)30193-8. [Epub ahead of print]27(6): 637-639
    Au YZ, Wang T, Sigua LH, Qi J.
      Qu et al. (2020) demonstrate a peptide-induced targeted degradation of the alpha-synuclein protein, a hallmark of Parkinson's disease. Using a modular three-component design, the target-protein-specific, cell-permeable peptide disposed of alpha-synuclein via the ubiquitin-proteasome pathway rather than the standard autophagy-lysosome pathway.
  15. Autophagy. 2020 Jun 16. 1-2
    Wen X, Klionsky DJ.
      There is a type of noncanonical autophagy, which is independent of ATG5 (autophagy related 5), also referred to as alternative autophagy. Both canonical and ATG5-independent alternative autophagy require the initiator ULK1 (unc-51 like kinase 1), but how ULK1 regulates these two types of autophagy differently remains unclear. A recent paper from Torii et al. demonstrates that phosphorylation of ULK1 at Ser746 by RIPK3 (receptor interacting serine/threonine kinase 3) is the key difference between these two types of autophagy; this phosphorylation is exclusively found during alternative autophagy.
    Keywords:  Alternative autophagy; Golgi; RIPK3; ULK1; phosphorylation
  16. Autophagy. 2020 Jun 19.
    Mookherjee D, Das S, Mukherjee R, Bera M, Jana SC, Chakrabarti S, Chakrabarti O.
      Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate "mitoplasts". This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The "mitoplasts" generated are degraded by reticulo-mito-phagy - simultaneously affecting dual organelle turnover.
    Keywords:  AMFR/GP78; OPA1; RETREG1/FAM134B; autophagy; mitoplast; reticulo-mito-phagy
  17. Autophagy. 2020 Jun 16. 1-2
    Torii S, Shimizu S.
      Alternative autophagy is an ATG5 (autophagy related 5)-independent, Golgi membrane-derived form of macroautophagy. ULK1 (unc-51 like kinase 1) is an essential initiator not only for canonical autophagy but also for alternative autophagy. However, the mechanism as to how ULK1 differentially regulates both types of autophagy has remained unclear. Recently, we identified a novel phosphorylation site of ULK1 at Ser746, which is required for alternative autophagy, but not canonical autophagy. We also identify RIPK3 (receptor-interacting serine-threonine kinase 3) as the kinase responsible for genotoxic stress-induced ULK1 S746 phosphorylation. These findings indicate that RIPK3-dependent ULK1 S746 phosphorylation plays a pivotal role in genotoxic stress-induced alternative autophagy.
    Keywords:  Alternative autophagy; RIPK3; ULK1; necroptosis; phosphorylation
  18. Biochem Biophys Res Commun. 2020 Jun 15. pii: S0006-291X(20)31044-5. [Epub ahead of print]
    Morshed S, Shibata T, Naito K, Miyasato K, Takeichi Y, Takuma T, Tasnin MN, Ushimaru T.
      The yeast E2F functional homologs MBF (Mbp1/Swi6) and SBF (Swi4/Swi6) complexes are critical transcription factors for G1/S transition. The target of rapamycin complex 1 (TORC1) kinase promotes G1/S transition via upregulation of the G1 cyclin Cln3 that activates MBF and SBF in favorable nutrient conditions. Here, we show evidence that TORC1 directly regulates G1/S transition via MBF and SBF. Various proteins involved in G1/S transition, including Mbp1 and Swi4, but not Swi6, were largely lost after rapamycin treatment. TORC1 inactivation facilitated degradation of Mbp1 and Swi4. Mbp1 degradation was dependent on Skp1-Cullin1-F-box (SCF)-Grr1 and proteasomes. We identified a PEST-like degron in Mbp1. Mutant cells with an unstable Mbp1 protein were hypersensitive to rapamycin and more accumulated G1 cells in the absence and presence of rapamycin. This study revealed that TORC1 directly controls MBF/SBF-mediated G1/S transition in response to nutrient availability.
    Keywords:  E2F; MBF; Mbp1; SBF; Swi4; TORC1
  19. Front Cell Dev Biol. 2020 ;8 343
    Morel E.
      Autophagy mobilizes a variety of intracellular endomembranes to ensure a proper stress response and the maintenance of cellular homeostasis. While the process of de novo biogenesis of pre-autophagic structures is not yet fully characterized, the role of the endoplasmic reticulum (ER) appears to be crucial in early steps of autophagic process. Here, I review and discuss various aspects of ER and ER-driven membrane contact site requirements and effects on mammalian organelles and endomembrane biogenesis, in particular during the early steps of autophagy-related membrane dynamics.
    Keywords:  ER; autophogosome; biogenesis; lipids; membrane contact site
  20. Cells. 2020 Jun 16. pii: E1468. [Epub ahead of print]9(6):
    Sanwald JL, Poschmann G, Stühler K, Behrends C, Hoffmann S, Willbold D.
      The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP-which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells-we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP's proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.
    Keywords:  APEX2; ATG8; GABARAP; autophagy; exosome; extracellular vesicle; proximity; secretion
  21. Mol Neurobiol. 2020 Jun 16.
    Ahsan AU, Sharma VL, Wani A, Chopra M.
      Deposition of an amyloid-β peptide is one of the first events in the pathophysiology of Alzheimer's disease (AD) and is clinically characterized by Aβ plaques, tau tangles, and behavioral impairments that lead to neuronal death. A substantial number of studies encourage targeting the skewness in the production and degradation of amyloid-β could be among the promising therapies in the disease. Neuronal autophagy has emerged for an essential role in the degradation of such toxic aggregate-prone proteins in various neurodegenerative diseases. We profiled a small library of common dietary compounds and identified those that can enhance autophagy in neuronal cells. Here we noted naringenin in silico exhibits a robust affinity with AMP-activated protein kinase (AMPK) and upregulated AMPK-mediated autophagy signaling in neurons. Naringenin can induce autophagy promoting proteins such as ULK1, Beclin1, ATG5, and ATG7 in Neuro2a cells and primary mouse neurons as well. The knockdown of AMPK by siRNA-AMPK was complemented by naringenin that restored transcript levels of AMPK. Further, naringenin can reduce the levels of Aβ at a nontoxic concentration from neuronal cells. Moreover, it maintained the mitochondrial membrane potential and resisted reactive oxygen species production, which led to the protection against Aβ1-42 evoked neurotoxicity. This highlights the neuroprotective potential of naringenin that can be developed as an anti-amyloidogenic nutraceutical.
    Keywords:  AICAR; AMPK; Aβ; GFAP; LC3B; MAP 2; Naringenin; ULK1; mTOR
  22. Cell Death Dis. 2020 Jun 15. 11(6): 460
    Biel TG, Aryal B, Gerber MH, Trevino JG, Mizuno N, Rao VA.
      Disrupting functional protein homeostasis is an established therapeutic strategy for certain tumors. Ongoing studies are evaluating autophagy inhibition for overcoming chemotherapeutic resistance to such therapies by neutralizing lysosomal pH. New and sensitive methods to monitor autophagy in patients are needed to improve trial design and interpretation. We report that mitochondrial-damaged breast cancer cells and rat breast tumors accumulate p53-positive protein aggregates that resist lysosomal degradation. These aggregates were localized to enzymatically-active autolysosomes that were degrading autophagosomes and the autophagic receptor proteins TAX1BP1 and NDP52. NDP52 was identified to associate with aggregated proteins and knocking down NDP52 led to the accumulation of protein aggregates. TAX1BP1 was identified to partly localize with aggregates, and knocking down TAX1BP1 enhanced aggregate formation, suppressed autophagy, impaired NDP52 autophagic degradation and induced cell death. We propose that quantifying aggregates and autophagic receptors are two potential methods to evaluate autophagy and lysosomal degradation, as confirmed using primary human tumor samples. Collectively, this report establishes protein aggregates and autophagy receptors, TAX1BP1 and NDP52, as potential endpoints for monitoring autophagy during drug development and clinical studies.
  23. Plant Physiol. 2020 Jun 17. pii: pp.00237.2020. [Epub ahead of print]
    Kikuchi Y, Nakamura S, Woodson JD, Ishida H, Ling Q, Hidema J, Jarvis P, Hagihara S, Izumi M.
      Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. Although ubiquitination acts as a signal inducing organelle-targeting autophagy, the interaction between ubiquitination and autophagy in chloroplast turnover has not been addressed. In this study, we found that two chloroplast-associated E3 enzymes, SUPPRESSOR OF PPI1 LOCUS 1 (SP1) and PLANT U-BOX 4 (PUB4), are not necessary for the induction of either piecemeal autophagy of chloroplast stroma or chlorophagy of whole damaged chloroplasts in Arabidopsis (Arabidopsis thaliana). Double mutations of an autophagy gene and PUB4 caused synergistic phenotypes relative to single mutations. The double mutants developed accelerated leaf chlorosis linked to the overaccumulation of reactive oxygen species (ROS) during senescence and had reduced seed production. Biochemical detection of ubiquitinated proteins indicated that both autophagy and PUB4-associated ubiquitination contributed to protein degradation in the senescing leaves. Furthermore, the double mutants had enhanced susceptibility to carbon or nitrogen starvation relative to single mutants. Together, these results indicate that autophagy and chloroplast-associated E3s cooperate for protein turnover, management of ROS accumulation, and adaptation to starvation.
  24. J Mol Cell Cardiol. 2020 Jun 14. pii: S0022-2828(20)30213-3. [Epub ahead of print]
    Qi L, Zang H, Wu W, Nagarkatti P, Nagarkatti M, Liu Q, Robbins J, Wang X, Cui T.
      Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.
    Keywords:  Autolysosome efflux; CYLD; Cardiomyocytes; Deubiquitinating enzymes; Pressure overload
  25. Autophagy. 2020 Jun 16.
    Chu Y, Kang Y, Yan C, Yang C, Zhang T, Huo H, Liu Y.
      Macroautophagy/autophagy is a membrane-mediated intracellular degradation pathway, through which bulky cytoplasmic content is digested in lysosomes. How the autophagy initiation and maturation steps are regulated is not clear. In this study, we found an E3 ubiquitin ligase complex, linear ubiquitin chain assembly complex (LUBAC) and a de-ubiquitinating enzyme (DUB) OTULIN localize to the phagophore area to control autophagy initiation and maturation. LUBAC key component RNF31/HOIP translocates to the LC3 puncta area when autophagy is induced. RNF31 knockdown inhibits autophagy initiation, and cells are more sensitive to bacterial infection. OTULIN knockdown, however, promotes autophagy initiation but blocks autophagy maturation. In OTULIN knockdown cells, excessive ubiquitinated ATG13 protein was recruited to the phagophore for prolonged expansion, and therefore inhibits autophagosome maturation. Together, our study provides evidence that LUBAC and OTULIN cooperatively regulate autophagy initiation and autophagosome maturation by mediating the linear ubiquitination and the stabilization of ATG13.
    Keywords:  ATG13; LUBAC; OTULIN; RNF31; autophagosome; autophagy; linear ubiquitination
  26. Sci Rep. 2020 Jun 15. 10(1): 9653
    Barthez M, Poplineau M, Elrefaey M, Caruso N, Graba Y, Saurin AJ.
      Autophagy is an essential cellular process that maintains homeostasis by recycling damaged organelles and nutrients during development and cellular stress. ZKSCAN3 is the sole identified master transcriptional repressor of autophagy in human cell lines. How ZKSCAN3 achieves autophagy repression at the mechanistic or organismal level however still remains to be elucidated. Furthermore, Zkscan3 knockout mice display no discernable autophagy-related phenotypes, suggesting that there may be substantial differences in the regulation of autophagy between normal tissues and tumor cell lines. Here, we demonstrate that vertebrate ZKSCAN3 and Drosophila M1BP are functionally homologous transcription factors in autophagy repression. Expression of ZKSCAN3 in Drosophila prevents premature autophagy onset due to loss of M1BP function and conversely, M1BP expression in human cells can prevent starvation-induced autophagy due to loss of nuclear ZKSCAN3 function. In Drosophila ZKSCAN3 binds genome-wide to sequences targeted by M1BP and transcriptionally regulates the majority of M1BP-controlled genes, demonstrating the evolutionary conservation of the transcriptional repression of autophagy. This study thus  allows the potential for transitioning the mechanisms, gene targets and plethora metabolic processes controlled by M1BP onto ZKSCAN3 and opens up Drosophila as a tool in studying the function of ZKSCAN3 in autophagy and tumourigenesis.
  27. J Cell Sci. 2020 Jun 16. pii: jcs.241356. [Epub ahead of print]
    Tamir TY, Bowman BM, Agajanian MJ, Goldfarb D, Schrank TP, Stohrer T, Hale AE, Siesser PF, Weir SJ, Murphy RM, LaPak KM, Weissman BE, Moorman NJ, Major MB.
      NFE2L2/NRF2 is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, and conversely NRF2 activity is diminished with age and in neurodegenerative as well as metabolic disorders. Though NRF2 activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here we used a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the understudied protein kinase Brain Specific Kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives AMPK signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppressed ribosome-RNA complexes, global protein synthesis, and NRF2 protein levels. Collectively our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis may prove useful for therapeutically targeting NRF2 in human disease.
    Keywords:  AMPK; BRSK1; BRSK2; Functional genomics; Kinase; MTOR; NRF2; Oxidative stress response; Phosphoproteomics
  28. Oncogene. 2020 Jun 15.
    Shang C, Zhou H, Liu W, Shen T, Luo Y, Huang S.
      The mammalian target of rapamycin (mTOR) functions as two complexes (mTORC1 and mTORC2), regulating cell growth and metabolism. Aberrant mTOR signaling occurs frequently in cancers, so mTOR has become an attractive target for cancer therapy. Iron chelators have emerged as promising anticancer agents. However, the mechanisms underlying the anticancer action of iron chelation are not fully understood. Particularly, reports on the effects of iron chelation on mTOR complexes are inconsistent or controversial. Here, we found that iron chelators consistently inhibited mTORC1 signaling, which was blocked by pretreatment with ferrous sulfate. Mechanistically, iron chelation-induced mTORC1 inhibition was not related to ROS induction, copper chelation, or PP2A activation. Instead, activation of AMPK pathway mainly and activation of both HIF-1/REDD1 and Bnip3 pathways partially contribute to iron chelation-induced mTORC1 inhibition. Our findings indicate that iron chelation inhibits mTORC1 via multiple pathways and iron is essential for mTORC1 activation.