bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2019‒12‒15
fifteen papers selected by
Viktor Korolchuk
Newcastle University


  1. BMB Rep. 2019 Dec 10. pii: 4841. [Epub ahead of print]
    Shin WH, Park JH, Chung KC.
      The ubiquitin-proteasome system (UPS) and autophagy are two major degradative pathways of proteins in eukaryotic cells. As about 30% of newly synthesized proteins are known to be misfolded under normal cell conditions, the precise and timely operation of the UPS and autophagy to remove them as well as their tightly controlled regulation, is so important for proper cell function and survival. In the UPS, target proteins are labeled by small proteins called ubiquitin, which are then transported to the proteasome complex for degradation. Alternatively, many greatly damaged proteins are believed to be delivered to the lysosome for autophagic degradation. Although these autophagy and UPS pathways have not been considered to be directly related, many recent studies proposed their close link and dynamic interconversion. In this review, we'll focus on the several regulatory molecules that function in both UPS and autophagy and their crosstalk. Among the proposed multiple modulators, we will take a closer look at the so-called main connector of UPS-autophagy regulation, p62. Last, the functional role of p62 in the mitophagy and its implication for the pathogenesis of Parkinson's disease, one of the major neurodegenerative diseases, will be briefly reviewed.
  2. Front Aging Neurosci. 2019 ;11 311
    Chakravorty A, Jetto CT, Manjithaya R.
      Neurons are highly specialized post-mitotic cells that are inherently dependent on mitochondria owing to their high bioenergetic demand. Mitochondrial dysfunction is therefore associated with various age-related neurodegenerative disorders such as Alzheimer's disease (AD), wherein accumulation of damaged and dysfunctional mitochondria has been reported as an early symptom further contributing to disease progression. In AD, impairment of mitochondrial function causes bioenergetic deficiency, intracellular calcium imbalance and oxidative stress, thereby aggravating the effect of Aβ and tau pathologies, leading to synaptic dysfunction, cognitive impairment and memory loss. Although there are reports suggesting intricate parallelism between mitochondrial dysfunction and AD pathologies such as Aβ aggregation and hyperphosphorylated tau accumulation, the factors that drive the pathogenesis of either are unclear. In addition, emerging evidence suggest that mitochondrial quality control (QC) mechanisms such as mitophagy are impaired in AD. As an important mitochondrial QC mechanism, mitophagy plays a critical role in maintaining neuronal health and function. Studies show that various proteins involved in mitophagy, mitochondrial dynamics, and mitochondrial biogenesis are affected in AD. Compromised mitophagy may also be attributed to impairment in autophagosome-lysosome fusion and defects in lysosomal acidification. Therapeutic interventions aiming to restore mitophagy functions can be used as a strategy for ameliorating AD pathogenesis. Recent evidence implicates the role of microglial activation via mitophagy induction in reducing amyloid plaque load. This review summarizes the current developments in the field of mitophagy and mitochondrial dysfunction in AD.
    Keywords:  Alzheimer’s disease; amyloid beta; microglia; mitochondrial dysfunction; mitophagy; tau
    DOI:  https://doi.org/10.3389/fnagi.2019.00311
  3. Autophagy. 2019 Dec 10. 1-20
    An HK, Chung KM, Park H, Hong J, Gim JE, Choi H, Lee YW, Choi J, Mun JY, Yu SW.
      CASP9 (caspase 9) is a well-known initiator caspase which triggers intrinsic apoptosis. Recent studies also suggest various non-apoptotic roles of CASP9, including macroautophagy/autophagy regulation. However, the involvement of CASP9 in autophagy and its molecular mechanisms are not well understood. Here we report the non-apoptotic function of CASP9 in positive regulation of autophagy through maintenance of mitochondrial homeostasis. Growth factor or amino acid deprivation-induced autophagy activated CASP9, but without apoptotic features. Pharmacological inhibition or genetic ablation of CASP9 decreased autophagy flux, while ectopic expression of CASP9 rescued autophagy defects. In CASP9 knockout (KO) cells, initiation and elongation of phagophore membranes were normal, but sealing of the membranes and autophagosome maturation were impaired, and the lifetime of autophagosomes was prolonged. Ablation of CASP9 caused an accumulation of inactive ATG3 and decreased lipidation of the Atg8-family members, most severely that of GABARAPL1. Moreover, it resulted in abnormal mitochondrial morphology with depolarization of the membrane potential, reduced reactive oxygen species production, and aberrant accumulation of mitochondrial fusion-fission proteins. CASP9 expression or exogenously added H2O2 in the CASP9 KO cells corrected the ATG3 level and lipidation status of Atg8-family members, and restored autophagy flux. Of note, only CASP9 expression but not H2O2 rescued mitochondrial defects, revealing regulation of mitochondrial homeostasis by CASP9. Our findings suggest a new regulatory link between mitochondria and autophagy through CASP9 activity, especially for the proper operation of the Atg8-family conjugation system and autophagosome closure and maturation.Abbreviations: AA: amino acid; ACD: autophagic cell death; ACTB: actin beta; ANXA5: annexin A5; APAF1: apoptotic peptidase activating factor 1; Atg: autophagy related; ATG16L1: autophagy related 16 like 1; BafA1: bafilomycin A1; BCL2: BCL2 apoptosis regulator; BECN1: beclin 1; CARD: caspase recruitment domain containing; CASP: caspase; CM-H2DCFDA: chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; Δψm: mitochondrial membrane potential; DN: dominant-negative; DNM1L/DRP1: dynamin 1 like; EBSS: Earle's balanced salt solution; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; HCN: hippocampal neural stem cells; IAM: inner autophagosome membrane; INS: insulin; KO: knockout; LEHD: Z-LEHD-fmk; MAP1LC3: microtubule associated protein 1 light chain 3; MFN1: mitofusin 1; MFN2: mitofusin 2; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP1: poly(ADP-ribose) polymerase 1; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; ROS: reactive oxygen species; sgRNA: single guide RNA; SR-SIM: super-resolution structured illumination microscopy; SQSTM1: sequestosome 1; STS: staurosporine; STX17: syntaxin 17; TMRE: tetramethylrhodamine ethyl ester; TUBB: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.
    Keywords:  ATG3; autophagosome maturation; caspase 9; membrane closure; mitochondria; reactive oxygen species
    DOI:  https://doi.org/10.1080/15548627.2019.1695398
  4. Nature. 2019 Dec 11.
    Ramirez C, Hauser AD, Vucic EA, Bar-Sagi D.
      Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis1. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions2. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic RAS. Here we identify vacuolar ATPase (V-ATPase) as an essential regulator of RAS-induced macropinocytosis. Oncogenic RAS promotes the translocation of V-ATPase from intracellular membranes to the plasma membrane via a pathway that requires the activation of protein kinase A by a bicarbonate-dependent soluble adenylate cyclase. Accumulation of V-ATPase at the plasma membrane is necessary for the cholesterol-dependent plasma-membrane association of RAC1, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations establish a link between V-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of RAS-mutant tumour cells.
    DOI:  https://doi.org/10.1038/s41586-019-1831-x
  5. Dev Cell. 2019 Nov 29. pii: S1534-5807(19)30903-7. [Epub ahead of print]
    Jia J, Claude-Taupin A, Gu Y, Choi SW, Peters R, Bissa B, Mudd MH, Allers L, Pallikkuth S, Lidke KA, Salemi M, Phinney B, Mari M, Reggiori F, Deretic V.
      Endomembrane damage elicits homeostatic responses including ESCRT-dependent membrane repair and autophagic removal of damaged organelles. Previous studies have suggested that these systems may act separately. Here, we show that galectin-3 (Gal3), a β-galactoside-binding cytosolic lectin, unifies and coordinates ESCRT and autophagy responses to lysosomal damage. Gal3 and its capacity to recognize damage-exposed glycans were required for efficient recruitment of the ESCRT component ALIX during lysosomal damage. Both Gal3 and ALIX were required for restoration of lysosomal function. Gal3 promoted interactions between ALIX and the downstream ESCRT-III effector CHMP4 during lysosomal repair. At later time points following lysosomal injury, Gal3 controlled autophagic responses. When this failed, as in Gal3 knockout cells, lysosomal replacement program took over through TFEB. Manifestations of this staged response, which includes membrane repair, removal, and replacement, were detected in model systems of lysosomal damage inflicted by proteopathic tau and during phagosome parasitism by Mycobacterium tuberculosis.
    Keywords:  ESCRT; TFEB; autophagy; endosome; galectins; lysosome; membrane damage homeostasis
    DOI:  https://doi.org/10.1016/j.devcel.2019.10.025
  6. Autophagy. 2019 Dec 11.
    Wang H, Wang N, Xu D, Ma Q, Chen Y, Xu S, Xia Q, Zhang Y, Prehn JHM, Wang G, Ying Z.
      Significant evidences indicate that reactive oxygen species (ROS) can induce macroautophagy/autophagy under both physiological and pathological conditions. Although the relationship between ROS and autophagy regulation has been well studied, the basic mechanism by which ROS affects autophagy and the biological role of this regulation are still not fully understood. In the present study we show that multiple MiT-TFE transcription factors including TFEB, TFE3 and MITF, which are master regulators of autophagy and lysosomal biogenesis, can be activated upon direct cysteine oxidation by ROS. Oxidation promotes the nuclear translocation of these MiT-TFE transcription factors by inhibiting the association of them with RRAG GTPases, which in turn leads to enhanced global gene expression level in autophagy-lysosome system. Our study highlights the role of oxidation of MiT-TFE transcription factors in ROS-linked autophagy, and provides novel mechanism that MiT-TFE transcription factors-mediated transcriptional control of autophagy may govern cell homeostasis in response to oxidative stress, a biological process tightly linked to human diseases including neurodegenerative diseases and cancer.
    Keywords:  MTORC1; RRAG GTPases; TFEB; autophagy; oxidation
    DOI:  https://doi.org/10.1080/15548627.2019.1704104
  7. Nat Commun. 2019 Dec 11. 10(1): 5648
    Kumsta C, Chang JT, Lee R, Tan EP, Yang Y, Loureiro R, Choy EH, Lim SHY, Saez I, Springhorn A, Hoppe T, Vilchez D, Hansen M.
      Autophagy can degrade cargos with the help of selective autophagy receptors such as p62/SQSTM1, which facilitates the degradation of ubiquitinated cargo. While the process of autophagy has been linked to aging, the impact of selective autophagy in lifespan regulation remains unclear. We have recently shown in Caenorhabditis elegans that transcript levels of sqst-1/p62 increase upon a hormetic heat shock, suggesting a role of SQST-1/p62 in stress response and aging. Here, we find that sqst-1/p62 is required for hormetic benefits of heat shock, including longevity, improved neuronal proteostasis, and autophagy induction. Furthermore, overexpression of SQST-1/p62 is sufficient to induce autophagy in distinct tissues, extend lifespan, and improve the fitness of mutants with defects in proteostasis in an autophagy-dependent manner. Collectively, these findings illustrate that increased expression of a selective autophagy receptor is sufficient to induce autophagy, enhance proteostasis and extend longevity, and demonstrate an important role for sqst-1/p62 in proteotoxic stress responses.
    DOI:  https://doi.org/10.1038/s41467-019-13540-4
  8. Nat Commun. 2019 Dec 10. 10(1): 5630
    Scotto Rosato A, Montefusco S, Soldati C, Di Paola S, Capuozzo A, Monfregola J, Polishchuk E, Amabile A, Grimm C, Lombardo A, De Matteis MA, Ballabio A, Medina DL.
      The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKβ and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders.
    DOI:  https://doi.org/10.1038/s41467-019-13572-w
  9. Sci Rep. 2019 Dec 09. 9(1): 18597
    García-Macia M, Santos-Ledo A, Caballero B, Rubio-González A, de Luxán-Delgado B, Potes Y, Rodríguez-González SM, Boga JA, Coto-Montes A.
      Sexual dimorphism has been reported in many processes. However, sexual bias in favour of the use of males is very present in science. One of the main reasons is that the impact of hormones in diverse pathways and processes such as autophagy have not been properly addressed in vivo. The Harderian gland is a perfect model to study autophagic modulation as it exhibits important changes during the oestrous cycle. The aim of this study is to identify the main processes behind Harderian gland differences under oestrous cycle and their modulator. In the present study we show that redox-sensitive transcription factors have an essential role: NF-κB may activate SQSTM1/p62 in oestrus, promoting selective types of autophagy: mitophagy and lipophagy. Nrf2 activation in dioestrus, leads the retrieval phase and restoration of mitochondrial homeostasis. Melatonin's receptors show higher expression in dioestrus, leading to decreases in pro-inflammatory mediators and enhanced Nrf2 expression. Consequently, autophagy is blocked, and porphyrin release is reduced. All these results point to melatonin as one of the main modulators of the changes in autophagy during the oestrous cycle.
    DOI:  https://doi.org/10.1038/s41598-019-54743-5
  10. Cells. 2019 Dec 10. pii: E1603. [Epub ahead of print]8(12):
    Kazibwe Z, Liu AY, MacIntosh GC, Bassham DC.
      Ribosomes are essential for protein synthesis in all organisms and their biogenesis and number are tightly controlled to maintain homeostasis in changing environmental conditions. While ribosome assembly and quality control mechanisms have been extensively studied, our understanding of ribosome degradation is limited. In yeast or animal cells, ribosomes are degraded after transfer into the vacuole or lysosome by ribophagy or nonselective autophagy, and ribosomal RNA can also be transferred directly across the lysosomal membrane by RNautophagy. In plants, ribosomal RNA is degraded by the vacuolar T2 ribonuclease RNS2 after transport by autophagy-related mechanisms, although it is unknown if a selective ribophagy pathway exists in plants. In this review, we describe mechanisms of turnover of ribosomal components in animals and yeast, and, then, discuss potential pathways for degradation of ribosomal RNA and protein within the vacuole in plants.
    Keywords:  RNA; autophagy; lysosome; ribonuclease; ribophagy; ribosome; target of rapamycin (TOR); vacuole
    DOI:  https://doi.org/10.3390/cells8121603
  11. Cells. 2019 Dec 09. pii: E1597. [Epub ahead of print]8(12):
    Wang H, Liu Y, Wang D, Xu Y, Dong R, Yang Y, Lv Q, Chen X, Zhang Z.
      Autophagy, originally found in liver experiments, is a cellular process that degrades damaged organelle or protein aggregation. This process frees cells from various stress states is a cell survival mechanism under stress stimulation. It is now known that dysregulation of autophagy can cause many liver diseases. Therefore, how to properly regulate autophagy is the key to the treatment of liver injury. mechanistic target of rapamycin (mTOR)is the core hub regulating autophagy, which is subject to different upstream signaling pathways to regulate autophagy. This review summarizes three upstream pathways of mTOR: the phosphoinositide 3-kinase (PI3K)/protein kinase (AKT) signaling pathway, the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and the rat sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-extracellular activated protein kinase kinase (MEK)/ extracellular-signal-regulated kinase (ERK) signaling pathway, specifically explored their role in liver fibrosis, hepatitis B, non-alcoholic fatty liver, liver cancer, hepatic ischemia reperfusion and other liver diseases through the regulation of mTOR-mediated autophagy. Moreover, we also analyzed the crosstalk between these three pathways, aiming to find new targets for the treatment of human liver disease based on autophagy.
    Keywords:  AKT; AMPK; ERK; MEK; PI3K; Raf; Ras; autophagy; liver diseases; mTOR
    DOI:  https://doi.org/10.3390/cells8121597
  12. EMBO J. 2019 Dec 10. e100875
    Toyofuku T, Okamoto Y, Ishikawa T, Sasawatari S, Kumanogoh A.
      Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Impaired mitochondrial function is suspected to play a major role in PD. Nonetheless, the underlying mechanism by which impaired LRRK2 activity contributes to PD pathology remains unclear. Here, we identified the role of LRRK2 in endoplasmic reticulum (ER)-mitochondrial tethering, which is essential for mitochondrial bioenergetics. LRRK2 regulated the activities of E3 ubiquitin ligases MARCH5, MULAN, and Parkin via kinase-dependent protein-protein interactions. Kinase-active LRRK2(G2019S) dissociated from these ligases, leading to their PERK-mediated phosphorylation and activation, thereby increasing ubiquitin-mediated degradation of ER-mitochondrial tethering proteins. By contrast, kinase-dead LRRK2(D1994A)-bound ligases blocked PERK-mediated phosphorylation and activation of E3 ligases, thereby increasing the levels of ER-mitochondrial tethering proteins. Thus, the role of LRRK2 in the ER-mitochondrial interaction represents an important control point for cell fate and pathogenesis in PD.
    Keywords:   PERK ; LRRK2; endoplasmic reticulum; mitochondria; ubiquitin ligase
    DOI:  https://doi.org/10.15252/embj.2018100875
  13. Cell Rep. 2019 Dec 10. pii: S2211-1247(19)31494-9. [Epub ahead of print]29(11): 3620-3635.e7
    Kouloulia S, Hallin EI, Simbriger K, Amorim IS, Lach G, Amvrosiadis T, Chalkiadaki K, Kampaite A, Truong VT, Hooshmandi M, Jafarnejad SM, Skehel P, Kursula P, Khoutorsky A, Gkogkas CG.
      The translation initiation repressor 4E-BP2 is deamidated in the brain on asparagines N99/N102 during early postnatal brain development. This post-translational modification enhances 4E-BP2 association with Raptor, a central component of mTORC1 and alters the kinetics of excitatory synaptic transmission. We show that 4E-BP2 deamidation is neuron specific, occurs in the human brain, and changes 4E-BP2 subcellular localization, but not its disordered structure state. We demonstrate that deamidated 4E-BP2 is ubiquitinated more and degrades faster than the unmodified protein. We find that enhanced deamidated 4E-BP2 degradation is dependent on Raptor binding, concomitant with increased association with a Raptor-CUL4B E3 ubiquitin ligase complex. Deamidated 4E-BP2 stability is promoted by inhibiting mTORC1 or glutamate receptors. We further demonstrate that deamidated 4E-BP2 regulates the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria, and NF-κB activity, and thus may be crucial for postnatal brain development in neurodevelopmental disorders, such as ASD.
    Keywords:  4E-BP2; CUL4B; NF-κB; Raptor; asparagine deamidation; mTORC1; postnatal brain; proteasome; translational control
    DOI:  https://doi.org/10.1016/j.celrep.2019.11.023
  14. J Biol Chem. 2019 Dec 12. pii: jbc.RA119.011774. [Epub ahead of print]
    Wang H, Loerke D, Bruns C, Müller R, Koch PA, Puchkov D, Schultz C, Haucke V.
      Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates such as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P2 by phosphatidylinositol 3-kinase C2α (PI3KC2α) at plasma membrane clathrin-coated pits (CCPs) is spatially segregated from its hydrolysis by the PI(3,4)P2-specific inositol polyphosphate 4-phosphatase 4A (INPP4A). We observed that INPP4A is dispensable for clathrin-mediated endocytosis (CME) and is undetectable in endocytic CCPs. Instead, we find that INPP4A partially localizes to endosomes and that loss of INPP4A in HAP1 cancer cells perturbs signaling via AKT kinase (AKT) and mTOR complex 1 (mTORC1). These results reveal a function for INPP4-mediated PI(3,4)P2 hydrolysis in the local regulation of growth factor and nutrient signals at endosomes in cancer cells. They further suggest a model according to which the synthesis and turnover of PI(3,4)P2 are spatially segregated within the endocytic pathway to couple endocytic membrane traffic to growth factor and nutrient signaling.
    Keywords:  clathrin; endocytosis; endosome; imaging; lysosome; mTOR complex (mTORC); nutrient signaling; phosphatidylinositol kinase (PI Kinase); phosphatidylinositol phosphatase; phosphoinositide
    DOI:  https://doi.org/10.1074/jbc.RA119.011774
  15. Mol Cell. 2019 Nov 27. pii: S1097-2765(19)30839-1. [Epub ahead of print]
    Klann K, Tascher G, Münch C.
      Regulation of translation is essential during stress. However, the precise sets of proteins regulated by the key translational stress responses-the integrated stress response (ISR) and mTORC1-remain elusive. We developed multiplexed enhanced protein dynamics (mePROD) proteomics, adding signal amplification to dynamic-SILAC and multiplexing, to enable measuring acute changes in protein synthesis. Treating cells with ISR/mTORC1-modulating stressors, we showed extensive translatome modulation with ∼20% of proteins synthesized at highly reduced rates. Comparing translation-deficient sub-proteomes revealed an extensive overlap demonstrating that target specificity is achieved on protein level and not by pathway activation. Titrating cap-dependent translation inhibition confirmed that synthesis of individual proteins is controlled by intrinsic properties responding to global translation attenuation. This study reports a highly sensitive method to measure relative translation at the nascent chain level and provides insight into how the ISR and mTORC1, two key cellular pathways, regulate the translatome to guide cellular survival upon stress.
    Keywords:  SILAC; TMT; cap-dependent translation; integrated stress response; mTOR; proteomics; pulse labeling; stress response; translation; unfolded protein response
    DOI:  https://doi.org/10.1016/j.molcel.2019.11.010