bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒11‒01
38 papers selected by
Viktor Korolchuk, Newcastle University
  1. Ischemia-induced Upregulation of Autophagy Preludes Dysfunctional Lysosomal Storage and Associated Synaptic Impairments in Neurons.
  2. Defective lysosome reformation during autophagy causes skeletal muscle disease.
  3. The multifaceted functions of ATG16L1 in autophagy and related processes.
  4. Aberrant autophagosome formation occurs upon small molecule inhibition of ULK1 kinase activity.
  5. Sensitive ELISA-based detection method for the mitophagy marker p-S65-Ub in human cells, autopsy brain, and blood samples.
  6. MEKK3-MEK5-ERK5 signaling promotes mitochondrial degradation.
  7. Maintaining the balance of TDP-43, mitochondria, and autophagy: a promising therapeutic strategy for neurodegenerative diseases.
  8. Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery.
  9. Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion.
  10. Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.
  11. Selective Autophagy by Close Encounters of the Ubiquitin Kind.
  12. Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways.
  13. Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression.
  14. Autophagy and heat: a potential role for heat therapy to improve autophagic function in health and disease.
  15. A novel reticulophagy receptor, Epr1: a bridge between the phagophore protein Atg8 and ER transmembrane VAP proteins.
  16. Activation of mTORC1 by LSECtin in macrophages directs intestinal repair in inflammatory bowel disease.
  17. Extracellular SQSTM1 as an inflammatory mediator.
  18. Structure, lipid scrambling activity and role in autophagosome formation of ATG9A.
  19. PTPN9-mediated dephosphorylation of VTI1B promotes ATG16L1 precursor fusion and autophagosome formation.
  20. Cp1/cathepsin L is required for autolysosomal clearance in Drosophila.
  21. Programmed cell death 4 modulates lysosomal function by inhibiting TFEB translation.
  22. ATF4 links ER stress with reticulophagy in glioblastoma cells.
  23. Aging, senescence and mitochondria: the PGC-1/ERR axis.
  24. USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.
  25. Upregulation of Cellular Palmitoylation Mitigates α-Synuclein Accumulation and Neurotoxicity.
  26. The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death.
  27. Autophagy plays a role in the prolongation of the lifespan of Caenorhabditis elegans by astaxanthin.
  28. mTOR-dependent translation amplifies microglia priming in aging mice.
  29. Insights into Lysosomal PI(3,5)P2 Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex.
  30. Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.
  31. Upregulation of CFTR Protects against Palmitate-Induced Endothelial Dysfunction by Enhancing Autophagic Flux.
  32. Disruption of Atg7-dependent autophagy causes electromotility disturbances, outer hair cell loss, and deafness in mice.
  33. Transcription factor EB: an emerging drug target for neurodegenerative disorders.
  34. ER-phagy in human atherosclerosis: an exploratory ultrastructural study.
  35. Synergistic Carcinogenesis of HPV18 and MNNG in Het-1A Cells through p62-KEAP1-NRF2 and PI3K/AKT/mTOR Pathway.
  36. BECN2 (beclin 2)-mediated non-canonical autophagy in innate immune signaling and tumor development.
  37. Dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibits human renal cell carcinoma cell growth.
  38. MTORC1 and the Rebirth of Stemness.
  1. Autophagy. 2020 Oct 28.
    Ischemia-induced Upregulation of Autophagy Preludes Dysfunctional Lysosomal Storage and Associated Synaptic Impairments in Neurons.
    Xia Zhang, Mengping Wei, Jiahui Fan, Weijie Yan, Xu Zha, Huimeng Song, Rongqi Wan, Yanling Yin, Wei Wang.
      Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins.
    Keywords:  functional lysosomal storage; lysosomal-associated membrane protein 1 (LAMP1); lysosome; middle cerebral artery occlusion (MCAO); neurons; oxygen-glucose deprivation (OGD); synaptic plasticity
    DOI:  https://doi.org/10.1080/15548627.2020.1840796
  2. J Clin Invest. 2020 Oct 29. pii: 135124. [Epub ahead of print]
    Defective lysosome reformation during autophagy causes skeletal muscle disease.
    Meagan J McGrath, Matthew J Eramo, Rajendra Gurung, Absorn Sriratana, Stefan M Gehrig, Gordon S Lynch, Sonia Raveena Lourdes, Frank Koentgen, Sandra J Feeney, Michael Lazarou, Catriona A McLean, Christina A Mitchell.
      The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We show the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyses phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol 4-phosphate (PI(4)P) and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report loss of INPP5K in muscle causes severe disease, autophagy inhibition and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k-/- muscle suppresses autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k-/- myoblasts was characterised by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane-recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules which we propose interferes with ALR completion. Inhibition of PI(4,5)P2 synthesis, or expression of wild-type, but not INPP5K-disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes is integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k-/- muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy.
    Keywords:  Autophagy; Cell Biology; Lysosomes; Muscle Biology; Skeletal muscle
    DOI:  https://doi.org/10.1172/JCI135124
  3. J Cell Sci. 2020 Oct 30. pii: jcs249227. [Epub ahead of print]133(20):
    The multifaceted functions of ATG16L1 in autophagy and related processes.
    Noor Gammoh.
      Autophagy requires the formation of membrane vesicles, known as autophagosomes, that engulf cellular cargoes and subsequently recruit lysosomal hydrolases for the degradation of their contents. A number of autophagy-related proteins act to mediate the de novo biogenesis of autophagosomes and vesicular trafficking events that are required for autophagy. Of these proteins, ATG16L1 is a key player that has important functions at various stages of autophagy. Numerous recent studies have begun to unravel novel activities of ATG16L1, including interactions with proteins and lipids, and how these mediate its role during autophagy and autophagy-related processes. Various domains have been identified within ATG16L1 that mediate its functions in recognising single and double membranes and activating subsequent autophagy-related enzymatic activities required for the recruitment of lysosomes. These recent findings, as well as the historical discovery of ATG16L1, pathological relevance, unresolved questions and contradictory observations, will be discussed here.
    Keywords:  ATG16L1; Atg16; Autophagosome; Autophagy; Lipid binding; Membrane recruitment; Phagophore; Single-membrane lipidation
    DOI:  https://doi.org/10.1242/jcs.249227
  4. Life Sci Alliance. 2020 Dec;pii: e202000815. [Epub ahead of print]3(12):
    Aberrant autophagosome formation occurs upon small molecule inhibition of ULK1 kinase activity.
    Maria Zachari, Marianna Longo, Ian G Ganley.
      Autophagy is a crucial homeostatic mechanism that mediates the degradation of damaged or excess intracellular components. Such components are engulfed and sequestered into double membrane autophagosomes, which deliver their contents to lysosomes for degradation. Autophagy plays a role in numerous human disorders and its pharmacological targeting by small molecules offers therapeutic potential. The serine/threonine kinase ULK1 (and its homologue ULK2) is the most upstream component of the autophagic machinery and is required for autophagy initiation. Here, we use the most selective and potent published ULK1 inhibitors to gain insights into ULK1 kinase function during autophagy. Treatment with all inhibitors blocked autophagy but also resulted in the limited formation of initial autophagosome-like structures, which appeared abnormal in size and did not traffic to lysosomes. We found that upon ULK1 inhibition, phosphatidylinositol-3-phosphate-binding proteins are still recruited to forming autophagosomes, implying that ULK1 activity is not essential for VPS34 activation. We conclude that the kinase activity of ULK1 is important in regulating autophagosome maturation, by the phosphorylation of currently unidentified key substrates.
    DOI:  https://doi.org/10.26508/lsa.202000815
  5. Autophagy. 2020 Oct 28. 1-16
    Sensitive ELISA-based detection method for the mitophagy marker p-S65-Ub in human cells, autopsy brain, and blood samples.
    Jens O Watzlawik, Xu Hou, Dominika Truban, Chloe Ramnarine, Sandeep K Barodia, Tania F Gendron, Michael G Heckman, Michael DeTure, Joanna Siuda, Zbigniew K Wszolek, Clemens R Scherzer, Owen A Ross, Guojun Bu, Dennis W Dickson, Matthew S Goldberg, Fabienne C Fiesel, Wolfdieter Springer.
      Mitochondrial dysfunction is an early, imminent event in neurodegenerative disorders including Parkinson disease (PD) and Alzheimer disease (AD). The enzymatic pair PINK1 and PRKN/Parkin recognize and transiently label damaged mitochondria with ubiquitin (Ub) phosphorylated at Ser65 (p-S65-Ub) as a signal for degradation via the autophagy-lysosome system (mitophagy). Despite its discovery in cell culture several years ago, robust and quantitative detection of altered mitophagy in vivo has remained challenging. Here we developed a sandwich ELISA targeting p-S65-Ub with the goal to assess mitophagy levels in mouse brain and in human clinical and pathological samples. We characterized five total Ub and four p-S65-Ub antibodies by several techniques and found significant differences in their ability to recognize phosphorylated Ub. The most sensitive antibody pair detected recombinant p-S65-Ub chains in the femtomolar to low picomolar range depending on the poly-Ub chain linkage. Importantly, this ELISA was able to assess very low baseline mitophagy levels in unstressed human cells and in brains from wild-type and prkn knockout mice as well as elevated p-S65-Ub levels in autopsied frontal cortex from AD patients vs. control cases. Moreover, the assay allowed detection of p-S65-Ub in blood plasma and was able to discriminate between PINK1 mutation carriers and controls. In summary, we developed a robust and sensitive tool to measure mitophagy levels in cells, tissue, and body fluids. Our data strongly support the idea that the stress-activated PINK1-PRKN mitophagy pathway is constitutively active in mice and humans under unstimulated, physiological and elevated in diseased, pathological conditions. Abbreviations: Ab: antibody; AD: Alzheimer disease; AP: alkaline phosphatase; CV: coefficient of variation; ECL: electrochemiluminescence; KO: knockout; LoB: Limit of Blank; LoD: Limit of Detection; LoQ: Limit of Quantification; MSD: meso scale discovery; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated ubiquitin at serine 65; Std.Dev.: standard deviation; Ub: ubiquitin; WT: wild type.
    Keywords:  Alzheimer disease; PINK1; PRKN; Parkin; Parkinson disease; autophagy; mitophagy; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2020.1834712
  6. Cell Death Discov. 2020 ;6 107
    MEKK3-MEK5-ERK5 signaling promotes mitochondrial degradation.
    Jane E Craig, Joseph N Miller, Raju R Rayavarapu, Zhenya Hong, Gamze B Bulut, Wei Zhuang, Sadie Miki Sakurada, Jamshid Temirov, Jonathan A Low, Taosheng Chen, Shondra M Pruett-Miller, Lily Jun-Shen Huang, Malia B Potts.
      Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.
    Keywords:  Mitophagy; Stress signalling
    DOI:  https://doi.org/10.1038/s41420-020-00342-7
  7. Transl Neurodegener. 2020 Oct 30. 9(1): 40
    Maintaining the balance of TDP-43, mitochondria, and autophagy: a promising therapeutic strategy for neurodegenerative diseases.
    Chunhui Huang, Sen Yan, Zaijun Zhang.
      Mitochondria are the energy center of cell operations and are involved in physiological functions and maintenance of metabolic balance and homeostasis in the body. Alterations of mitochondrial function are associated with a variety of degenerative and acute diseases. As mitochondria age in cells, they gradually become inefficient and potentially toxic. Acute injury can trigger the permeability of mitochondrial membranes, which can lead to apoptosis or necrosis. Transactive response DNA-binding protein 43 kDa (TDP-43) is a protein widely present in cells. It can bind to RNA, regulate a variety of RNA processes, and play a role in the formation of multi-protein/RNA complexes. Thus, the normal physiological functions of TDP-43 are particularly important for cell survival. Normal TDP-43 is located in various subcellular structures including mitochondria, mitochondrial-associated membrane, RNA particles and stress granules to regulate the endoplasmic reticulum-mitochondrial binding, mitochondrial protein translation, and mRNA transport and translation. Importantly, TDP-43 is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia and Alzheimer's disease, which are characterized by abnormal phosphorylation, ubiquitination, lysis or nuclear depletion of TDP-43 in neurons and glial cells. Although the pathogenesis of TDP-43 proteinopathy remains unknown, the presence of pathological TDP-43 inside or outside of mitochondria and the functional involvement of TDP-43 in the regulation of mitochondrial morphology, transport, and function suggest that mitochondria are associated with TDP-43-related diseases. Autophagy is a basic physiological process that maintains the homeostasis of cells, including targeted clearance of abnormally aggregated proteins and damaged organelles in the cytoplasm; therefore, it is considered protective against neurodegenerative diseases. However, the combination of abnormal TDP-43 aggregation, mitochondrial dysfunction, and insufficient autophagy can lead to a variety of aging-related pathologies. In this review, we describe the current knowledge on the associations of mitochondria with TDP-43 and the role of autophagy in the clearance of abnormally aggregated TDP-43 and dysfunctional mitochondria. Finally, we discuss a novel approach for neurodegenerative treatment based on the knowledge.
    Keywords:  Autophagy/mitophagy; Mitochondria; Neurodegeneration; TDP-43
    DOI:  https://doi.org/10.1186/s40035-020-00219-w
  8. Int J Mol Sci. 2020 Oct 28. pii: E8051. [Epub ahead of print]21(21):
    Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery.
    Yiu Wing Sunny Cheung, Sung-Eun Nam, Calvin K Yip.
      Macroautophagy (also known as autophagy) is a major pathway for selective degradation of misfolded/aggregated proteins and damaged organelles and non-selective degradation of cytoplasmic constituents for the generation of power during nutrient deprivation. The multi-step degradation process, from sequestering cytoplasmic cargo into the double-membrane vesicle termed autophagosome to the delivery of the autophagosome to the lysosome or lytic vacuole for breakdown, is mediated by the core autophagy machinery composed of multiple Atg proteins, as well as the divergent sequence family of selective autophagy receptors. Single-particle electron microscopy (EM) is a molecular imaging approach that has become an increasingly important tool in the structural characterization of proteins and macromolecular complexes. This article summarizes the contributions single-particle EM have made in advancing our understanding of the core autophagy machinery and selective autophagy receptors. We also discuss current technical challenges and roadblocks, as well as look into the future of single-particle EM in autophagy research.
    Keywords:  Atg proteins; autophagy; cryo-EM; selective autophagy; single-particle electron microscopy
    DOI:  https://doi.org/10.3390/ijms21218051
  9. Nat Struct Mol Biol. 2020 Oct 26.
    Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion.
    Kazuaki Matoba, Tetsuya Kotani, Akihisa Tsutsumi, Takuma Tsuji, Takaharu Mori, Daisuke Noshiro, Yuji Sugita, Norimichi Nomura, So Iwata, Yoshinori Ohsumi, Toyoshi Fujimoto, Hitoshi Nakatogawa, Masahide Kikkawa, Nobuo N Noda.
      The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
    DOI:  https://doi.org/10.1038/s41594-020-00518-w
  10. Autophagy. 2020 Oct 30.
    Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein.
    Hui-Yun Hwang, Joong Sup Shim, Dasol Kim, Ho Jeong Kwon.
      Macroautophagy/autophagy (hereafter autophagy), the process of mass degradation of unnecessary elements within the cell, is often dysregulated in many diseases such as cancer, atherosclerosis, and neurodegenerative diseases. Hence, autophagy modulating agents have a great potential to be therapeutic agents for the autophagy-related diseases. Here we report that an anti-depressant drug sertraline (Sert) is an autophagy-inducing agent. Mechanistically, Sert potentially binds to and antagonizes the mitochondrial VDAC1 (voltage dependent anion channel 1), resulting in reduced cellular ATP (adenosine triphosphate) level, activation of AMP-activated protein kinase (AMPK) and inhibition of its downstream, MTOR (mechanistic target of rapamycin kinase)-RPS6KB1 (ribosomal protein S6 kinase B1) signaling pathway. Cells lacking VDAC1 expression completely abrogate the modulatory effect of Sert on AMPK-MTOR pathway and autophagy-inducing activity. We further show that Sert suppresses tauopathy by promoting the autophagic degradation of MAPT (microtubule associated protein tau) protein via inducing autophagy. Our study demonstrates the potential of Sert as a novel small molecule autophagy-inducing agent and provides a new drug candidate to treat autophagy related diseases by targeting VDAC1.
    Keywords:  AMPK; DARTS; MAPT; MTOR; Sert; VDAC1; antidepressant; tauopathy
    DOI:  https://doi.org/10.1080/15548627.2020.1841953
  11. Cells. 2020 Oct 24. pii: E2349. [Epub ahead of print]9(11):
    Selective Autophagy by Close Encounters of the Ubiquitin Kind.
    Anna Vainshtein, Paolo Grumati.
      Autophagy, a bulk degradation process within eukaryotic cells, is responsible for cellular turnover and nutrient liberation during starvation. Increasing evidence indicate that this process can be extremely discerning. Selective autophagy segregates and eliminates protein aggregates, damaged organelles, and invading organisms. The specificity of this process is largely mediated by post-translational modifications (PTMs), which are recognized by autophagy receptors. These receptors grant autophagy surgical precision in cargo selection, where only tagged substrates are engulfed within autophagosomes and delivered to the lysosome for proteolytic breakdown. A growing number of selective autophagy receptors have emerged including p62, NBR1, OPTN, NDP52, TAX1BP1, TOLLIP, and more continue to be uncovered. The most well-documented PTM is ubiquitination and selective autophagy receptors are equipped with a ubiquitin binding domain and an LC3 interacting region which allows them to physically bridge cargo to autophagosomes. Here, we review the role of ubiquitin and ubiquitin-like post-translational modifications in various types of selective autophagy.
    Keywords:  ER-phagy; aggrephagy; cargo receptors; lipophagy; lysophagy; mitophagy; nucleophagy; selective autophagy; ubiquitin; xenophagy
    DOI:  https://doi.org/10.3390/cells9112349
  12. Cell Death Differ. 2020 Oct 27.
    Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways.
    Jin Hwa Cho, Kidae Kim, Sung Ah Kim, Sungryul Park, Bi-Oh Park, Jong-Hwan Kim, Seon-Young Kim, Min Jee Kwon, Myeong Hoon Han, Sung Bae Lee, Byoung Chul Park, Sung Goo Park, Jeong-Hoon Kim, Sunhong Kim.
      The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized β-transducin repeat-containing protein 1 (βTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.
    DOI:  https://doi.org/10.1038/s41418-020-00649-z
  13. Cell Rep. 2020 Oct 27. pii: S2211-1247(20)31310-3. [Epub ahead of print]33(4): 108321
    Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression.
    María Clara Ingaramo, Juan Andrés Sánchez, Norbert Perrimon, Andrés Dekanty.
      The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, we show that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status.
    Keywords:  AMPK; Drosophila; Leptin; Upd2; fat body; insulin-producing cells; inter-organ communication; metabolism; p53; starvation
    DOI:  https://doi.org/10.1016/j.celrep.2020.108321
  14. J Appl Physiol (1985). 2020 Oct 29.
    Autophagy and heat: a potential role for heat therapy to improve autophagic function in health and disease.
    James J McCormick, Karol Dokladny, Pope L Moseley, Glen P Kenny.
      Autophagy is a crucial cell survival mechanism that involves the degradation and recycling of old or damaged organelles and proteins to maintain cellular homeostasis. Impairments in autophagy are central to the pathogenesis of many conditions including metabolic and neurodegenerative disorders, cardiovascular and pulmonary diseases, diabetes, and aging. While various pharmacological agents may be able to stimulate autophagic function, to the best of our knowledge few interventions exist which have been deemed safe and effective in humans. An emerging body of evidence suggests that targeting the autophagic pathway via passive heating (heat therapy) may stimulate autophagic function. Therefore, the primary focus of the present review is to analyze the mechanisms in which passive heating induces autophagy as defined by in vitro and in vivo (animals and humans) models. Our secondary focus is to examine the implications of utilizing passive heating to restore dysfunctional autophagy in chronic disease and aging. Finally, we discuss potential therapeutic strategies to implement passive heating to stimulate autophagic function in humans.
    Keywords:  aging; autophagy; chronic disease; heat stress; heat therapy
    DOI:  https://doi.org/10.1152/japplphysiol.00542.2020
  15. Autophagy. 2020 Oct 29. 1-2
    A novel reticulophagy receptor, Epr1: a bridge between the phagophore protein Atg8 and ER transmembrane VAP proteins.
    Ying Yang, Daniel J Klionsky.
      Reticulophagy, a type of selective autophagy that specifically targets and degrades parts of the endoplasmic reticulum (ER) network (sheets or tubules), plays a crucial role in the responses to ER stress. The selectivity of the ER cargo recognition relies on the unique reticulophagy receptors, which tether and deliver cargos to phagophores, the precursors to autophagosomes. Various integral membrane proteins have been well characterized as reticulophagy receptors, including Atg39, Atg40, RETREG1/FAM134B, SEC62, RTN3L, CCPG1, TEX264, and ATL3, in both yeast and mammals in the past five years. In a recent paper, Zhao et al. discovered in fission yeast a novel reticulophagy receptor, Epr1, which bridges the ER and phagophore by binding to Atg8 and VAPs, a mechanism different from the aforementioned reticulophagy receptors.
    Keywords:  Autophagy; endoplasmic reticulum; stress; vacuole; yeast
    DOI:  https://doi.org/10.1080/15548627.2020.1837457
  16. Cell Death Dis. 2020 Oct 26. 11(10): 918
    Activation of mTORC1 by LSECtin in macrophages directs intestinal repair in inflammatory bowel disease.
    Qian Li, Hanxing Cheng, Yuanping Liu, Xiaowen Wang, Fuchu He, Li Tang.
      Damage to intestinal epithelial cells and the induction of cellular apoptosis are characteristics of inflammatory bowel disease. The C-type lectin receptor family member LSECtin promotes apoptotic cell clearance by macrophages and induces the production of anti-inflammatory/tissue growth factors, which direct intestinal repair in experimental colitis. However, the mechanisms by which the phagocytosis of apoptotic cells triggers the pro-repair function of macrophages remain largely undefined. Here, using immunoprecipitation in combination with mass spectrometry to identify LSECtin-interacting proteins, we found that LSECtin interacted with mTOR, exhibiting a role in activating mTORC1. Mechanistically, apoptotic cells enhance the interaction between LSECtin and mTOR, and increase the activation of mTORC1 induced by LSECtin in macrophages. Elevated mTORC1 signaling triggers macrophages to produce anti-inflammatory/tissue growth factors that contribute to the proliferation of epithelial cells and promote the reestablishment of tissue homeostasis. Collectively, our findings suggest that LSECtin-dependent apoptotic cell clearance by macrophages activates mTORC1, and thus contributes to intestinal regeneration and the remission of colitis.
    DOI:  https://doi.org/10.1038/s41419-020-03114-4
  17. Autophagy. 2020 Oct 28.
    Extracellular SQSTM1 as an inflammatory mediator.
    Borong Zou, Jiao Liu, Daniel J Klionsky, Daolin Tang, Rui Kang.
      Excessive inflammation may lead to irreparable injury and even death, but the key mediators and underlying mechanisms remain unclear. Our recent findings indicate that SQSTM1/p62 (sequestosome 1), a well-known macroautophagy/autophagy receptor, is a lethal inflammatory mediator of sepsis and septic shock. The release of SQSTM1 occurs during tissue damage or microbial invasion through two main ways: one is passive and the other is active. Passive release occurs in the context of GSDMD-mediated pyroptosis. Active SQSTM1 secretion requires two basic steps: the first step is the expression and phosphorylation of SQSTM1 mediated by STING1/STING/TMEM173, and then the unconventional secretion of SQSTM1 by secretory lysosomes. After release, the extracellular SQSTM1 binds to membrane receptor INSR to activate glycolysis, leading to subsequent production of pro-inflammatory cytokines in a transcription factor NFKB-dependent manner. Functionally, genetic deletion or pharmacological inhibition of the SQSTM1-INSR pathway limits tissue damage, systemic inflammation, organ failure, and death in experimental sepsis models in mice. Moreover, the activation of the SQSTM1-INSR pathway is related to the severity of sepsis in patients. These findings highlight a pathological role of extracellular SQSTM1 in infection, inflammation, and immunity.
    Keywords:  DAMP; INSR; SQSTM1; STING1; TLR4; autophagy; immunometabolism; inflammasome; sepsis
    DOI:  https://doi.org/10.1080/15548627.2020.1843253
  18. Nat Struct Mol Biol. 2020 Oct 26.
    Structure, lipid scrambling activity and role in autophagosome formation of ATG9A.
    Shintaro Maeda, Hayashi Yamamoto, Lisa N Kinch, Christina M Garza, Satoru Takahashi, Chinatsu Otomo, Nick V Grishin, Stefano Forli, Noboru Mizushima, Takanori Otomo.
      De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.
    DOI:  https://doi.org/10.1038/s41594-020-00520-2
  19. Autophagy. 2020 Oct 28. 1-16
    PTPN9-mediated dephosphorylation of VTI1B promotes ATG16L1 precursor fusion and autophagosome formation.
    He-Yen Chou, Yi-Tang Lee, Yuchieh Jay Lin, Jung-Kun Wen, Wen-Hsin Peng, Pei-Lien Hsieh, Shu-Yu Lin, Chin-Chun Hung, Guang-Chao Chen.
      Macroautophagy/autophagy is an evolutionarily conserved intracellular pathway for the degradation of cytoplasmic materials. Under stress conditions, autophagy is upregulated and double-membrane autophagosomes are formed by the expansion of phagophores. The ATG16L1 precursor fusion contributes to development of phagophore structures and is critical for the biogenesis of autophagosomes. Here, we discovered a novel role of the protein tyrosine phosphatase PTPN9 in the regulation of homotypic ATG16L1 vesicle fusion and early autophagosome formation. Depletion of PTPN9 and its Drosophila homolog Ptpmeg2 impaired autophagosome formation and autophagic flux. PTPN9 colocalized with ATG16L1 and was essential for homotypic fusion of ATG16L1+ vesicles during starvation-induced autophagy. We further identified the Q-SNARE VTI1B as a substrate target of PTPN9 phosphatase. Like PTPN9, the VTI1B nonphosphorylatable mutant but not the phosphomimetic mutant enhanced SNARE complex assembly and autophagic flux. Our findings highlight the important role of PTPN9 in the regulation of ATG16L1+ autophagosome precursor fusion and autophagosome biogenesis through modulation of VTI1B phosphorylation status. Abbreviations: csw: corkscrew; EBSS: Earle's balanced salt solution; ERGIC: ER-Golgi intermediate compartment; ESCRT: endosomal sorting complexes required for transport; mop: myopic; NSF: N-ethylmaleimide-sensitive factor; PAS: phagophore assembly site; PolyQ: polyglutamine; PtdIns3P: phosphatidylinositol-3-phosphate; PTK: protein tyrosine kinase; PTM: posttranslational modification; PTP: protein tyrosine phosphatase; PTPN23/HD-PTP: protein tyrosine phosphatase non-receptor type 23; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; STX7: syntaxin 7; STX8: syntaxin 8; STX17: syntaxin 17; VAMP3: vesicle associated membrane protein 3; VAMP7: vesicle associated membrane protein 7; VTI1B: vesicle transport through interaction with t-SNAREs 1B; YKT6: YKT6 v-SNARE homolog; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.
    Keywords:  ATG16l1; Autophagosome; PTPN9; SNARE; VTI1B
    DOI:  https://doi.org/10.1080/15548627.2020.1838117
  20. Autophagy. 2020 Oct 28. 1-16
    Cp1/cathepsin L is required for autolysosomal clearance in Drosophila.
    Tianqi Xu, Shannon Nicolson, Jarrod J Sandow, Sonia Dayan, Xin Jiang, Jantina A Manning, Andrew I Webb, Sharad Kumar, Donna Denton.
      Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of our current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Here, using proteomic analysis of Drosophila larval midguts during degradation, we identify a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. We show that Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, we find that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. Our findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover.
    Keywords:   Drosophila ; Autophagy; cell death; lysosome; midgut; proteome
    DOI:  https://doi.org/10.1080/15548627.2020.1838105
  21. Cell Death Differ. 2020 Oct 25.
    Programmed cell death 4 modulates lysosomal function by inhibiting TFEB translation.
    Xiaotong Chen, Yetong Guan, Yi Zhang, Yufeng Jia, Wen Li, Chun Guo, Yuan Li, Xiaoyan Wang, Yongyu Shi, Qun Wang, Faliang Zhu, Yan Li, Lining Zhang.
      Transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis. The post-translational phosphorylation modulations of TFEB by mTOR and ERK signaling can determine its nucleocytoplasmic shuttling and activity in response to nutrient availability. However, regulations of TFEB at translational level are rarely known. Here, we found that programmed cell death 4 (PDCD4), a tumor suppressor, decreased levels of nuclear TFEB to inhibit lysosome biogenesis and function. Mechanistically, PDCD4 reduces global pool of TFEB by suppressing TFEB translation in an eIF4A-dependent manner, rather than influencing mTOR- and ERK2-dependnet TFEB nucleocytoplasmic shuttling. Both of MA3 domains within PDCD4 are required for TFEB translation inhibition. Furthermore, TFEB is required for PDCD4-mediated lysosomal function suppression. In the tumor microenvironment, PDCD4 deficiency promotes the anti-tumor effect of macrophage via enhancing TFEB expression. Our research reveals a novel PDCD4-dependent TFEB translational regulation and supports PDCD4 as a potential therapeutic target for lysosome dysfunction related diseases.
    DOI:  https://doi.org/10.1038/s41418-020-00646-2
  22. Autophagy. 2020 Oct 28. 1-17
    ATF4 links ER stress with reticulophagy in glioblastoma cells.
    Svenja Zielke, Simon Kardo, Laura Zein, Muriel Mari, Adriana Covarrubias-Pinto, Maximilian N Kinzler, Nina Meyer, Alexandra Stolz, Simone Fulda, Fulvio Reggiori, Donat Kögel, Sjoerd J L van Wijk.
      Selective degradation of the endoplasmic reticulum (ER; reticulophagy) is a type of autophagy involved in the removal of ER fragments. So far, amino acid starvation as well as ER stress have been described as inducers of reticulophagy, which in turn restores cellular energy levels and ER homeostasis. Here, we explored the autophagy-inducing mechanisms that underlie the autophagic cell death (ACD)-triggering compound loperamide (LOP) in glioblastoma cells. Interestingly, LOP triggers upregulation of the transcription factor ATF4, which is accompanied by the induction of additional ER stress markers. Notably, knockout of ATF4 significantly attenuated LOP-induced autophagy and ACD. Functionally, LOP also specifically induces the engulfment of large ER fragments within autophagosomes and lysosomes as determined by electron and fluorescence microscopy. LOP-induced reticulophagy and cell death are predominantly mediated through the reticulophagy receptor RETREG1/FAM134B and, to a lesser extent, TEX264, confirming that reticulophagy receptors can promote ACD. Strikingly, apart from triggering LOP-induced autophagy and ACD, ATF4 is also required for LOP-induced reticulophagy. These observations highlight a key role for ATF4, RETREG1 and TEX264 in response to LOP-induced ER stress, reticulophagy and ACD, and establish a novel mechanistic link between ER stress and reticulophagy, with possible implications for additional models of drug-induced ER stress. Abbreviations: ACD: autophagic cell death; ATF6: activating transcription factor 6; ATL3: atlastin 3; BafA1: bafilomycin A1; CCPG1: cell cycle progression gene 1; co-IP: co-immunoprecipitation; DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; GBM: glioblastoma multiforme; HSPA5/BiP: heat shock protein family (Hsp70) member 5; LOP: loperamide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; RETREG1/FAM134B: reticulophagy regulator 1; RTN3L: reticulon 3 long; SEC62: SEC62 homolog, protein translocation factor; TEX264: testis-expressed 264, reticulophagy receptor; UPR: unfolded protein response.
    Keywords:  HSPA5/BiP; MEFs; MZ-54; RETREG1/FAM134B; TEX264; autophagic cell death; loperamide; p-eIF2α; selective autophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1827780
  23. J Mol Endocrinol. 2020 Oct 01. pii: JME-20-0196.R1. [Epub ahead of print]
    Aging, senescence and mitochondria: the PGC-1/ERR axis.
    Vincent Giguere, Mathieu Vernier.
      Aging is a degenerative process that results from the accumulation of cellular and tissue lesions, leading progressively to organ dysfunction and death. Although the biological basis of human aging remains unclear, a large amount of data points to deregulated mitochondrial function as a central regulator of this process. Mounting years of research on aging support the notion that the engendered age-related decline of mitochondria is associated with alterations in key pathways that regulate mitochondrial biology. Particularly, several studies in the last decade have emphasized the importance of the estrogen-related receptor (ERR) family of nuclear receptors, master regulators of mitochondrial function, and their transcriptional coactivators PGC-1s in this context. In this review, we summarize key discoveries implicating the PGC-1/ERR axis in age-associated mitochondrial deregulation and tissue dysfunction. Also, we highlight the pharmacological potential of targeting the PGC-1/ERR axis to alleviate the onset of aging and its adverse effects.
    DOI:  https://doi.org/10.1530/JME-20-0196
  24. Proc Natl Acad Sci U S A. 2020 Oct 26. pii: 202014349. [Epub ahead of print]
    USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.
    Tao Zhang, Goran Periz, Yu-Ning Lu, Jiou Wang.
      An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here, we report the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.
    Keywords:  NEDD4L; SMAD; USP7; protein misfolding; protein quality control
    DOI:  https://doi.org/10.1073/pnas.2014349117
  25. Mov Disord. 2020 Oct 26.
    Upregulation of Cellular Palmitoylation Mitigates α-Synuclein Accumulation and Neurotoxicity.
    Gary P H Ho, Nagendran Ramalingam, Thibaut Imberdis, Erin C Wilkie, Ulf Dettmer, Dennis J Selkoe.
      BACKGROUND: Synucleinopathies, including Parkinson's disease (PD), are characterized by α-synuclein (αS) cytoplasmic inclusions. αS-dependent vesicle-trafficking defects are important in PD pathogenesis, but their mechanisms are not well understood. Protein palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, promotes trafficking by anchoring specific proteins to the vesicle membrane. αS itself cannot be palmitoylated as it lacks cysteines, but it binds to membranes, where palmitoylation occurs, via an amphipathic helix. We hypothesized that abnormal αS membrane-binding impairs trafficking by disrupting palmitoylation. Accordingly, we investigated the therapeutic potential of increasing cellular palmitoylation.OBJECTIVES: We asked whether upregulating palmitoylation by inhibiting the depalmitoylase acyl-protein-thioesterase-1 (APT1) ameliorates pathologic αS-mediated cellular phenotypes and sought to identify the mechanism.
    METHODS: Using human neuroblastoma cells, rat neurons, and iPSC-derived PD patient neurons, we examined the effects of pharmacologic and genetic downregulation of APT1 on αS-associated phenotypes.
    RESULTS: APT1 inhibition or knockdown decreased αS cytoplasmic inclusions, reduced αS serine-129 phosphorylation (a PD neuropathological marker), and protected against αS-dependent neurotoxicity. We identified the APT1 substrate microtubule-associated-protein-6 (MAP6), which binds to vesicles in a palmitoylation-dependent manner, as a key mediator of these effects. Mechanistically, we found that pathologic αS accelerated palmitate turnover on MAP6, suggesting that APT1 inhibition corrects a pathological αS-dependent palmitoylation deficit. We confirmed the disease relevance of this mechanism by demonstrating decreased MAP6 palmitoylation in neurons from αS gene triplication patients.
    CONCLUSIONS: Our findings demonstrate a novel link between the fundamental process of palmitoylation and αS pathophysiology. Upregulating palmitoylation represents an unexplored therapeutic strategy for synucleinopathies. © 2020 International Parkinson and Movement Disorder Society.
    Keywords:  palmitoylation; vesicle trafficking; α-synuclein
    DOI:  https://doi.org/10.1002/mds.28346
  26. Elife. 2020 10 27. pii: e55694. [Epub ahead of print]9
    The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death.
    Nicole L Nuckolls, Anthony C Mok, Jeffrey J Lange, Kexi Yi, Tejbir S Kandola, Andrew M Hunn, Scott McCroskey, Julia L Snyder, María Angélica Bravo Núñez, Melainia McClain, Sean A McKinney, Christopher Wood, Randal Halfmann, Sarah E Zanders.
      Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism to selectively kill meiotic products (spores) that do not inherit wtf4. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate trafficking, as mutations that disrupt either of these processes result in cell death in the presence of the Wtf4 proteins. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy spores.
    Keywords:  Meiotic drive; S. cerevisiae; S. pombe; autophagy; cell biology; evolutionary biology; meiosis; protein aggregation; proteostasis; wtf
    DOI:  https://doi.org/10.7554/eLife.55694
  27. Rejuvenation Res. 2020 Oct 29.
    Autophagy plays a role in the prolongation of the lifespan of Caenorhabditis elegans by astaxanthin.
    Min Fu, Xumei Zhang, Xuguang Zhang, Liu Yang, Suhui Luo, Huan Liu.
      Astaxanthin, a xanthophyll belonging to the family of carotenoids, is a potent antioxidant. The effect of astaxanthin on longevity and its physiological and molecular mechanism are still unclear. In this study, we proved that astaxanthin could prolong the lifespan of Caenorhabditis elegans. To uncover whether astaxanthin delay aging by up-regulating autophagy, we assessed the expression of autophagy genes and found that the expression of autophagy genes was up-regulated after feeding with astaxanthin compared with the control group, and the difference was statistically significant. In order to explore the molecular mechanism of astaxanthin-induced autophagy upregulation, we knocked out the key genes daf-16 or hlh-30 of Insulin/IGF-1 signal pathway and target of rapamycin signal pathway (two common conservative signal pathways capable of regulating autophagy) by RNA interference, and the expression of autophagy gene lgg-1 decreased. Collectively, our results strongly suggest that autophagy, which is both the Insulin/IGF-1 signal pathway-dependent and target of rapamycin signal pathway-dependent, plays a role in the prolongation of the lifespan of Caenorhabditis elegans by astaxanthin.
    DOI:  https://doi.org/10.1089/rej.2020.2355
  28. J Clin Invest. 2020 Oct 27. pii: 132727. [Epub ahead of print]
    mTOR-dependent translation amplifies microglia priming in aging mice.
    Lily Keane, Ignazio Antignano, Sean-Patrick Riechers, Raphael Zollinger, Anaelle A Dumas, Nina Offermann, Maria E Bernis, Jenny Russ, Frederike J Graelmann, Patrick N McCormick, Julia Esser, Dario Tejera, Ai Nagano, Jun Wang, Claude Chelala, Yvonne Biederbick, Annett Halle, Paolo Salomoni, Michael Thomas Heneka, Melania Capasso.
      Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice upregulated mammalian target of rapamycin (mTOR) complex 1 signaling regulating translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual, yet contrasting effect on microglia priming: it caused an NF-kB-dependent upregulation of priming genes at mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating upregulation of mTOR-dependent translation is an essential step licensing microglia priming in aging and neurodegeneration.
    Keywords:  Aging; Cytokines; Inflammation; Macrophages; Translation
    DOI:  https://doi.org/10.1172/JCI132727
  29. Mol Cell. 2020 Oct 15. pii: S1097-2765(20)30686-9. [Epub ahead of print]
    Insights into Lysosomal PI(3,5)P2 Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex.
    Joshua A Lees, PeiQi Li, Nikit Kumar, Lois S Weisman, Karin M Reinisch.
      The phosphoinositide PI(3,5)P2, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P2 levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P2 from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P2 production by PIKfyve in vivo.
    Keywords:  lipid kinase; lipid phosphatase; phosphoinositide homeostasis
    DOI:  https://doi.org/10.1016/j.molcel.2020.10.003
  30. J Cell Sci. 2020 Oct 26. pii: jcs.252015. [Epub ahead of print]
    Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.
    Dale P Corkery, Aftab Nadeem, Kyaw Min Aung, Ahmed Hassan, Tao Liu, Ramón Cervantes-Rivera, Alf Håkon Lystad, Hui Wang, Karina Persson, Andrea Puhar, Anne Simonsen, Bernt Eric Uhlin, Sun Nyunt Wai, Yao-Wen Wu.
      Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.
    Keywords:  Bacterial toxin; IL-1 beta; MakA; Membrane aggregate; Noncanonical autophagy; Unconventional secretion
    DOI:  https://doi.org/10.1242/jcs.252015
  31. Oxid Med Cell Longev. 2020 ;2020 8345246
    Upregulation of CFTR Protects against Palmitate-Induced Endothelial Dysfunction by Enhancing Autophagic Flux.
    Hongqi Chen, Wenliang Chen, Yinlian Yao, Naobei Ye, Ning Hou, Jiandong Luo.
      Saturated free fatty acids (FFAs) elevate in metabolic symptom leading to endothelial dysfunction. Cystic fibrosis transmembrane regulator (CFTR) functionally expresses in endothelial cells. The role of CFTR in FFA-induced endothelial dysfunction remains unclear. This study is aimed at exploring the effects of CFTR on palmitate- (PA-) induced endothelial dysfunction and its underlying mechanisms. We found that PA-induced endothelial dysfunction is characterized by a decrease of cell viability, reduction of NO generation and mitochondrial membrane potential, impairment of the tube formation, but an increase of ROS generation and cell apoptosis. Simultaneously, PA decreased CFTR protein expression. CFTR agonist Forskolin upregulated CFTR protein expression and protected against PA-induced endothelial dysfunction, while CFTR knockdown exacerbated endothelial dysfunction induced by PA and blunted the protective effects of Forskolin. In addition, PA impaired autophagic flux, and autophagic flux inhibitors aggravated PA-induced endothelial apoptosis. CFTR upregulation significantly restored autophagic flux in PA-insulted endothelial cells, which was involved in increasing the protein expression of Atg16L, Atg12-Atg5 complex, cathepsin B, and cathepsin D. In contrast, CFTR knockdown significantly inhibited the effects of Forskolin on autophagic flux and the expression of the autophagy-regulated proteins. Our findings illustrate that CFTR upregulation protects against PA-induced endothelial dysfunction by improving autophagic flux and underlying mechanisms are involved in enhancing autophagic signaling mediated by the Atg16L-Atg12-Atg5 complex, cathepsin B, and cathepsin D. CFTR might serve as a novel drug target for endothelial protection in cardiovascular diseases with a characteristic of elevation of FFAs.
    DOI:  https://doi.org/10.1155/2020/8345246
  32. Cell Death Dis. 2020 Oct 24. 11(10): 913
    Disruption of Atg7-dependent autophagy causes electromotility disturbances, outer hair cell loss, and deafness in mice.
    Han Zhou, Xiaoyun Qian, Nana Xu, Shasha Zhang, Guangjie Zhu, Yuan Zhang, Dingding Liu, Cheng Cheng, Xiaocheng Zhu, Yongze Liu, Ling Lu, Jie Tang, Renjie Chai, Xia Gao.
      Atg7 is an indispensable factor that plays a role in canonical nonselective autophagy. Here we show that genetic ablation of Atg7 in outer hair cells (OHCs) in mice caused stereocilium damage, somatic electromotility disturbances, and presynaptic ribbon degeneration over time, which led to the gradual wholesale loss of OHCs and subsequent early-onset profound hearing loss. Impaired autophagy disrupted OHC mitochondrial function and triggered the accumulation of dysfunctional mitochondria that would otherwise be eliminated in a timely manner. Atg7-independent autophagy/mitophagy processes could not compensate for Atg7 deficiency and failed to rescue the terminally differentiated, non-proliferating OHCs. Our results show that OHCs orchestrate intricate nonselective and selective autophagic/mitophagy pathways working in concert to maintain cellular homeostasis. Overall, our results demonstrate that Atg7-dependent autophagy plays a pivotal cytoprotective role in preserving OHCs and maintaining hearing function.
    DOI:  https://doi.org/10.1038/s41419-020-03110-8
  33. Drug Discov Today. 2020 Oct 21. pii: S1359-6446(20)30430-X. [Epub ahead of print]
    Transcription factor EB: an emerging drug target for neurodegenerative disorders.
    Ju-Xian Song, Jia Liu, Yimin Jiang, Zi-Ying Wang, Min Li.
      The discovery of transcription factor EB (TFEB) as a master regulator of the autophagy-lysosomal pathway (ALP) has triggered increasing numbers of studies that aim to explore the therapeutic potential of targeting TFEB to treat neurodegenerative disorders (NDs) such as Alzheimer's disease and Parkinson's disease. So far, the findings are exciting and promising. Here, we delineate the dysfunction of the TFEB-mediated ALP in NDs, and we summarize small molecules that have been identified as TFEB activators, along with their protective effects in NDs. We discuss the molecular mechanisms and targets, and the pros and cons of these TFEB activators from the perspective of drug development. Specific and potent small-molecule TFEB activators with ideal brain bioavailability could provide a method for treating NDs.
    Keywords:  Autophagy‐lysosomal pathway; Neurodegenerative disorders; Small molecules; Transcription factor EB
    DOI:  https://doi.org/10.1016/j.drudis.2020.10.013
  34. Ultrastruct Pathol. 2020 Oct 29. 1-7
    ER-phagy in human atherosclerosis: an exploratory ultrastructural study.
    Ida Perrotta.
      Autophagy is a vacuolar self-digesting mechanism responsible for the removal of damaged organelles, indigestible aggregates, and nonfunctional long-lived proteins by lysosome. Autophagy is dynamically connected to the endoplasmic reticulum (ER) in several ways. It is capable to counteract the possible harmful effects linked to the impairment of protein folding in the ER; the ER has been proposed as the source for autophagosomal membranes. Also, the ER itself can undergo a selective form of autophagy (called ER-phagy) which ensures the maintenance of ER's morphology and function. Autophagy has been widely investigated in the cardiovascular system however there is no evidence to date regarding the occurrence of ER-phagy into the blood vessel wall. This study has been undertaken to explore the existence of this selective control mechanism in the cells of human atherosclerotic plaques. Transmission Electron Microscopy (TEM) analysis revealed that in the plaque cells the smooth ER profiles reorganized into concentric whorls and closely packed membranes arranged in curved and parallel arrays. Circular, often ring-shaped, ER membranes studded with ribosomes and enclosed in a sequestering vesicle have been also frequently observed. This preliminary study demonstrates the existence of a distinct machinery for the specific turnover of ER membranes in human atherosclerosis and provides the first ultrastructural description of ER-phagy in the diseased vascular tissue. These results may open new perspectives for future investigation in the cardiovascular field.
    Keywords:  Atherosclerosis; ER-phagy; electron microscopy; endoplasmic reticulum
    DOI:  https://doi.org/10.1080/01913123.2020.1840468
  35. Oxid Med Cell Longev. 2020 ;2020 6352876
    Synergistic Carcinogenesis of HPV18 and MNNG in Het-1A Cells through p62-KEAP1-NRF2 and PI3K/AKT/mTOR Pathway.
    Ying Zhang, Yue Ma, Chao Zhao, Hu Zhang, Yuepu Pu, Lihong Yin.
      N-methyl-N´-nitro-N-nitrosoguanidine is a clear carcinogen, increasing evidence that indicates an etiological role of human papillomavirus in esophageal carcinoma. Studies have reported the synergistic effect on environmental carcinogens and viruses in recent years. On the basis of establishing the malignant transformation model of Het-1A cells induced by synergistic of HPV18 and MNNG, this study was to explore the synergistic carcinogenesis of MNNG and HPV. Our research indicated that HPV&MNNG led to a significant increase in the protein-expression levels of c-Myc, cyclinD1, BCL-2, BAX, E-cadherin, N-cadherin, mTOR, LC3II, and p62, with concomitant decreases in p21 and LC3I. HPV18 and MNNG induced accumulation of p62 and its interaction with KEAP1, which promoted NRF2 nuclear translocation. p62 loss prevents growth and increases autophagy of malignant cells by activating KEAP1/NRF2-dependent antioxidative response. In addition, PI3K and p-AKT were stimulated by HPV&MNNG, and PI3K/AKT/mTOR is positively associated with cell proliferation, migration, invasion, and autophagy during malignant transformation. Taken together, MNNG&HPV regulates autophagy and further accelerates cell appreciation by activating the p62/KEAP1/NRF2 and PI3K/AKT/mTOR pathway. MNNG&HPV may improve Het-1A cell autophagy to contribute to excessive cell proliferation, reduced apoptosis, and protection from oxidative damage, thus accelerating the process of cell malignant transformation and leading to cancerous cells.
    DOI:  https://doi.org/10.1155/2020/6352876
  36. Autophagy. 2020 Oct 29. 1-3
    BECN2 (beclin 2)-mediated non-canonical autophagy in innate immune signaling and tumor development.
    Motao Zhu, Guangtong Deng, Changsheng Xing, Guangjun Nie, Rong-Fu Wang.
      BECN2 (beclin 2) is a newly identified mammalian-specific macroautophagy/autophagy family member, and plays a critical role in the control of obesity and insulin sensitivity. However, its role in innate immune signaling and inflammation remains elusive. In our recent study, we show that BECN2 functions as a negative regulator in innate immune signaling and tumor development through non-canonical autophagy. Loss of Becn2 causes splenomegaly, lymphadenopathy, elevated proinflammatory cytokine production and spontaneous lymphoma development in mice. Mechanistically, BECN2 mediates the degradation of MAP3K7/TAK1 and MAP3K3/MEKK3 through an ATG9A- and ULK1-dependent but ATG16L1-BECN1-MAP1LC3B/LC3B-independent autophagy pathway to control systemic inflammation. BECN2 interacts with MAP3K7 and MAP3K3 through the engagement of ATG9A+ vesicles upon ULK1 activation, and promotes the fusion of MAP3K3- or MAP3K7-associated ATG9A+ vesicles with phagophores for subsequent degradation. Our findings have identified a previously unrecognized role of BECN2 in innate immune signaling and tumor development through non-canonical autophagy, thus providing a potential target for inflammatory disease and cancer therapy.
    Keywords:  ATG9A; BECN2; MAP3K3; MAPK1/ERK2-MAPK3/ERK1 and NFKB signaling; inflammation-associated cancer; non-canonical autophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1839277
  37. Aging (Albany NY). 2020 Oct 27. 12
    Dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibits human renal cell carcinoma cell growth.
    Bing Zheng, Xu Sun, Xin-Feng Chen, Zhan Chen, Wei-Li Zhu, Hua Zhu, Dong-Hua Gu.
      CC-115 is a dual inhibitor of DNA-PKcs and mTOR, both are valuable therapeutic targets for renal cell carcinoma (RCC). Our results showed that CC-115 inhibited survival and proliferation of established RCC cell lines (786-O and A489) and primary human RCC cells. The dual inhibitor induced selective apoptosis activation in RCC cells, as compared to no cytotoxicity nor apoptotic effects toward normal renal epithelial cells. CC-115 inhibited DNA-PKcs and mTORC1/2 activation in RCC cells. It was however ineffective in DNA-PKcs-mTOR double knockout (DKO) 786-O cells. CC-115 induced feedback autophagy activation in RCC cells. Autophagy inhibitors or Beclin-1/Light chain 3 (LC3) silencing potentiated CC-115-induced anti-RCC cell activity. Conversely, ectopic overexpression of Beclin-1 inhibited CC-115-induced cytotoxicity. At last CC-115 oral administration inhibited 786-O subcutaneous xenograft growth in nude mice. Taken together, dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibited RCC cell growth.
    Keywords:  CC-115; DNA-PKcs; autophagy; mTOR; renal cell carcinoma
    DOI:  https://doi.org/10.18632/aging.103847
  38. Dev Cell. 2020 Oct 26. pii: S1534-5807(20)30763-2. [Epub ahead of print]55(2): 113-115
    MTORC1 and the Rebirth of Stemness.
    Nicolette M Johnson, Christopher J Lengner.
      MTORC1 activity is critical for tissue regeneration in multiple organs and contexts. In this issue of Developmental Cell, Miao et al. describe upstream regulators of mTORC1 activity which promote paligenosis, a process where mature cells de-differentiate to acquire stem cell activity in the face of injury.
    DOI:  https://doi.org/10.1016/j.devcel.2020.10.001
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