bims-auttor 2019-12-29 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2019–12–29
eleven papers selected by
Viktor Korolchuk, Newcastle University

Differential regulation of autophagy during metabolic stress in astrocytes and neurons.
ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.
RAB18 loss interferes with lipid droplet catabolism and provokes autophagy network adaptations.
Podocytes maintain high basal levels of autophagy independent of mtor signaling.
Regulation of LC3B levels by ubiquitination and proteasomal degradation.
Loss of autophagy impairs physiological steatosis by accumulation of NCoR1.
Multifaceted roles of COPII subunits in autophagy.
The protease activity of human ATG4B is regulated by reversible oxidative modification.
On ATG4B as Drug Target for Treatment of Solid Tumours-The Knowns and the Unknowns.
The Synaptic Autophagy Cycle.
Autophagy in hepatitis B or C virus infection: An incubator and a potential therapeutic target.

Autophagy. 2019 Dec 26. 1-17

Differential regulation of autophagy during metabolic stress in astrocytes and neurons.

Aditi Kulkarni, Audrey Dong, Vineet Vinay Kulkarni, Jessica Chen, Olivia Laxton, Anip Anand, Sandra Maday.

  Macroautophagy/autophagy is a key homeostatic process that targets cytoplasmic components to the lysosome for breakdown and recycling. Autophagy plays critical roles in glia and neurons that affect development, functionality, and viability of the nervous system. The mechanisms that regulate autophagy in glia and neurons, however, are poorly understood. Here, we define the molecular underpinnings of autophagy in primary cortical astrocytes in response to metabolic stress, and perform a comparative study in primary hippocampal neurons. We find that inducing metabolic stress by nutrient deprivation or pharmacological inhibition of MTOR (mechanistic target of rapamycin kinase) robustly activates autophagy in astrocytes. While both paradigms of metabolic stress dampen MTOR signaling, they affect the autophagy pathway differently. Further, we find that starvation-induced autophagic flux is dependent on the buffering system of the starvation solution. Lastly, starvation conditions that strongly activate autophagy in astrocytes have less pronounced effects on autophagy in neurons. Combined, our study reveals the complexity of regulating autophagy in different paradigms of metabolic stress, as well as in different cell types of the brain. Our findings raise important implications for how neurons and glia may collaborate to maintain homeostasis in the brain.Abbreviations ACSF: artificial cerebrospinal fluid; baf A1: bafilomycin A1; EBSS: earle's balanced salt solution; GFAP: glial fibrillary acidic protein; Glc: glucose; GM: glial media; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; p-RPS6: phospho-RPS6; p-ULK1: phospho-ULK1; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; ULK1: unc-51-like kinase 1.

Keywords:  Astrocytes; LC3; MTOR; SQSTM1; autophagy; glia; neurons; starvation

DOI:  https://doi.org/10.1080/15548627.2019.1703354
J Nutr. 2019 Dec 25. pii: nxz309. [Epub ahead of print]

ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.

Dandan Xu, Weiwei Dai, Lydia Kutzler, Holly A Lacko, Leonard S Jefferson, Michael D Dennis, Scot R Kimball.

   BACKGROUND: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase.
OBJECTIVE: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2.
METHODS: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates.
RESULTS: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted.
CONCLUSIONS: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

Keywords:  ATF4; REDD1; Sestrin2; leucine; mTOR

DOI:  https://doi.org/10.1093/jn/nxz309
J Mol Biol. 2019 Dec 21. pii: S0022-2836(19)30740-5. [Epub ahead of print]

RAB18 loss interferes with lipid droplet catabolism and provokes autophagy network adaptations.

Fazilet Bekbulat, Daniel Schmitt, Anne Feldmann, Heike Huesmann, Stefan Eimer, Thomas Juretschke, Petra Beli, Christian Behl, Andreas Kern.

Autophagy is dependent on appropriate lipid supply for autophagosome formation. The regulation of lipid acquisition and the autophagy network response to lipid-limiting conditions are mostly elusive. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet catabolism, causing an impaired fatty acid release. The resulting reduced lipid droplet-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network. These adjustments include increased expression and phosphorylation of ATG2B as well as augmented formation of the ATG12-ATG5 conjugate. Moreover, ATG9A shows an enhanced phosphorylation at amino acid residues tyrosine 8 and serine 14, resulting in an increased ATG9A trafficking. Via pharmacological inhibition of Y8 phosphorylation, we demonstrate that this ATG9A modification is important to maintain basal autophagy under RAB18 knockout conditions. However, while the network adaptations are sufficient to maintain basal autophagic activity, they are incapable of ensuring autophagy induction upon starvation, which is characterized by an enhanced lipid demand. Thus, here, we define the molecular role of RAB18 in connecting lipid droplets and autophagy, emphasize the significance of lipid droplets as lipid sources for the degradative pathway, and uncover a remarkable autophagy network plasticity, including phosphorylation-dependent ATG9A activation, to compensate reduced lipid availability in order to rescue basal autophagic activity.

DOI: https://doi.org/10.1016/j.jmb.2019.12.031
Autophagy. 2019 Dec 23. 1-17

Podocytes maintain high basal levels of autophagy independent of mtor signaling.

Tillmann Bork, Wei Liang, Kosuke Yamahara, Philipp Lee, Zhejia Tian, Shuya Liu, Christoph Schell, Kathrin Thedieck, Bjoern Hartleben, Ketan Patel, Pierre-Louis Tharaux, Olivia Lenoir, Tobias B Huber.

  While constant basal levels of macroautophagy/autophagy are a prerequisite to preserve long-lived podocytes at the filtration barrier, MTOR regulates at the same time podocyte size and compensatory hypertrophy. Since MTOR is known to generally suppress autophagy, the apparently independent regulation of these two key pathways of glomerular maintenance remained puzzling. We now report that long-term genetic manipulation of MTOR activity does in fact not influence high basal levels of autophagy in podocytes either in vitro or in vivo. Instead we present data showing that autophagy in podocytes is mainly controlled by AMP-activated protein kinase (AMPK) and ULK1 (unc-51 like kinase 1). Pharmacological inhibition of MTOR further shows that the uncoupling of MTOR activity and autophagy is time dependent. Together, our data reveal a novel and unexpected cell-specific mechanism, which permits concurrent MTOR activity as well as high basal autophagy rates in podocytes. Thus, these data indicate manipulation of the AMPK-ULK1 axis rather than inhibition of MTOR as a promising therapeutic intervention to enhance autophagy and preserve podocyte homeostasis in glomerular diseases.Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy related; BW: body weight; Cq: chloroquine; ER: endoplasmic reticulum; ESRD: end stage renal disease; FACS: fluorescence activated cell sorting; GFP: green fluorescent protein; i.p.: intra peritoneal; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PLA: proximity-ligation assay; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; RPTOR/RAPTOR: regulatory associated protein of MTOR, complex 1; RFP: red fluorescent protein; TSC1: tuberous sclerosis 1; ULK1: unc-51 like kinase 1.

Keywords:  AMPK; LC3; MTOR; Raptor; Tsc1; autophagy; glomerulus; kidney; podocyte; rapamycin; signaling

DOI:  https://doi.org/10.1080/15548627.2019.1705007
Autophagy. 2019 Dec 27.

Regulation of LC3B levels by ubiquitination and proteasomal degradation.

Rui Jia, Juan S Bonifacino.

  Like other biological processes, macroautophagy/autophagy must be tightly controlled for maintenance of cellular homeostasis and for proper response to changing cellular conditions. To gain insights into the regulation of autophagy, we recently conducted a genome-wide CRISPR-Cas9 knockout screen using cells expressing endogenous LC3B tagged with GFP-mCherry as a reporter. This approach allowed us to identify the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as novel autophagy regulators. We found that these enzymes cooperate to mediate monoubiquitination and proteasomal degradation of LC3B, thus limiting the pool of LC3B available for autophagy. Depletion of UBA6 or BIRC6 increased the level of cytosolic LC3B, enhancing the degradation of autophagy adaptors and the clearance of intracellular proteins aggregates. This finding could be the basis for the development of pharmacological inhibitors of UBA6 or BIRC6 for the treatment of protein aggregation disorders. Recent work by another group showed that BIRC6 itself is subject to ubiquitination and proteasomal degradation, highlighting the existence of a complex regulatory network for the control of LC3B levels.

Keywords:  Apollon; Autophagy; BIRC6; BRUCE; CRISPR-Cas9 screen; LC3; UBA6; intracellular protein aggregates; proteasomal degradation; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2019.1709766
Life Sci Alliance. 2020 Jan;pii: e201900513. [Epub ahead of print]3(1):

Loss of autophagy impairs physiological steatosis by accumulation of NCoR1.

Shun-Saku Takahashi, Yu-Shin Sou, Tetsuya Saito, Akiko Kuma, Takayuki Yabe, Yuki Sugiura, Hyeon-Cheol Lee, Makoto Suematsu, Takehiko Yokomizo, Masato Koike, Shuji Terai, Noboru Mizushima, Satoshi Waguri, Masaaki Komatsu.

Lipid droplets (LDs) are dynamic organelles that store neutral lipids during times of energy excess, such as after a meal. LDs serve as an energy reservoir during fasting and have a buffering capacity that prevents lipotoxicity. Autophagy and the autophagic machinery have been proposed to play a role in LD biogenesis, but the underlying molecular mechanism remains unclear. Here, we show that when nuclear receptor co-repressor 1 (NCoR1), which inhibits the transactivation of nuclear receptors, accumulates because of autophagy suppression, LDs decrease in size and number. Ablation of ATG7, a gene essential for autophagy, suppressed the expression of gene targets of liver X receptor α, a nuclear receptor responsible for fatty acid and triglyceride synthesis in an NCoR1-dependent manner. LD accumulation in response to fasting and after hepatectomy was hampered by the suppression of autophagy. These results suggest that autophagy controls physiological hepatosteatosis by fine-tuning NCoR1 protein levels.

DOI: https://doi.org/10.26508/lsa.201900513
Biochim Biophys Acta Mol Cell Res. 2019 Dec 19. pii: S0167-4889(19)30235-6. [Epub ahead of print]1867(4): 118627

Multifaceted roles of COPII subunits in autophagy.

Zhongyu Li, Wei Huang, Wei Wang.

  COPII vesicles mediate anterograde ER-Golgi traffic of newly synthesized proteins in nutrient rich conditions. An accumulating body of results indicates that the secretory COPII vesicles can be shifted to the roles in autophagosome formation and selective ER-phagy (autophagy of ER), depending on their specific subunits, in response to environmental stresses. In this mini-review, we summarize and discuss the multifaceted roles of COPII vesicles in autophagy and the underlying molecular mechanisms.

Keywords:  Autophagy; COPII; ER-Golgi traffic; ER-phagy

DOI:  https://doi.org/10.1016/j.bbamcr.2019.118627
Autophagy. 2019 Dec 27.

The protease activity of human ATG4B is regulated by reversible oxidative modification.

Xueping Zheng, Zuolong Yang, Qianqian Gu, Fan Xia, Yuanyuan Fu, Peiqing Liu, Xiao-Ming Yin, Min Li.

  Macroautophagy/autophagy plays a pivotal role in cytoplasmic material recycling and metabolic turnover, in which ATG4B functions as a "scissor" for processing pro-LC3 and lipidated LC3 to drive the autophagy progress. Mounting evidence has demonstrated the tight connection between ROS and autophagy during various pathological situations. Coincidentally, several studies have shown that ATG4B is potentially regulated by redox modification, but the underlying molecular mechanism and its relationship with autophagy is ambiguous. In this study, we verified that ATG4B activity was definitely regulated in a reversible redox manner. We also determined that Cys292 and Cys361 are essential sites of ATG4B to form reversible intramolecular disulfide bonds that respond to oxidative stress. Interestingly, we unraveled a new phenomenon that ATG4B concurrently formed disulfide-linked oligomers at Cys292 and Cys361, and that both sites underwent redox modifications thereby modulating ATG4B activity. Finally, increased autophagic flux and decreased oxidation sensitivity were observed in Cys292 and Cys361 double site-mutated cells under normal growth conditions. In conclusion, our research reveals a novel molecular mechanism that oxidative modification at Cys292 and Cys361 sites regulates ATG4B function, which modulates autophagy.

Keywords:  ATG4B; autophagy; disulfide bond; oligomer; oxidative modification; redox

DOI:  https://doi.org/10.1080/15548627.2019.1709763
Cells. 2019 Dec 24. pii: E53. [Epub ahead of print]9(1):

On ATG4B as Drug Target for Treatment of Solid Tumours-The Knowns and the Unknowns.

Alexander Agrotis, Robin Ketteler.

  Autophagy is an evolutionary conserved stress survival pathway that has been shown to play an important role in the initiation, progression, and metastasis of multiple cancers; however, little progress has been made to date in translation of basic research to clinical application. This is partially due to an incomplete understanding of the role of autophagy in the different stages of cancer, and also to an incomplete assessment of potential drug targets in the autophagy pathway. While drug discovery efforts are on-going to target enzymes involved in the initiation phase of the autophagosome, e.g., unc51-like autophagy activating kinase (ULK)1/2, vacuolar protein sorting 34 (Vps34), and autophagy-related (ATG)7, we propose that the cysteine protease ATG4B is a bona fide drug target for the development of anti-cancer treatments. In this review, we highlight some of the recent advances in our understanding of the role of ATG4B in autophagy and its relevance to cancer, and perform a critical evaluation of ATG4B as a druggable cancer target.

Keywords:  ATG4; autophagy; biomarker; drug screening; pancreatic ductal adenocarcinoma (PDAC); screening assay; small molecule compound

DOI:  https://doi.org/10.3390/cells9010053
J Mol Biol. 2019 Dec 19. pii: S0022-2836(19)30737-5. [Epub ahead of print]

The Synaptic Autophagy Cycle.

Ori J Lieberman, David Sulzer.

  Macroautophagy (hereafter referred to as autophagy) is a evolutionarily conserved pathway in which proteins and organelles are delivered to the lysosome for degradation. In neurons, autophagy was originally described as associated with disease states and neuronal survival. Over the last decade, however, evidence has accumulated that autophagy controls synaptic function in both the axon and dendrite. Here, we review this literature, highlighting the role of autophagy in the pre- and postsynapse, synaptic plasticity, and behavior. We end by discussing open questions in the field of synaptic autophagy.

Keywords:  Autophagy; endocytosis; protein degradation; synaptic plasticity; synaptic transmission

DOI:  https://doi.org/10.1016/j.jmb.2019.12.028
Life Sci. 2019 Dec 19. pii: S0024-3205(19)31134-8. [Epub ahead of print] 117206

Autophagy in hepatitis B or C virus infection: An incubator and a potential therapeutic target.

Lu Zhang.

  Autophagy is a metabolic process that maintains the homeostasis of the intracellular environment by degrading damaged proteins and organelles in lysosomes. Recently, independent studies have indicated that some hepatitis viruses develop abilities to hijack autophagy for their replication, such as hepatitis B (HBV) and C (HCV); these two viruses are structurally unrelated, but both of them can cause hepatitis, liver cirrhosis, and even hepatocellular carcinoma (HCC). In this review, we will summarize the current knowledge about the mechanism of autophagy induction in HBV- and HCV-infected hepatocytes, discuss how induced autophagy promotes HBV and HCV replication and describe some autophagy-related therapeutic strategies, which may provide perspective with which to cure HBV and HCV infections and their related liver diseases.

Keywords:  Autophagy; Hepatitis B; Hepatitis C

DOI:  https://doi.org/10.1016/j.lfs.2019.117206