bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒08‒30
thirty-six papers selected by
Viktor Korolchuk
Newcastle University

  1. Genes (Basel). 2020 Aug 25. pii: E989. [Epub ahead of print]11(9):
    Melick CH, Jewell JL.
      The mammalian target of rapamycin (mTOR) is an evolutionary conserved Ser/Thr protein kinase that senses multiple upstream stimuli to control cell growth, metabolism, and autophagy. mTOR is the catalytic subunit of mTOR complex 1 (mTORC1). A significant amount of research has uncovered the signaling pathways regulated by mTORC1, and the involvement of these signaling cascades in human diseases like cancer, diabetes, and ageing. Here, we review advances in mTORC1 regulation by upstream stimuli. We specifically focus on how growth factors, amino acids, G-protein coupled receptors (GPCRs), phosphorylation, and small GTPases regulate mTORC1 activity and signaling.
    Keywords:  G-protein coupled receptors; amino acids; and autophagy; cell growth; kinases; mTORC1; metabolism; phosphorylation; small GTPases
  2. Int J Mol Sci. 2020 Aug 25. pii: E6130. [Epub ahead of print]21(17):
    Kim H, Jeon BT, Kim IM, Bennett SJ, Lorch CM, Viana MP, Myers JF, Trupp CJ, Whipps ZT, Kundu M, Chung S, Sun X, Khalimonchuk O, Lee J, Ro SH.
      Selective autolysosomal degradation of damaged mitochondria, also called mitophagy, is an indispensable process for maintaining integrity and homeostasis of mitochondria. One well-established mechanism mediating selective removal of mitochondria under relatively mild mitochondria-depolarizing stress is PINK1-Parkin-mediated or ubiquitin-dependent mitophagy. However, additional mechanisms such as LC3-mediated or ubiquitin-independent mitophagy induction by heavy environmental stress exist and remain poorly understood. The present study unravels a novel role of stress-inducible protein Sestrin2 in degradation of mitochondria damaged by transition metal stress. By utilizing proteomic methods and studies in cell culture and rodent models, we identify autophagy kinase ULK1-mediated phosphorylation sites of Sestrin2 and demonstrate Sestrin2 association with mitochondria adaptor proteins in HEK293 cells. We show that Ser-73 and Ser-254 residues of Sestrin2 are phosphorylated by ULK1, and a pool of Sestrin2 is strongly associated with mitochondrial ATP5A in response to Cu-induced oxidative stress. Subsequently, this interaction promotes association with LC3-coated autolysosomes to induce degradation of mitochondria damaged by Cu-induced ROS. Treatment of cells with antioxidants or a Cu chelator significantly reduces Sestrin2 association with mitochondria. These results highlight the ULK1-Sestrin2 pathway as a novel stress-sensing mechanism that can rapidly induce autophagic degradation of mitochondria under severe heavy metal stress.
    Keywords:  ATP5A; Sestrin2; ULK1; autophagy; mitochondria; phosphorylation
  3. Mol Cell Neurosci. 2020 Aug 21. pii: S1044-7431(20)30162-7. [Epub ahead of print] 103539
    Foster AD, Downing P, Figredo E, Polain N, Stott A, Layfield R, Rea SL.
      Mutations affecting SQSTM1 coding for p62 and TANK-Binding Kinase 1 (TBK1) have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TBK1 is a serine-threonine kinase that regulates p62's activity as an autophagy receptor via phosphorylation and also has roles in neuroinflammatory signalling pathways. The mechanisms underlying ALS and FTLD pathogenesis as a result of TBK1 mutations are incompletely understood, however, loss of TBK1 function can lead to dysregulated autophagy and mitophagy. Here, we report that an ALS-associated TBK1 variant affecting the kinase domain, p.G175S, is defective in phosphorylation of p62 at Ser-403, a modification critical for regulating its ubiquitin-binding function, as well as downstream phosphorylation at Ser-349. Consistent with these findings, expression of p.G175S TBK1 was associated with decreased induction of autophagy compared to wild type and reduced degradation of the ALS-linked protein TDP-43. Expression of wild type TBK1 increased NF-κB signalling ~300 fold in comparison to empty vector cells, whereas p.G175S TBK1 was unable to promote NF-κB signalling above levels observed in empty vector transfected cells. We also noted a hitherto unknown role for TBK1 as a suppressor of oxidative stress (Nrf2) signalling and show that p.G175S TBK1 expressing cells lose this inhibitory function. Our data suggest that TBK1 ALS mutations may broadly impair p62-mediated cell signalling, which ultimately may reduce neuronal survival, in addition TDP-43 was not efficiently degraded, together these effects may contribute to TBK1 mutation associated ALS and FTLD pathogenesis.
    Keywords:  ALS-FTLD; Autophagy; Cell signalling; Mitophagy; TBK1; p62/SQSTM1
  4. Bioessays. 2020 Aug 26. e2000122
    Ueno T, Komatsu M.
      Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi-step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome-dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been developed. In these assays, autophagosome marker proteins, microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) and gamma-aminobutyric acid receptor-associated protein-II (GABARAP-II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3-II and/or GABARAP-II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure.
    Keywords:  autolysosome; autophagosome; autophagy; autophagy-flux; gamma-aminobutyric acid receptor-associated protein; microtubule-associated proteins 1A/1B light chain 3B
  5. Front Cell Dev Biol. 2020 ;8 733
    Assar EA, Tumbarello DA.
      Autophagy is an essential catabolic intracellular pathway that maintains homeostasis by degrading long-lived proteins, damaged organelles, and provides an energy source during nutrient starvation. It is now understood that autophagy has discrete functions as a selective lysosomal degradation pathway targeting large cytosolic structural and signaling complexes to influence cell motility and adhesion. We provide evidence suggesting the primary autophagy regulators Atg5 and FIP200 both play a role in cell motility and extracellular matrix adhesion. However, their loss of function has a differential impact on focal adhesion composition and organization, as well as signaling in response to fibronectin induced cell spreading. This differential impact on focal adhesions is illustrated by smaller focal adhesion complexes and a decrease in FAK, paxillin, and vinculin expression associated with FIP200 loss of function. In contrast, Atg5 loss of function results in production of large and stable focal adhesions, characterized by their retention of phosphorylated FAK and Src, which correlates with increased vinculin and FAK protein expression. Importantly, autophagy is upregulated during processes associated with focal adhesion reorganization and their exhibits colocalization of autophagosomes with focal adhesion cargo. Interestingly, FIP200 localizes to vinculin-rich focal adhesions and its loss negatively regulates FAK phosphorylation. These data collectively suggest FIP200 and Atg5 may have both autophagy-dependent and -independent functions that provide distinct mechanisms and impacts on focal adhesion dynamics associated with cell motility.
    Keywords:  Src; actin cytoskeleton; autophagy; cell adhesion; focal adhesion kinase
  6. Prog Mol Biol Transl Sci. 2020 ;pii: S1877-1173(20)30037-5. [Epub ahead of print]174 263-305
    Popelka H.
      Autophagy is a major catabolic pathway that must be tightly regulated to maintain cellular homeostasis. Protein intrinsic disorder provides a very suitable conformation for regulation; accordingly, the molecular machinery of autophagy is significantly enriched in intrinsically disordered proteins and protein regions (IDPs/IDPRs). Despite experimental challenges that the characterization of IDPRs encounters, remarkable progress has been made in recent years in revealing various roles of IDPs/IDPRs in autophagy. This chapter describes the autophagy pathway from a specific point of view, that of IDPRs. It focuses in detail on structural and mechanistic functions in autophagy that are executed by disordered regions. Via a description of autophagosome biogenesis, linking the cargo to the autophagy machinery, as well as a discussion of certain post-translational regulations, this review reveals many indispensable roles of IDPRs in the functional autophagy pathway. Devastating pathologies such as neurodegeneration, cancer, or diabetes have been linked to a malfunction in IDPs/IDPRs. The same pathologies are associated with dysfunctional autophagy, indicating that autophagic IDPRs may be a paramount causative factor. Several disease-related mechanisms of the autophagy pathway involving protein intrinsic disorder are reported in this chapter, to illustrate a wide-ranging potential of IDPRs in the therapeutic modulation of autophagy.
    Keywords:  AIM/LIR; Atg8; Beclin 1; Human disease; Membrane; Pathogen
  7. Trends Biochem Sci. 2020 Aug 19. pii: S0968-0004(20)30192-4. [Epub ahead of print]
    Abildgaard MH, Brynjólfsdóttir SH, Frankel LB.
      Autophagy is a highly conserved degradation pathway that ensures nutrient recycling and removal of unwanted substrates. This process has a fundamental role in stress adaptation and maintenance of cellular homeostasis. Here, we discuss emerging aspects of the autophagy-RNA interplay, including autophagy-mediated degradation of RNA, RNA-binding proteins (RBPs), and ribonucleoprotein (RNP) complexes. Beyond degradation, we review new roles for autophagy players in the secretion and intracellular transport of RNA and related complexes. We discuss the physiological importance of these events for RNA homeostasis and gene expression programs, as well as their implications for disease, including cancer and neurodegeneration. Lastly, we examine how post-transcriptional regulation of autophagy, through specialized processing and selective translation of key transcripts, challenges and updates our current view of autophagy complexity.
    Keywords:  RNA homeostasis; RNA-binding proteins; autophagy; degradation; post-transcriptional regulation; translation
  8. Front Cell Dev Biol. 2020 ;8 715
    Kitada M, Xu J, Ogura Y, Monno I, Koya D.
      Nutrients are closely involved in the regulation of lifespan and metabolic health. Cellular activities, such as the regulation of metabolism, growth, and aging, are mediated by a network of nutrients and nutrient-sensing pathways. Among the nutrient-sensing pathways, the mechanistic target of rapamycin complex 1 (mTORC1) acts as the central regulator of cellular functions, which include autophagy. Autophagy plays a significant role in the removal of protein aggregates and damaged or excess organelles, including mitochondria, to maintain intracellular homeostasis, which is involved in lifespan extension and cardiometabolic health. Moreover, dietary methionine restriction may have a beneficial effect on lifespan extension and metabolic health. In contrast, methionine may activate mTORC1 and suppress autophagy. As the mechanism of methionine sensing on mTORC1, SAMTOR was identified as a sensor of S-adenosyl methionine (SAM), a metabolite of methionine, in the cytoplasm. Conversely, methionine may activate the mTORC1 signaling pathway through the activation of phosphatase 2A (PP2A) because of increased methylation in response to intracellular SAM levels. In this review, we summarized the recent findings regarding the mechanism via which methionine activates mTORC1.
    Keywords:  S-adenosyl methionine; SAMTOR; autophagy; mechanistic target of rapamycin complex 1; methionine; phosphatase 2A methylation
  9. J Lipid Res. 2020 Aug 26. pii: jlr.RA120000895. [Epub ahead of print]
    Bruiners N, Dutta N, Guerrini V, Salamon H, Yamaguchi KD, Karakousis PC, Gennaro ML.
      The rise of drug-resistant tuberculosis (TB) poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each essential branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, AMPK- mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.
    Keywords:  Cholesterol/Trafficking; Immunology; Lipids; Macrophages / monocytes; Mycobacterium tuberculosis; Statins; mTORC1 regulation
  10. Int Rev Neurobiol. 2020 ;pii: S0074-7742(20)30017-9. [Epub ahead of print]155 169-202
    Aman Y, Ryan B, Torsetnes SB, Knapskog AB, Watne LO, McEwan WA, Fang EF.
      Neurodegenerative diseases are highly debilitating illnesses and a growing cause of morbidity and mortality worldwide. Mitochondrial dysfunction and impairment of mitochondrial-specific autophagy, namely mitophagy, have emerged as important components of the cellular processes underlying neurodegeneration. Defective mitophagy has been highlighted as the cause of the accumulation of damaged mitochondria, which consequently leads to cellular dysfunction and/or death in neurodegenerative diseases. Here, we highlight the recent advances in the molecular mechanisms of mitochondrial homeostasis and mitophagy in neurodegenerative diseases. In particular, we evaluate how mitophagy is altered in Alzheimer's, Parkinson's, and Huntington's diseases, as well as in amyotrophic lateral sclerosis, and the potential of restoring mitophagy as a therapeutic intervention. We also discuss the interlinked connections between mitophagy and innate immunity (e.g., the involvement of Parkin, interferons and TRIM21) as well as the opportunity these pathways provide to develop combinational therapeutic strategies targeting them and related molecular mechanisms in such neurodegenerative diseases.
    Keywords:  Alzheimer's disease; Amyotrophic lateral sclerosis; Autophagy; Huntington's disease; Innate immunity; Mitophagy; Parkinson's disease
  11. Autophagy. 2020 Aug 24. 1-15
    Lauterbach MA, Saavedra V, Mangan MSJ, Penno A, Thiele C, Latz E, Kuerschner L.
      1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system.ABBREVIATIONS: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N3635P: azido-STAR635P; N3Cy3: azido-cyanine 3; N3picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.
    Keywords:  Autophagy; HSAN1; crystal; doxSA; innate immunity; lipid; macrophage
  12. Front Cell Dev Biol. 2020 ;8 727
    Banerjee C, Roy M, Mondal R, Chakraborty J.
      In the recent past, many of the deubiquitinases (DUB) were found to modulate mitochondrial clearance or mitophagy and thus they are currently projected as therapeutic targets against neurodegeneration. Among these DUBs, USP14 stands at a distinctive juncture, since it can influence both proteasome complex activity and autophagy process. USP14 interference can enhance mitochondrial clearance and thus can protect Parkinsonian phenotypes in Drosophila model. However, in higher animal models of neurodegenerative disorders, evaluation of the protective role of USP14 is yet to be done. In this perspective, we pointed out a few of the major considerations that should be classified before designing experiments to evaluate the therapeutic potential of this DUB in rodent models of neurodegeneration. These are mainly: level of USP14 in the concerned brain region and how the level alters in the model system. Because USP14 mediated mitophagy is Prohibitin2 dependent, the anticipated impact of this protein in this aspect is also discussed. To illustrate our view, we show that USP14 levels increases in adult rat brain substantia nigra (SN) and cerebellum compared to the young ones. We also depict that rotenone treatment can immediately lead to increased SN specific USP14 levels. Our perception thus portrays USP14 as a therapeutic target, especially for addressing SN specific neurodegeneration in adult rat brain, but may vary with the disease model.
    Keywords:  3-nitropropionic acid; Parkinson’s disease; Prohibitin2; USP14; mitophagy; neurodegeneration; rotenone; substantia nigra
  13. Proc Natl Acad Sci U S A. 2020 Aug 24. pii: 201913177. [Epub ahead of print]
    Zhou Z, Xu H, Li Y, Yang M, Zhang R, Shiraishi A, Kiyonari H, Liang X, Huang X, Wang Y, Xie Q, Liu S, Chen R, Bao L, Guo W, Wang Y, Meng W.
      The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.
    Keywords:  CAMSAP1; MARK2; cell migration; neuronal polarity; noncentrosomal microtubules
  14. Cell Rep. 2020 Aug 25. pii: S2211-1247(20)31064-0. [Epub ahead of print]32(8): 108079
    Tur J, Pereira-Lopes S, Vico T, Marín EA, Muñoz JP, Hernández-Alvarez M, Cardona PJ, Zorzano A, Lloberas J, Celada A.
      Mitofusin 2 (Mfn2) plays a major role in mitochondrial fusion and in the maintenance of mitochondria-endoplasmic reticulum contact sites. Given that macrophages play a major role in inflammation, we studied the contribution of Mfn2 to the activity of these cells. Pro-inflammatory stimuli such as lipopolysaccharide (LPS) induced Mfn2 expression. The use of the Mfn2 and Mfn1 myeloid-conditional knockout (KO) mouse models reveals that Mfn2 but not Mfn1 is required for the adaptation of mitochondrial respiration to stress conditions and for the production of reactive oxygen species (ROS) upon pro-inflammatory activation. Mfn2 deficiency specifically impairs the production of pro-inflammatory cytokines and nitric oxide. In addition, the lack of Mfn2 but not Mfn1 is associated with dysfunctional autophagy, apoptosis, phagocytosis, and antigen processing. Mfn2floxed;CreLysM mice fail to be protected from Listeria, Mycobacterium tuberculosis, or LPS endotoxemia. These results reveal an unexpected contribution of Mfn2 to ROS production and inflammation in macrophages.
    Keywords:  apoptotic bodies; inflammation; lipopolysaccharide (LPS); macrophages; mitochondria; mitofusin; phagocytosis; protein degradation; reactive oxygen species (ROS); septic shock
  15. Biochem Biophys Res Commun. 2020 Sep 10. pii: S0006-291X(20)31471-6. [Epub ahead of print]530(1): 285-291
    Kikuchi N, Soga T, Nomura M, Sato T, Sakamoto Y, Tanaka R, Abe J, Morita M, Shima H, Okada Y, Tanuma N.
      Recent advances in cancer biology reveal the importance of metabolic changes in cancer; however, less is known about how metabolic pathways in tumors are regulated in vivo. Here, we report analysis of the lung cancer metabolism based on different surgical procedures, namely lobectomy and partial resection. In lobectomy, but not in partial resection, pulmonary arteries and veins are ligated prior to removal of tissues, rendering tissues ischemic. We show that tumors indeed undergo ischemia upon lobectomy and that the tumor metabolome differs markedly from that of tumors removed by partial resection. Comparison of the responses to ischemia in tumor and normal lung tissues revealed that lung cancer tissue exhibits greater TCA cycle and autophagic activity than do normal lung tissues in vivo in patients. Finally, we report that deleting ATG7, which encodes a protein essential for autophagy, antagonizes growth of tumors derived from lung cancer cell lines, suggesting that autophagy confers metabolic advantages to lung cancer. Our findings shed light on divergent metabolic responses to ischemia seen in tumors and normal tissues.
    Keywords:  Autophagy; Cancer metabolism; Ischemia; Lung cancer; Metabolome
  16. Int Rev Neurobiol. 2020 ;pii: S0074-7742(20)30009-X. [Epub ahead of print]155 1-35
    Maiese K.
      Non-communicable diseases (NCDs) that involve neurodegenerative disorders and metabolic disease impact over 400 million individuals globally. Interestingly, metabolic disorders, such as diabetes mellitus, are significant risk factors for the development of neurodegenerative diseases. Given that current therapies for these NCDs address symptomatic care, new avenues of discovery are required to offer treatments that affect disease progression. Innovative strategies that fill this void involve the mechanistic target of rapamycin (mTOR) and its associated pathways of mTOR complex 1 (mTORC1), mTOR complex 2 (mTORC2), AMP activated protein kinase (AMPK), trophic factors that include erythropoietin (EPO), and the programmed cell death pathways of autophagy and apoptosis. These pathways are intriguing in their potential to provide effective care for metabolic and neurodegenerative disorders. Yet, future work is necessary to fully comprehend the entire breadth of the mTOR pathways that can effectively and safely translate treatments to clinical medicine without the development of unexpected clinical disabilities.
    Keywords:  AMPK; Alzheimer's disease; Apoptosis; Autophagy; Dementia; Diabetes mellitus; Erythropoietin; mTOR; mTORC1; mTORC2
  17. J Cell Biol. 2020 Sep 07. pii: e202008031. [Epub ahead of print]219(9):
    Ikeda F.
      Mitophagy has a critical role in maintaining cellular homeostasis by removing damaged mitochondria. In this issue, Yamano et al. (2020. J. Cell Biol. uncover that a novel complex of the autophagy adaptor optineurin and the membrane protein ATG9A specifically regulate ubiquitin-induced mitophagy.
  18. Nat Commun. 2020 Aug 27. 11(1): 4286
    Sudhakar JN, Lu HH, Chiang HY, Suen CS, Hwang MJ, Wu SY, Shen CN, Chang YM, Li FA, Liu FT, Shui JW.
      Intracellular galectins are carbohydrate-binding proteins capable of sensing and repairing damaged lysosomes. As in the physiological conditions glycosylated moieties are mostly in the lysosomal lumen but not cytosol, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions. Here, we show in gut epithelial cells, galectin-9 is enriched in lysosomes and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner. At the steady state, galectin-9 binding to glycosylated Asn175 of Lamp2 is essential for functionality of lysosomes and autophagy. Loss of N-glycan-binding capability of galectin-9 causes its complete depletion from lysosomes and defective autophagy, leading to increased endoplasmic reticulum (ER) stress preferentially in autophagy-active Paneth cells and acinar cells. Unresolved ER stress consequently causes cell degeneration or apoptosis that associates with colitis and pancreatic disorders in mice. Therefore, lysosomal galectins maintain homeostatic function of lysosomes to prevent organ pathogenesis.
  19. Oxid Med Cell Longev. 2020 ;2020 2896036
    Zhang ZY, Bao XL, Cong YY, Fan B, Li GY.
      Age-related macular degeneration (AMD) is a leading cause of severe visual loss and irreversible blindness in the elderly population worldwide. Retinal pigment epithelial (RPE) cells are the major site of pathological alterations in AMD. They are responsible for the phagocytosis of shed photoreceptor outer segments (POSs) and clearance of cellular waste under physiological conditions. Age-related, cumulative oxidative stimuli contribute to the pathogenesis of AMD. Excessive oxidative stress induces RPE cell degeneration and incomplete digestion of POSs, leading to the continuous accumulation of cellular waste (such as lipofuscin). Autophagy is a major system of degradation of damaged or unnecessary proteins. However, degenerative RPE cells in AMD patients cannot perform autophagy sufficiently to resist oxidative damage. Increasing evidence supports the idea that enhancing the autophagic process can properly alleviate oxidative injury in AMD and protect RPE and photoreceptor cells from degeneration and death, although overactivated autophagy may lead to cell death at early stages of retinal degenerative diseases. The crosstalk among the NFE2L2, PGC-1, p62, AMPK, and PI3K/Akt/mTOR pathways may play a crucial role in improving disturbed autophagy and mitigating the progression of AMD. In this review, we discuss how autophagy prevents oxidative damage in AMD, summarize potential neuroprotective strategies for therapeutic interventions, and provide an overview of these neuroprotective mechanisms.
  20. J Cell Sci. 2020 Aug 25. pii: jcs.243477. [Epub ahead of print]
    Eck F, Phuyal S, Smith MD, Kaulich M, Wilkinson S, Farhan H, Behrends C.
      While studies of ATG genes in knockout models led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP proteins are still poorly understood. A major drawback for their understanding is that the available interactome data was largely acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was exemplarily employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Further, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together, we describe ACSL3 as novel regulator of the enigmatic UFM1 conjugation pathway.
    Keywords:  ACSL3; ER-phagy; GABARAPL2; Lipid droplets; UBA5; Ufmylation
  21. Free Radic Biol Med. 2020 Aug 20. pii: S0891-5849(20)31205-3. [Epub ahead of print]
    Chen L, Xia YF, Shen SF, Tang J, Chen JL, Qian K, Chen Z, Qin ZH, Sheng R.
      Previous studies have shown that syntaxin 17 (STX17) is involved in mediating the fusion of autophagosomes and lysosomes. This study aimed to investigate the role and mechanism of STX17 in neuronal injury following cerebral ischemia/reperfusion. The ischemia/reperfusion (I/R) models were established by transient middle cerebral artery occlusion (tMCAO) in mice and oxygen glucose deprivation/reperfusion (O/R) in primary cultured cortical neurons and HT22 cells. Cerebral ischemia/reperfusion significantly up-regulated the expression of STX17 in neurons. Lentivirus mediated knockdown of STX17 in neurons reduced neuronal viability and increased LDH leakage. Injection of AAV9-shSTX17 into the brain of mice then subjected to tMCAO also significantly augmented the infarct area and exacerbated neurobehavioral deficits and mortality. Depletion of STX17 caused accumulation of autophagic marker/substrate LC3 II and p62, blockade of the autophagic flux, and the accumulation of dysfunctional lysosomes. Knockdown of STX17 also aggravated endoplasmic reticulum (ER) stress-dependent neuronal apoptosis induced by ischemia/reperfusion. Importantly, induction of autophagy-lysosomal pathway and alleviation of ER stress partially rescued STX17 knockdown-induced neuronal damage. These results suggest that STX17 may ameliorate ischemia/reperfusion-induced neuronal damage by enhancing autophagy flux and reducing ER stress-dependent neuronal apoptosis.
    Keywords:  Syntaxin 17; apoptosis; autophagy; cerebral ischemia; cortical neurons; endoplasmic reticulum stress; lysosome
  22. Adv Sci (Weinh). 2020 Aug;7(16): 1903323
    Xie Y, Jiang J, Tang Q, Zou H, Zhao X, Liu H, Ma D, Cai C, Zhou Y, Chen X, Pu J, Liu P.
      The combined treatment with nanoparticles and autophagy inhibitors, such as chloroquine (CQ) and hydroxychloroquine (HCQ), is extensively explored for cancer therapy. However, the toxicity of autophagy inhibitors and their unselective for tumoricidal autophagy have seriously hindered the application of the combined treatment. In this study, a carboxy-functional iron oxide nanoparticle (Fe2O3@DMSA) is designed and identified to significantly exert an antitumor effect without adding CQ or HCQ. Further investigation indicates that the effective inhibition effect of Fe2O3@DMSA alone on hepatoma growth is triggered by inhibiting the fusion of autophagosomes and lysosomes to enhance tumoricidal autophagy, which is induced by intracellular iron-retention-induced sustained reactive oxygen species (ROS) production. Furthermore, in two hepatoma-bearing mouse models, Fe2O3@DMSA alone effectively suppresses the growth of tumors without obvious toxic side effects. These studies offer a promising strategy for cancer therapy.
    Keywords:  hepatoma therapy; iron oxide nanoparticles; iron transport systems; tumoricidal autophagy
  23. Elife. 2020 Aug 27. pii: e58396. [Epub ahead of print]9
    Stephani M, Picchianti L, Gajic A, Beveridge R, Skarwan E, Sanchez de Medina Hernandez V, Mohseni A, Clavel M, Zeng Y, Naumann C, Matuszkiewicz M, Turco E, Loefke C, Li B, Dürnberger G, Schutzbier M, Chen HT, Abdrakhmanov A, Savova A, Chia KS, Djamei A, Schaffner I, Abel S, Jiang L, Mechtler K, Ikeda F, Martens S, Clausen T, Dagdas Y.
      Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.
    Keywords:  A. thaliana; cell biology; human; plant biology; viruses
  24. Trends Biochem Sci. 2020 Aug 21. pii: S0968-0004(20)30191-2. [Epub ahead of print]
    Mizushima N, Murphy LO.
      Autophagy is a lysosome-dependent intracellular degradation system required for various physiological processes and can be dysregulated in human disease. To understand its biological significance and underlying mechanisms, measuring autophagic activity (i.e., autophagic flux) is critical. However, navigating which assays to use, and when, is complicated and at times the results are often interpreted inappropriately. This review will summarize both advantages and disadvantages of currently available methods to monitor autophagy. In addition, we discuss how these assays should be used in high-throughput screens to identify autophagy-modulating drugs and genes and the general features needed for biomarkers to assess autophagy in humans.
    Keywords:  autophagic flux; autophagy biomarkers; high-throughput screens; selective autophagy
  25. FASEB J. 2020 Aug 26.
    Bomfim L, Ramos I.
      In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.
    Keywords:  ULK1/ATG1; choriogenesis; follicle cells; vector biology
  26. Mol Cell Neurosci. 2020 Aug 20. pii: S1044-7431(20)30163-9. [Epub ahead of print] 103540
    Kido J, Nakamura K, Era T.
      Lysosomal storage diseases (LSDs) are a group of metabolism inborn errors caused by defective enzymes in the lysosome, resulting in the accumulation of undegraded substrates. Many characteristic cell features have been revealed in LSDs, including abnormal autophagy and mitochondrial dysfunction. The development of induced pluripotent stem cells (iPSCs) dramatically boosted research on LSDs, particularly regarding novel opportunities to clarify the disease etiology based on the storage of macromolecules, such as sphingolipids in lysosomes. iPSCs made from LSD patients (LSD-iPSCs) have been differentiated into neurons, endothelial cells, cardiomyocytes, hepatocytes, and macrophages, with each cell type closely resembling the primary disease phenotypes, providing new tools to probe the disease pathogenesis and to test therapeutic strategies. Abnormally accumulated substrates impaired autophagy and mitochondrial and synapse functions in LSD-iPSC-derived neurons. Reducing the accumulation with the treatment of drug candidates improved LSD-iPSC-derived neuron functions. Additionally, iPSC technology can help probe the gene expressions, proteomics, and metabolomics of LSDs. Further, gene repair and the generation of new mutations in causative genes in LSD-iPSCs can be used to understand both the specific roles of causative genes and the contributions of other genetic factors to these phenotypes. Moreover, the development of iPSC-derived organoids as disease models has bridged the gap between studies using cell lines and in vivo animal models. There are some reproducibility issues in iPSC research, however, including genetic and epigenetic abnormalities, such as chromosomal abnormalities, DNA mutations, and gene modifications via methylation. In this review, we present the disease and treatment concepts gathered using selected LSD-iPSCs, discuss iPSC research limitations, and set our future research visions. Such studies are expected to further inform and generate insights into LSDs and are important in research and clinical practice.
    Keywords:  Induced pluripotent stem cell; Lysosomal storage diseases; Neuron; Pathogenesis
  27. Hum Cell. 2020 Aug 26.
    Matsubara S, Tsukasa K, Kuwahata T, Takao S.
      CD133 expression in pancreatic cancer correlates with poor prognosis and increased metastasis. CD133+ pancreatic cancer cells exhibit cancer stem cell (CSC)-like properties. We established a CD133+ cell-rich subline from Capan-1 pancreatic cancer cells as a pancreatic CSC model and compared the effects of KU-0063794, a dual mTORC1/mTORC2 inhibitor, against those of mTORC1-specific rapamycin. We found that KU-0063794 prevents sphere formation, a self-renewal index, at high concentrations. Rapamycin inhibited sphere formation but to a lesser degree. In the present study, we aimed to determine the mechanistic roles of mTOR complex 2 (mTORC2) in maintaining CSC-like properties. By examining the PI3K/Akt/mTOR signaling pathway, we observed lower Akt phosphorylation in KU-0063794-treated cells. Phosphorylation of mTORC1 downstream effectors was inhibited by both inhibitors. Thus, mTORC2 activates Akt and modulate stem-like properties, whereas mTORC1 downstream signaling correlates directly with stem-like properties.
    Keywords:  CD133; Mechanistic/mammalian target of rapamycin (mTOR); Pancreatic cancer stem cells; mTOR complex 1 (mTORC1); mTOR complex 2 (mTORC2)
  28. Stem Cell Rev Rep. 2020 Aug 24.
    Wei R, Zhang X, Cai Y, Liu H, Wang B, Zhao X, Zou K.
      In testis, a rare undifferentiated germ cell population with the capacity to regenerate robustly and support spermatogenesis, is defined as spermatogonial progenitor cells (SPCs) population. As a widely used drug for tumor therapy or bone marrow transplantation, busulfan has a severe side effect on SPCs population and causes a consequent infertility. Recently, accumulating evidence revealed the protective role of autophagy in stem cell maintenance under exogenous stress. To better understand the role of autophagy in SPCs fates, we investigated the potential function of autophagy in SPCs under busulfan stress, and found that treatment of busulfan induced the formation of autophagic vesicles and autophagosomes in mouse SPCs. Subsequently, a connection of autophagy and SPCs maintenance and survival was demonstrated in a dose-dependent manner. Moreover, mTOR was identified as an essential factor for autophagy in SPCs with a complicated mechanism: (1) mTOR is phosphorylated by AKT to activate its target genes, p70s6 kinase, resulting in the inhibition of autophagy during short-term busulfan treatment. (2) mTOR mediates autophagy with p53 together, to regulate the fate of SPCs. Collectively, observations from this study indicate that moderate autophagy effectively protects SPCs from the stress of chemotherapy, which may provide an important hint for fertility protection in clinic.
    Keywords:  Apoptosis; Busulfan; Cell fate; Differential gene expression; Fertility
  29. Sci Adv. 2020 Aug;6(33): eabb9036
    Heckmann BL, Teubner BJW, Boada-Romero E, Tummers B, Guy C, Fitzgerald P, Mayer U, Carding S, Zakharenko SS, Wileman T, Green DR.
      Noncanonical functions of autophagy proteins have been implicated in neurodegenerative conditions, including Alzheimer's disease (AD). The WD domain of the autophagy protein Atg16L is dispensable for canonical autophagy but required for its noncanonical functions. Two-year-old mice lacking this domain presented with robust β-amyloid (Aβ) pathology, tau hyperphosphorylation, reactive microgliosis, pervasive neurodegeneration, and severe behavioral and memory deficiencies, consistent with human disease. Mechanistically, we found this WD domain was required for the recycling of Aβ receptors in primary microglia. Pharmacologic suppression of neuroinflammation reversed established memory impairment and markers of disease pathology in this novel AD model. Therefore, loss of the Atg16L WD domain drives spontaneous AD in mice, and inhibition of neuroinflammation is a potential therapeutic approach for treating neurodegeneration and memory loss. A decline in expression of ATG16L in the brains of human patients with AD suggests the possibility that a similar mechanism may contribute in human disease.
  30. PLoS One. 2020 ;15(8): e0235551
    Kobylarz MJ, Goodwin JM, Kang ZB, Annand JW, Hevi S, O'Mahony E, McAllister G, Reece-Hoyes J, Wang Q, Alford J, Russ C, Lindeman A, Beibel M, Roma G, Carbone W, Knehr J, Loureiro J, Antczak C, Wiederschain D, Murphy LO, Menon S, Nyfeler B.
      VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.
  31. Biochem Biophys Res Commun. 2020 Sep 10. pii: S0006-291X(20)31426-1. [Epub ahead of print]530(1): 292-300
    Li M, Jia J, Zhang X, Dai H.
      Mitophagy regulates the metabolic level and cell fates by specifically degrading damaged or redundant mitochondria in these cells. During the formation of autophagosomes, autophagy receptors and adaptors, which usually contain a LC3-interacting region (LIR) domain, are recruited through their interactions with LC3/GABARAP family of proteins. Bcl-rambo is one of the mitophagy receptors that interact with LC3s/GABARAPs. In this study, we first measured the binding of Bcl-rambo to LC3s/GABARAPs in vitro and found Bcl-rambo has a selectivity to LC3C/GABARP/GABARAPL1. Further investigations with bioinformatics analyses and mutagenesis suggested that the interactions with the HP1 and HP2 sites of LC3s/GABARAPs and the residues at the X2 site of the LIR domain of Bcl-rambo are both critical for the selectivity. Moreover, assays in vivo showed that manipulating the selective binding of Bcl-rambo resulted in the changes of mitophagy inductions in the cells. Overall, our data revealed the selective binding between Bcl-rambo and LC3s/GABARAPs and its molecular mechanisms and biological significances, which will be helpful for future studies of mitophagy mediated by Bcl-rambo.
    Keywords:  Bcl-rambo; LC3s/GABARAPs; Mitophagy; Selective binding
  32. Front Cell Dev Biol. 2020 ;8 716
    Friesen EL, Zhang YT, Earnshaw R, De Snoo ML, O'Hara DM, Agapova V, Chau H, Ngana S, Chen KS, Kalia LV, Kalia SK.
      Molecular chaperones are critical to maintaining intracellular proteostasis and have been shown to have a protective role against alpha-synuclein-mediated toxicity. Co-chaperone proteins regulate the activity of molecular chaperones and connect the chaperone network to protein degradation and cell death pathways. Bcl-2 associated athanogene 5 (BAG5) is a co-chaperone that modulates proteostasis by inhibiting the activity of Heat shock protein 70 (Hsp70) and several E3 ubiquitin ligases, resulting in enhanced neurodegeneration in models of Parkinson's disease (PD). Here we identify a novel interaction between BAG5 and p62/sequestosome-1 (SQSTM1), suggesting that BAG5 may bridge the chaperone network to autophagy-mediated protein degradation. We found that BAG5 enhanced the formation of pathogenic alpha-synuclein oligomers and regulated the levels and subcellular distribution of p62. These results extend the role of BAG5 in alpha-synuclein processing and intracellular proteostasis.
    Keywords:  BAG5; alpha-synuclein; bcl-2 associated athanogene; chaperones; p62; proteostasis; sequestosome-1
  33. Neurotox Res. 2020 Aug 25.
    Yang Z, Zhou C, Shi H, Zhang N, Tang B, Ji N.
      Intracerebral hemorrhage (ICH) is a serious medical problem, and effective treatment is limited. Hemorrhaged blood is highly toxic to the brain, and heme, which is mainly released from hemoglobin, plays a vital role in neurotoxicity. However, the specific mechanism involved in heme-mediated neurotoxicity has not been well studied. In this study, we investigated the neurotoxicity of heme in neurons. Neurons were treated with heme, and cell death, autophagy, and endoplasmic reticulum (ER) stress were analyzed. In addition, the relationship between autophagy and apoptosis in heme-induced cell death and the downstream effects were also assessed. We showed that heme induced cell death and autophagy in neurons. The suppression of autophagy using either pharmacological inhibitors (3-methyladenine) or RNA interference of essential autophagy genes (BECN1 and ATG5) decreased heme-induced cell death in neurons. Moreover, the ER stress activator thapsigargin increased cell autophagy and the cell death ratio following heme treatment. Autophagy promoted heme-induced cell apoptosis and cell death through the BECN1/ATG5 pathway. Our findings suggest that heme potentiates neuronal autophagy via ER stress, which in turn induces cell death via the BECN1/ATG5 pathway. Targeting ER stress-mediated autophagy might be a promising therapeutic strategy for ICH.
    Keywords:  Autophagic death; ER stress; Heme; Neuron
  34. Neurobiol Dis. 2020 Aug 24. pii: S0969-9961(20)30331-4. [Epub ahead of print] 105056
    Cutillo G, Simon DK, Eleuteri S.
      Mutations in VPS35 (PARK17), a key molecule in the retromer complex, are a rare cause of autosomal dominant Parkinson's disease (PD), the second most common neurodegenerative disorder. VPS35 exerts crucial functions within the cell in terms of regulating endosomal trafficking. However new data suggest its relevance also in the regulation of mitochondrial dynamics and homeostasis. Herein, we review the crosstalk between VPS35 and the mitochondria, highlighting the potential relevance to PD pathogenesis. VPS35 is not only a critical player in pathways connected to α-synuclein accumulation and clearance, but also plays a key role in ensuring mitochondrial stability and function. The genetic links of VPS35 to PD and the involvement of VPS35 in different PD related pathological mechanisms highlight the potential for targeting VPS35 as a neuroprotective strategy for PD.
    Keywords:  Endosomal trafficking; Mitochondrial dynamic; Neuroprotection; Parkinson's disease; Therapeutic approach; VPS35
  35. BMC Biol. 2020 Aug 28. 18(1): 107
    Li D, Yang SG, He CW, Zhang ZT, Liang Y, Li H, Zhu J, Su X, Gong Q, Xie Z.
      BACKGROUND: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive.RESULTS: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response.
    CONCLUSIONS: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.
    Keywords:  Autophagy; Glycerolipid; Intracellular trafficking; Organelle; Phospholipid
  36. J Biol Chem. 2020 Aug 26. pii: jbc.RA120.013999. [Epub ahead of print]
    Chen F, Amgalan D, Kitsis RN, Pessin JE, Feng D.
      Previously we reported that adipocyte SNAP23 (synaptosome-associated protein of 23 kDa) deficiency blocks the activation of macroautophagy leading to an increased abundance of BAX, a pro-death Bcl-2 family member, and activation and adipocyte cell death both in vitro and in vivo. Here, we found that knockdown of SNAP23 inhibited the association of the autophagosome regulators ATG16L1 and ATG9 compartments by nutrient depletion and reduced the formation of ATG16L1 membrane puncta. ATG16L1 knockdown inhibited autophagy flux and increased BAX protein levels by suppressing BAX degradation. The elevation in BAX protein had no effect on BAX activation or cell death in the nutrient-replete state. However, following nutrient depletion BAX was activated with a concomitant induction of cell death. Co-immunoprecipitation analyses demonstrated that SNAP23 and ATG16L1 proteins form a stable complex independent of nutrient condition, while in the nutrient-deplete state BAX binds to SNAP23 to form a ternary BAX/SNAP23/ATG16L1 protein complex. Taken together, these data support a model in which SNAP23 plays a crucial function as a scaffold for ATG16L1 necessary for the suppression of BAX activation and induction of the intrinsic cell death program.
    Keywords:  ATG16L1; ATG9; Bax; SNAP23; SNARE proteins; adipocyte; apoptosis; autophagy