bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒04‒26
23 papers selected by
Viktor Korolchuk
Newcastle University


  1. Autophagy. 2020 Apr 22. 1-11
    Dalle Pezze P, Karanasios E, Kandia V, Manifava M, Walker SA, Gambardella Le Novère N, Ktistakis NT.
      During macroautophagy/autophagy, the ULK complex nucleates autophagic precursors, which give rise to autophagosomes. We analyzed, by live imaging and mathematical modeling, the translocation of ATG13 (part of the ULK complex) to the autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In nonselective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution, whereas wortmannin reduced this effect and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13 with increasing time between peaks were observed. We hypothesized that these multiple translocations arise because the engulfment of mitochondrial fragments required successive nucleation of phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behavior. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Thus, our data provide novel insights into the early dynamics of selective and nonselective autophagy.Abbreviations: ATG: autophagy related 13; CFP: cyan fluorescent protein; dsRED: Discosoma red fluorescent protein; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; IVM: ivermectin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: PtdIns-3-phosphate; ULK: unc-51 like autophagy activating kinase.
    Keywords:  ATG13; LC3; ULK; autophagy; mathematical modeling; mitophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1749401
  2. Autophagy. 2020 Apr 22. 1-3
    Luciani A, Devuyst O.
      Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism due to the deficiency of mitochondrial MMUT (methylmalonyl-CoA mutase) - an enzyme that mediates the cellular breakdown of certain amino acids and lipids. The loss of MMUT leads to the accumulation of toxic organic acids causing severe organ dysfunctions and life-threatening complications. The mechanisms linking MMUT deficiency, mitochondrial alterations and cell toxicity remain uncharacterized. Using cell and animal-based models, we recently unveiled that MMUT deficiency impedes the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, thereby halting their delivery and subsequent degradation by macroautophagy/autophagy-lysosome systems. In turn, this defective mitophagy process instigates the accumulation of dysfunctional mitochondria that spark epithelial distress and tissue damage. Correction of PINK1-directed mitophagy defects or mitochondrial dysfunctions rescues epithelial distress in MMA cells and alleviates disease-relevant phenotypes in mmut‒deficient zebrafish. Our findings suggest a link between primary MMUT deficiency and diseased mitochondria, mitophagy dysfunction and cell distress, offering potential therapeutic perspectives for MMA and other metabolic diseases.
    Keywords:  Cell damage; inherited metabolic disorders; kidney tubule; metabolism; mitochondria; mitophagy; oxidative stress
    DOI:  https://doi.org/10.1080/15548627.2020.1753927
  3. Acta Pharm Sin B. 2020 Apr;10(4): 569-581
    Xiang H, Zhang J, Lin C, Zhang L, Liu B, Ouyang L.
      Autophagy, defined as a scavenging process of protein aggregates and damaged organelles mediated by lysosomes, plays a significant role in the quality control of macromolecules and organelles. Since protein kinases are integral to the autophagy process, it is critically important to understand the role of kinases in autophagic regulation. At present, intervention of autophagic processes by small-molecule modulators targeting specific kinases has becoming a reasonable and prevalent strategy for treating several varieties of human disease, especially cancer. In this review, we describe the role of some autophagy-related kinase targets and kinase-mediated phosphorylation mechanisms in autophagy regulation. We also summarize the small-molecule kinase inhibitors/activators of these targets, highlighting the opportunities of these new therapeutic agents.
    Keywords:  4E-BP1, eukaryotic translation initiation factor 4E-binding protein; AKT1, AKT serine/threonine kinase 1; AMBRA1, autophagy/beclin-1 regulator 1; AMPK, AMP-activated protein kinase; ARF, auxin response factor gene; ATG, autophagy-related protein; Autophagy; Autophagy-related kinase; CaMKK2, calcium/calmodulin-dependent protein kinase kinase 2; DAPK, death associated protein kinase; FIP200, FAK family kinase-interacting protein of 200 kDa; GAP, GTPase-activating protein; GO, gene ontology; GSK3α, glycogen synthase kinase 3 alpha; HMGB1, high mobility group protein B1; Human disease therapy; JNK1, C-Jun N-terminal kinase; LC3, microtubule-associated protein 1 light chain 3; LKB1, serine/threonine-protein kinase stk11; LPS, lipopolysaccharide; LRRK2, leucine rich repeat kinase 2; PD, Parkinson's disease; PI, phosphatidylinositol; PI3 kinase, phosphoinositide 3-kinase; PI3P, phosphatidylinositol triphosphate; PIM2, proviral insertion in murine lymphomas 2; PINK1, PTEN-induced putative kinase 1; PIP2, phosphatidylinositol-4,5-bisphosphate; PKACα, a protein kinase cAMP-activated catalytic subunit alpha; PKCα, protein kinase C alpha type; PKD1, polycystin-1; PPIs, protein–protein interactions; PROTAC, proteolysis targeting chimeras; PTMs, post-translational modifications; Phosphorylation; Protein kinases; Rheb, the RAS homolog enriched in brain; Small-molecule kinase inhibitors/activators; TAK1, transforming growth factor activated kinase-1; TFEB, transcription factor EB; TNBC, triple-negative breast cancer; TSC1/2, tuberous sclerosis complex proteins 1/2; ULK complex, ULK1–mATG13–FIP200–ATG101 complex; ULK1, unc-51-like kinase 1; UVRAG, ultraviolet resistance-associated gene; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1
    DOI:  https://doi.org/10.1016/j.apsb.2019.10.003
  4. Front Cell Dev Biol. 2020 ;8 221
    Wu C, Yao W, Kai W, Liu W, Wang W, Li S, Chen Y, Wu X, Wang L, Li Y, Tong J, Qian J, Zhang L, Hong Z, Yi C.
      Mitochondria are highly dynamic organelles, which can form a network in cells through fusion, fission, and tubulation. Its morphology is closely related to the function of mitochondria. The damaged mitochondria can be removed by mitophagy. However, the relationship between mitochondrial morphology and non-selective autophagy is not fully understood. We found that mitochondrial fusion machinery, not fission or tubulation machinery, is essential for energy deprivation-induced autophagy. In response to glucose starvation, deletion of mitochondrial fusion proteins severely impaired the association of Atg1/ULK1 with Atg13, and then affected the recruitment of Atg1 and other autophagic proteins to PAS (phagophore assembly site). Furthermore, the deletion of fusion proteins blocks mitochondrial respiration, the binding of Snf1-Mec1, the phosphorylation of Mec1 by Snf1, and the dissociation of Mec1 from mitochondria under prolonged starvation. We propose that mitochondrial fusion machinery regulates energy deprivation-induced autophagy through maintaining mitochondrial respiration.
    Keywords:  autophagy; fusion machinery; glucose starvation; mitochondrial morphology; mitochondrial respiration
    DOI:  https://doi.org/10.3389/fcell.2020.00221
  5. J Biol Chem. 2020 Apr 20. pii: jbc.AC120.012595. [Epub ahead of print]
    Melick CH, Meng D, Jewell JL.
      The mammalian target of rapamycin complex 1 (mTORC1) senses nutrients to mediate anabolic processes within the cell. Exactly how mTORC1 promotes cell growth remains unclear. Here, we identified a novel mTORC1-interacting protein called A-kinase anchoring protein 8L (AKAP8L). Using biochemical assays, we found that the N-terminal region of AKAP8L binds to mTORC1 in the cytoplasm. Importantly, loss of AKAP8L decreased mTORC1-mediated processes such as translation, cell growth and cell proliferation. AKAPs anchor protein kinase A (PKA) through PKA regulatory subunits, and we show that AKAP8L can anchor PKA through regulatory subunit Ia. Reintroducing full-length AKAP8L into cells restored mTORC1-regulated activities, whereas reintroduction of AKAP8L missing the N-terminal region that confers the interaction with mTORC1 did not. Our results suggest a multifaceted role for AKAPs in the cell. We conclude that mTORC1 appears to regulate protein translation, perhaps in part through AKAP8L.
    Keywords:  AKAP; cell biology; cell proliferation; cell size; mRNA translation; mTOR complex (mTORC); nutrient sensing; protein kinase A (PKA); protein synthesis; scaffolding protein
    DOI:  https://doi.org/10.1074/jbc.AC120.012595
  6. Dev Cell. 2020 Apr 20. pii: S1534-5807(20)30230-6. [Epub ahead of print]53(2): 154-168.e6
    Puri C, Manni MM, Vicinanza M, Hilcenko C, Zhu Y, Runwal G, Stamatakou E, Menzies FM, Mamchaoui K, Bitoun M, Rubinsztein DC.
      Autophagy involves engulfment of cytoplasmic contents by double-membraned autophagosomes, which ultimately fuse with lysosomes to enable degradation of their substrates. We recently proposed that the tubular-vesicular recycling endosome membranes were a core platform on which the critical early events of autophagosome formation occurred, including LC3-membrane conjugation to autophagic precursors. Here, we report that the release of autophagosome precursors from recycling endosomes is mediated by DNM2-dependent scission of these tubules. This process is regulated by DNM2 binding to LC3 and is increased by autophagy-inducing stimuli. This scission is defective in cells expressing a centronuclear-myopathy-causing DNM2 mutant. This mutant has an unusual mechanism as it depletes normal-functioning DNM2 from autophagosome formation sites on recycling endosomes by causing increased binding to an alternative plasma membrane partner, ITSN1. This "scission" step is, thus, critical for autophagosome formation, is defective in a human disease, and influences the way we consider how autophagosomes are formed.
    Keywords:  DNM2; ITSN1; RAB11; autophagosome; autophagy; centronuclear myopathy; phagophore; recycling endosome
    DOI:  https://doi.org/10.1016/j.devcel.2020.03.018
  7. Autophagy. 2020 Apr 24. 1-2
    Leidal AM, Debnath J.
      Accumulating evidence implicates various autophagy-related (ATG) proteins in cellular secretion. Recently, we identified a new secretory autophagy pathway in which components of LC3 conjugation machinery specify the incorporation of RNA binding proteins (RBPs) and small non-coding RNAs into extracellular vesicles (EVs), resulting in their secretion outside of cells. We term this process LC3-Dependent EV Loading and Secretion (LDELS). Importantly, LDELS is distinct from classical macroautophagy/autophagy because it requires components of the LC3 conjugation machinery, but not other ATGs involved in autophagosome formation. Because EVs have emerged as mediators of intracellular communication, our results provide new insight into how the autophagy machinery may influence the non-cell autonomous exchange of information between cells.
    Keywords:  Extracellular vesicles; RNA-binding proteins; autophagy; exosomes
    DOI:  https://doi.org/10.1080/15548627.2020.1756557
  8. Mech Ageing Dev. 2020 Apr 21. pii: S0047-6374(20)30048-8. [Epub ahead of print] 111252
    Schiavi A, Strappazzon F, Ventura N.
      Aging is characterized by the deterioration of different cellular and organismal structures and functions. A typical hallmark of the aging process is the accumulation of dysfunctional mitochondria and excess iron, leading to a vicious cycle that promotes cell and tissue damage, which ultimately contribute to organismal aging. Accordingly, altered mitochondrial quality control pathways such as mitochondrial autophagy (mitophagy) as well as altered iron homeostasis, with consequent iron overload, can accelerate the aging process and the development and progression of different age-associated disorders. In this review we first briefly introduce the aging process and summarize molecular mechanisms regulating mitophagy and iron homeostasis. We then provide an overview on how dysfunction of these two processes impact on aging and age-associated neurodegenerative disorders with a focus on Alzheimer's disease, Parkinson's disease and Amyotrophic Lateral Sclerosis. Finally, we summarize some recent evidence showing mechanistic links between iron metabolism and mitophagy and speculate on how regulating the crosstalk between the two processes may provide protective effects against aging and age-associated neuronal pathologies.
    Keywords:  Alzheimer; Amyotrophic Lateral Sclerosis; Parkinson; aging; iron; mitochondria
    DOI:  https://doi.org/10.1016/j.mad.2020.111252
  9. Cell Mol Life Sci. 2020 Apr 22.
    Besemer AS, Maus J, Ax MDA, Stein A, Vo S, Freese C, Nalbach K, von Hilchen C, Pfalzgraf IF, Koziollek-Drechsler I, Silva B, Huesmann H, Boukhallouk F, Florin L, Kern A, Behl C, Clement AM.
      The cellular protein homeostasis (proteostasis) network responds effectively to insults. In a functional screen in C. elegans, we recently identified the gene receptor-mediated endocytosis 8 (rme-8; human ortholog: DNAJC13) as a component of the proteostasis network. Accumulation of aggregation-prone proteins, such as amyloid-β 42 (Aβ), α-synuclein, or mutant Cu/Zn-superoxide dismutase (SOD1), were aggravated upon the knockdown of rme-8/DNAJC13 in C. elegans and in human cell lines, respectively. DNAJC13 is involved in endosomal protein trafficking and associated with the retromer and the WASH complex. As both complexes have been linked to autophagy, we investigated the role of DNAJC13 in this degradative pathway. In knockdown and overexpression experiments, DNAJC13 acts as a positive modulator of autophagy. In contrast, the overexpression of the Parkinson's disease-associated mutant DNAJC13(N855S) did not enhance autophagy. Reduced DNAJC13 levels affected ATG9A localization at and its transport from the recycling endosome. As a consequence, ATG9A co-localization at LC3B-positive puncta under steady-state and autophagy-induced conditions is impaired. These data demonstrate a novel function of RME-8/DNAJC13 in cellular homeostasis by modulating ATG9A trafficking and autophagy.
    Keywords:  ATG9A; Autophagy; C. elegans; DNAJC13; Proteostasis; RME-8; Recycling endosome
    DOI:  https://doi.org/10.1007/s00018-020-03521-y
  10. J Am Chem Soc. 2020 Apr 22.
    Pavlinov I, Salkovski M, Aldrich LN.
      Autophagy, a catabolic recycling process, has been implicated as a critical pathway in cancer. Its role in maintaining cellular homeostasis helps to nourish hypoxic, nutrient-starved tumors and protects them from chemotherapy-induced death. Recent efforts to target autophagy in cancer have focused on kinase inhibition, which has led to molecules that lack specificity due to the multiple roles of key kinases in this pathway. For example, the lipid kinase VPS34 is present in two multiprotein complexes responsible for the generation of phosphatidylinositol-3-phosphate. Complex I generates the autophagosome, and Complex II is crucial for endosomal trafficking. Molecules targeting VPS34 inhibit both complexes, which inhibits autophagy but causes undesirable defects in vesicle trafficking. The lack of specific autophagy modulators has limited the utility of autophagy inhibition as a therapeutic strategy. We hypothesize that disruption of the Beclin 1-ATG14L protein-protein interaction, which is required for the formation, proper localization, and function of VPS34 Complex I but not Complex II, will disrupt Complex I formation and selectively inhibit autophagy. To this end, a high-throughput, cellular NanoBRET assay was developed targeting this interaction. An initial screen of 2560 molecules yielded 19 hits that effectively disrupted the interaction, and it was confirmed that one hit disrupted VPS34 Complex I formation and inhibited autophagy. In addition, the molecule did not disrupt the Beclin 1-UVRAG interaction, critical for VPS34 Complex II, and thus had little impact on vesicle trafficking. This molecule is a promising new tool that is critical for understanding how modulation of the Beclin 1-ATG14L interaction affects autophagy. More broadly, its discovery demonstrates that targeting protein-protein interactions found within the autophagy pathway is a viable strategy for the discovery of autophagy-specific probes and therapeutics.
    DOI:  https://doi.org/10.1021/jacs.9b12705
  11. J Cell Biol. 2020 May 04. pii: e202002179. [Epub ahead of print]219(5):
    Tan JX, Finkel T.
      Mitochondria, long viewed solely in the context of bioenergetics, are increasingly emerging as critical hubs for intracellular signaling. Due to their bacterial origin, mitochondria possess their own genome and carry unique lipid components that endow these organelles with specialized properties to help orchestrate multiple signaling cascades. Mitochondrial signaling modulates diverse pathways ranging from metabolism to redox homeostasis to cell fate determination. Here, we review recent progress in our understanding of how mitochondria serve as intracellular signaling platforms with a particular emphasis on lipid-mediated signaling, innate immune activation, and retrograde signaling. We further discuss how these signaling properties might potentially be exploited to develop new therapeutic strategies for a range of age-related conditions.
    DOI:  https://doi.org/10.1083/jcb.202002179
  12. Cell Rep. 2020 Apr 21. pii: S2211-1247(20)30457-5. [Epub ahead of print]31(3): 107547
    Wu W, Wang X, Berleth N, Deitersen J, Wallot-Hieke N, Böhler P, Schlütermann D, Stuhldreier F, Cox J, Schmitz K, Seggewiß S, Peter C, Kasof G, Stefanski A, Stühler K, Tschapek A, Gödecke A, Stork B.
      Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.
    Keywords:  MLKL; RIPK1; RIPK3; TNF; ULK1; autophagy; complex I; complex II; necroptosis; necrosome
    DOI:  https://doi.org/10.1016/j.celrep.2020.107547
  13. Nat Commun. 2020 Apr 22. 11(1): 1935
    Wakita M, Takahashi A, Sano O, Loo TM, Imai Y, Narukawa M, Iwata H, Matsudaira T, Kawamoto S, Ohtani N, Yoshimori T, Hara E.
      Although cellular senescence acts primarily as a tumour suppression mechanism, the accumulation of senescent cells in vivo eventually exerts deleterious side effects through inflammatory/tumour-promoting factor secretion. Thus, the development of new drugs that cause the specific elimination of senescent cells, termed senolysis, is anticipated. Here, by an unbiased high-throughput screening of chemical compounds and a bio-functional analysis, we identify BET family protein degrader (BETd) as a promising senolytic drug. BETd provokes senolysis through two independent but integrated pathways; the attenuation of non-homologous end joining (NHEJ), and the up-regulation of autophagic gene expression. BETd treatment eliminates senescent hepatic stellate cells in obese mouse livers, accompanied by the reduction of liver cancer development. Furthermore, the elimination of chemotherapy-induced senescent cells by BETd increases the efficacy of chemotherapy against xenograft tumours in immunocompromised mice. These results reveal the vulnerability of senescent cells and open up possibilities for its control.
    DOI:  https://doi.org/10.1038/s41467-020-15719-6
  14. Trends Biochem Sci. 2020 May;pii: S0968-0004(20)30050-5. [Epub ahead of print]45(5): 367-369
    Park JH, Lee G, Blenis J.
      Using cryo-electron microscopy and molecular characterization, David Sabatini and colleagues provide crucial new insights that validate and expand their model of how amino acids are sensed and signal at the lysosome to activate mechanistic target of rapamycin complex 1 (mTORC1) and cell growth-regulating processes. This work also reveals new therapeutic opportunities for mTORC1-driven diseases.
    DOI:  https://doi.org/10.1016/j.tibs.2020.02.004
  15. BMB Rep. 2020 Apr 22. pii: 4861. [Epub ahead of print]
    Kim K, Park SG, Park BC, Kim JH, Kim S.
      Phosphorylation of the signaling component by protein kinase often leads to a kinase cascade or feedback loop. 3-Phosphoinositide-dependent kinase 1 (PDK1) signaling pathway diverges into various kinases including Akt and p70 S6 kinase (p70S6k). However, the PDK1 feedback mechanism remains elusive. Here, we demonstrated that UNC-51-like kinase (ULK1), an autophagy initiator kinase downstream of mechanistic target of rapamycin (mTOR), directly phosphorylated PDK1 on serine 389 at the linker region. Furthermore, our data showed that this phosphorylation affected the kinase activity of PDK1 toward downstream substrates. These results suggest a possible negative feedback loop between PDK1 and ULK1.
  16. BMB Rep. 2020 Apr 22. pii: 4966. [Epub ahead of print]
    Jang DE, Song J, Park JW, Yoon SH, Bae YS.
      Protein kinase CK2 downregulation induces premature senescence in various human cell types via activation of the reactive oxygen species (ROS)-p53-p21Cip1/WAF1 pathway. The transcription factor "nuclear factor erythroid 2-related factor 2" (Nrf2) plays an important role in maintaining intracellular redox homeostasis. In this study, Nrf2 overexpression attenuated CK2 downregulation-induced ROS production and senescence markers including SA-β-gal staining and activation of p53-p21Cip1/WAF1 in human breast (MCF-7) and colon (HCT116) cancer cells. CK2 downregulation reduced the transcription of Nrf2 target genes, such as glutathione S-transferase, glutathione peroxidase 2, and glutathione reductase 1. Furthermore, CK2 downregulation destabilized Nrf2 protein via inhibiting autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1). Finally, CK2 downregulation decreased the nuclear import of Nrf2 by deactivating AMP-activated protein kinase (AMPK). Collectively, our data suggest that both Keap1 stabilization and AMPK inactivation are associated with decreased activity of Nrf2 in CK2 downregulation-induced cellular senescence.
  17. Biol Cell. 2020 Apr 19.
    Maayan DF, Chapnik N, Froy O.
      BACKGROUND INFORMATION: Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a master regulator of cell and whole-body energy homeostasis. REV-ERBα is a nuclear receptor that plays an important role in metabolism. While mTORC1 activation is necessary for muscle differentiation, the role of REV-ERBα is less clear.RESULTS: We studied the effect of REV-ERBα overexpression and silencing as well as mTORC1 activation and inhibition on the differentiation of C2C12 myoblasts to myotubes. mTOR, myogenin and REV-ERBα were induced during differentiation of myoblasts into myotubes. REV-ERBα was found to activate mTORC1 during the differentiation process even in the absence of the differentiation medium. This activation was presumably through the downregulation of the expression of TSC1, an mTORC1 inhibitor.
    CONCLUSION: Herein we show that REV-ERBα promotes myoblasts differentiation via the activation of the mTORC1 signaling pathway.
    SIGNIFICANCE: REV-ERBα modulation can activate mTORC1 signaling and promote myoblasts differentiation. This article is protected by copyright. All rights reserved.
    Keywords:  C2C12; Differentiation; Myoblasts; Myotubes; REV-ERVα; mTOR
    DOI:  https://doi.org/10.1111/boc.201900091
  18. Cells. 2020 Apr 18. pii: E1015. [Epub ahead of print]9(4):
    Uzhytchak M, Smolková B, Lunova M, Jirsa M, Frtús A, Kubinová Š, Dejneka A, Lunov O.
      Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies.
    Keywords:  autophagy; iron oxide nanoparticles; lysosomes; magnetic resonance imaging; nano-bio interactions; p53
    DOI:  https://doi.org/10.3390/cells9041015
  19. Food Chem Toxicol. 2020 Apr 16. pii: S0278-6915(20)30202-7. [Epub ahead of print] 111314
    Anand SK, Sharma A, Singh N, Kakkar P.
      Bisphenol-A, an endocrine disruptive chemical widely used to manufacture polycarbonate plastics and epoxy resins, acts via multiple mechanisms that perturb cellular and molecular functions. BPA has the potential to induce hepatotoxicity via generation of ROS and oxidative stress. However, the mechanism of BPA induced oxidative stress and autophagy is still ambiguous at molecular and cellular levels. This study aims to elucidate the impact of BPA exposure (50 and 100 μM) in primary rat hepatocytes. AMP kinase, an intracellular energy sensor and key regulator in cellular signaling were found to be activated during BPA exposure. The increased AMP/ATP ratio and subsequent phosphorylation by its upstream mediator Liver Kinase B1 (LKB1) activates AMPK. BPA down-regulated AMPK downstream molecule i.e. mammalian target of rapamycin (mTOR) by inhibiting its phosphorylation, eventually enhances expression of autophagic markers LC3B, Beclin-1 while lowers p62. Results also revealed that BPA induces mitophagy by promoting accumulation of PINK1 and translocation of Parkin to damaged mitochondria culminating in decreased mitochondrial mass. Ultra-structural changes also confirmed mitochondrial disintegration, enhanced autophagic induction as evident from autophagosome formation. Findings confirm that BPA caused oxidative stress which eventually triggered LKB1/AMPK mediated autophagy and maintains cellular energy balance by mitophagic removal of unhealthy mitochondria in primary rat hepatocytes.
    Keywords:  AMPK; Autophagy; LKB1; Mitophagy; Oxidative stress; mTOR
    DOI:  https://doi.org/10.1016/j.fct.2020.111314
  20. EMBO J. 2020 Apr 20. e102539
    Ryan TA, Phillips EO, Collier CL, Jb Robinson A, Routledge D, Wood RE, Assar EA, Tumbarello DA.
      Multiple mitochondrial quality control pathways exist to maintain the health of mitochondria and ensure cell homeostasis. Here, we investigate the role of the endosomal adaptor Tollip during the mitochondrial stress response and identify its interaction and colocalisation with the Parkinson's disease-associated E3 ubiquitin ligase Parkin. The interaction between Tollip and Parkin is dependent on the ubiquitin-binding CUE domain of Tollip, but independent of Tom1 and mitophagy. Interestingly, this interaction is independent of Parkin mitochondrial recruitment and ligase activity but requires an intact ubiquitin-like (UBL) domain. Importantly, Tollip regulates Parkin-dependent endosomal trafficking of a discrete subset of mitochondrial-derived vesicles (MDVs) to facilitate delivery to lysosomes. Retromer function and an interaction with Tom1 allow Tollip to facilitate late endosome/lysosome trafficking in response to mitochondrial stress. We find that upregulation of TOM20-positive MDVs upon mitochondrial stress requires Tollip interaction with ubiquitin, endosomal membranes and Tom1 to ensure their trafficking to the lysosomes. Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial-derived cargo to the lysosomes.
    Keywords:  Parkinson's disease; lysosome; membrane trafficking; mitochondria; vesicle transport
    DOI:  https://doi.org/10.15252/embj.2019102539
  21. FASEB J. 2020 Apr 23.
    Ghosh R, Wood-Kaczmar A, Dobson L, Smith EJ, Sirinathsinghji EC, Kriston-Vizi J, Hargreaves IP, Heaton R, Herrmann F, Abramov AY, Lam AJ, Heales SJ, Ketteler R, Bates GP, Andre R, Tabrizi SJ.
      Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.
    Keywords:  Huntington's; aggregation; mitochondria; model; respiration
    DOI:  https://doi.org/10.1096/fj.201902277RR
  22. Mol Biol Rep. 2020 Apr 21.
    Biray Avci C, Sezgin B, Goker Bagca B, Karci HB, Gode S.
      Although there are many studies on the role of PI3K/AKT/mTOR pathway and autophagy genes in the mechanism of head and neck cancer formation and prognostic significance, there is no study investigating the role of the genes in paranasal sinus carcinomas. The aim of the study was to assess the role of the PI3K/AKT/mTOR pathway and autophagy related gene expression changes in squamous cell carcinoma of paranasal sinuses with and without neck metastasis. Eight paranasal squamous cell carcinoma patients (five without and three with neck metastasis) were included. Tissues were obtained during the surgery. Total RNA was isolated from the tissues and cDNA synthesis was performed. Expression levels of the genes were determined using qRT-PCR method. The results were evaluated using the 2-∆∆Ct method, and fold changes of the gene expression levels in primary tumor and neck metastasis tissues were calculated according to the normal tissue. Expression levels of both PI3K/AKT/mTOR pathway and positive regulators of autophagy were significantly increased in metastasis-related two groups, especially in neck metastasis tissues. The increase in PI3K/AKT/mTOR pathway and autophagy related gene expression levels may support the metastatic character in paranasal squamous cell carcinomas. This is the first study to assess autophagy related genes in paranasal sinus cancer at transcriptome-level. Support of the transcriptome-level findings by the further protein analyses will contribute to the illumination of the rare paranasal sinus cancer molecular biology.
    Keywords:  Autophagy; Metastasis; PI3K/AKT/mTOR; Paranasal squamous cell carcinoma
    DOI:  https://doi.org/10.1007/s11033-020-05458-8
  23. J Biol Chem. 2020 Apr 20. pii: jbc.RA119.011864. [Epub ahead of print]
    Burton TD, Fedele AO, Xie J, Sandeman L, Proud CG.
      Autophagy and lysosomal activities play a key role in the cell by initiating and carrying out the degradation of misfolded proteins. Transcription factor EB (TFEB) functions as a master controller of lysosomal biogenesis and function during lysosomal stress, controlling most, but, importantly, not all lysosomal genes. Here, we sought to better understand the regulation of lysosomal genes whose expression does not appear to be controlled by TFEB. Sixteen of these genes were screened for transactivation in response to diverse cellular insults. mRNA levels for lysosomal-associated membrane protein 3 (LAMP3), a gene that is highly up-regulated in many forms of cancer, including breast and cervical cancers, were significantly increased during the integrated stress response (ISR), which occurs in eukaryotic cells in response to accumulation of unfolded and misfolded proteins. Of note, results from siRNA-mediated knockdown of activating transcription factor 4 (ATF4) and overexpression of exogenous ATF4 cDNA, indicated that ATF4 up-regulates LAMP3 mRNA levels. Finally, ChIP assays verified an ATF4-binding site in the LAMP3 gene promoter, and a dual luciferase assay confirmedthat this ATF4-binding site is indeed required for transcriptional up-regulation of LAMP3 These results reveal that ATF4 directly regulates LAMP3, representing the first identification of a gene for a lysosomal component whose expression is directly controlled by ATF4. This finding may provide a key link between stresses such as accumulation of unfolded proteins and modulation of autophagy, which removes them.
    Keywords:  activating transcription factor 4 (ATF4); autophagy; cell stress; eukaryotic initiation factor 2 (eIF2); lysosomal-associated membrane protein 3 (LAMP3); lysosome; mammalian target of rapamycin complex 1 (mTORC1); protein misfolding; transcription factor EB (TFEB); unfolded protein response (UPR)
    DOI:  https://doi.org/10.1074/jbc.RA119.011864