bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒12‒06
forty-two papers selected by
Viktor Korolchuk, Newcastle University
  1. Atg1 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling.
  2. Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila.
  3. BPIFB3 interacts with ARFGAP1 and TMED9 to regulate non-canonical autophagy and RNA virus infection.
  4. Autophagy Brings an END to Aberrant Endocytosis.
  5. Autophagy and Autophagy-Related Diseases: A Review.
  6. An Interplay Between Autophagy and Immunometabolism for Host Defense Against Mycobacterial Infection.
  7. The Role of Autophagy in Liver Cancer: Crosstalk in Signaling Pathways and Potential Therapeutic Targets.
  8. Ubiquitin conjugating enzymes in the regulation of the autophagy-dependent degradation pathway.
  9. Cellular and physiological functions of C9orf72 and implications for ALS/FTD.
  10. CDK1, the Other 'Master Regulator' of Autophagy.
  11. Mitophagy Receptors in Tumor Biology.
  12. Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus.
  13. AMP-activated protein kinase regulates β-catenin protein synthesis by phosphorylating serine/arginine-rich splicing factor 9.
  14. RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.
  15. mTORC1-mediated amino acid signaling is critical for cell fate determination under transplant-induced stress.
  16. The Aging Stress Response and Its Implication for AMD Pathogenesis.
  17. Visualization of Autophagy Progression by a Red-Green-Blue Autophagy Sensor.
  18. Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation.
  19. Nitrogen Starvation and Stationary Phase Lipophagy Have Distinct Molecular Mechanisms.
  20. Loss of Furin in β cells Induces an mTORC1-ATF4 Anabolic Pathway that Leads to β cell Dysfunction.
  21. Trehalose induces SQSTM1/p62 expression and enhances lysosomal activity and antioxidative capacity in adipocytes.
  22. Broad and Complex Roles of NBR1-Mediated Selective Autophagy in Plant Stress Responses.
  23. Cathepsin B is a mediator of organelle-specific initiation of ferroptosis.
  24. Protein quality control by the proteasome and autophagy: A regulatory role of ubiquitin and liquid-liquid phase separation.
  25. Triangular Relationship between p53, Autophagy, and Chemotherapy Resistance.
  26. A non-canonical Hedgehog pathway initiates ciliogenesis and autophagy.
  27. SUMO wrestles with mitophagy to extend lifespan.
  28. Prohibitin regulates mTOR pathway via interaction with FKBP8.
  29. Autophagy is not involved in lipid accumulation and the development of insulin resistance in skeletal muscle.
  30. Evaluating and modulating TFEB in the control of autophagy: towards new treatments in CNS disorders.
  31. Stability of tuberous sclerosis complex 2 is controlled by methylation at R1457 and R1459.
  32. Overexpression of c-Fos reverses osteoprotegerin-mediated suppression of osteoclastogenesis by increasing the Beclin1-induced autophagy.
  33. Mitochondria Control mTORC1 Activity Linked Compartmentalization of eIF4E to Regulate Extracellular Export of microRNAs.
  34. Isoquercitrin induces apoptosis and autophagy in hepatocellular carcinoma cells via AMPK/mTOR/p70S6K signaling pathway.
  35. mTORC2 negatively controls the maturation process of medullary thymic epithelial cells by inhibiting the LTβR/RANK-NF-κB axis.
  36. mTORC1 Overactivation Leads to Abnormalities in Skeletal Development.
  37. The Protective Effects of the Autophagic and Lysosomal Machinery in Vascular and Valvular Calcification: A Systematic Review.
  38. Oxidized LDL Causes Endothelial Apoptosis by Inhibiting Mitochondrial Fusion and Mitochondria Autophagy.
  39. A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway.
  40. Characterization of constitutive ER-phagy of excess membrane proteins.
  41. Premises among SARS-CoV-2, dysbiosis and diarrhea: Walking through the ACE2/mTOR/autophagy route.
  42. Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles.
  1. EMBO Rep. 2020 Dec 03. e51869
    Atg1 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling.
    Saskia Barz, Franziska Kriegenburg, Anna Henning, Anuradha Bhattacharya, Hector Mancilla, Pablo Sánchez-Martín, Claudine Kraft.
      Autophagy mediates the degradation of cytoplasmic material. Upon autophagy induction, autophagosomes form a sealed membrane around the cargo and fuse with the lytic compartment to release the cargo for degradation. In order to avoid premature fusion of immature autophagosomal membranes with the lytic compartment, this process needs to be tightly regulated. Several factors mediating autophagosome-vacuole fusion have recently been identified. In budding yeast, autophagosome-vacuole fusion requires the R-SNARE Ykt6 on the autophagosome, together with the three Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. However, how these SNAREs are regulated during the fusion process is poorly understood. In this study, we investigate the regulation of Ykt6. We found that Ykt6 is directly phosphorylated by Atg1 kinase, which keeps this SNARE in an inactive state. Ykt6 phosphorylation prevents SNARE bundling by disrupting its interaction with the vacuolar SNAREs Vam3 and Vti1, thereby preventing premature autophagosome-vacuole fusion. These findings shed new light on the regulation of autophagosome-vacuole fusion and reveal a further step in autophagy controlled by the Atg1 kinase.
    Keywords:  Atg1; SNARE; Ykt6; autophagosome; autophagy
    DOI:  https://doi.org/10.15252/embr.202051869
  2. PLoS Genet. 2020 Nov 30. 16(11): e1009083
    Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila.
    Ivana Bjedov, Helena M Cochemé, Andrea Foley, Daniela Wieser, Nathaniel S Woodling, Jorge Iván Castillo-Quan, Povilas Norvaisas, Celia Lujan, Jenny Regan, Janne M Toivonen, Michael P Murphy, Janet Thornton, Kerri J Kinghorn, Thomas P Neufeld, Filipe Cabreiro, Linda Partridge.
      Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention.
    DOI:  https://doi.org/10.1371/journal.pgen.1009083
  3. J Cell Sci. 2020 Dec 04. pii: jcs.251835. [Epub ahead of print]
    BPIFB3 interacts with ARFGAP1 and TMED9 to regulate non-canonical autophagy and RNA virus infection.
    Azia S Evans, Nicholas J Lennemann, Carolyn B Coyne.
      Autophagy is a degradative cellular pathway that targets cytoplasmic contents and organelles for turnover by the lysosome. Various autophagy pathways play key roles in the clearance of viral infections, and many families of viruses have developed unique methods for avoiding degradation. Some positive stranded RNA viruses, such as enteroviruses and flaviviruses, usurp the autophagic pathway to promote their own replication. We previously identified the endoplasmic reticulum-localized protein BPIFB3 as an important negative regulator of non-canonical autophagy that uniquely impacts the replication of enteroviruses and flaviviruses. Here, we find that many components of the canonical autophagy machinery are not required for BPIFB3 depletion induced autophagy and identify the host factors that facilitate its role in the replication of enteroviruses and flaviviruses. Using proximity-dependent biotinylation (BioID) followed by mass spectrometry, we identify ARFGAP1 and TMED9 as two cellular components that interact with BPIFB3 to regulate autophagy and viral replication. Importantly, our data demonstrate that non-canonical autophagy in mammalian cells can be controlled outside of the traditional pathway regulators and define the role of two proteins in BPIFB3 depletion mediated non-canonical autophagy.
    Keywords:  Autophagy; BPI-like proteins; BPIFB3; Enterovirus; Flavivirus
    DOI:  https://doi.org/10.1242/jcs.251835
  4. Mol Cell. 2020 Dec 03. pii: S1097-2765(20)30798-X. [Epub ahead of print]80(5): 758-759
    Autophagy Brings an END to Aberrant Endocytosis.
    Agata N Makar, Noor Gammoh.
      Wilfling et al. (2020) characterize a selective autophagy pathway in yeast for early clathrin-mediated endocytosis (CME) proteins facilitated by the phase separation of the CME protein, Ede1, which acts as an intrinsic autophagy receptor for the degradation of Ede1-dependent endocytic protein deposits (ENDs).
    DOI:  https://doi.org/10.1016/j.molcel.2020.11.020
  5. Int J Mol Sci. 2020 Nov 26. pii: E8974. [Epub ahead of print]21(23):
    Autophagy and Autophagy-Related Diseases: A Review.
    Tadashi Ichimiya, Tsukasa Yamakawa, Takehiro Hirano, Yoshihiro Yokoyama, Yuki Hayashi, Daisuke Hirayama, Kohei Wagatsuma, Takao Itoi, Hiroshi Nakase.
      Autophagy refers to the process involving the decomposition of intracellular components via lysosomes. Autophagy plays an important role in maintaining and regulating cell homeostasis by degrading intracellular components and providing degradation products to cells. In vivo, autophagy has been shown to be involved in the starvation response, intracellular quality control, early development, and cell differentiation. Recent studies have revealed that autophagy dysfunction is implicated in neurodegenerative diseases and tumorigenesis. In addition to the discovery of certain disease-causing autophagy-related mutations and elucidation of the pathogenesis of conditions resulting from the abnormal degradation of selective autophagy substrates, the activation of autophagy is essential for prolonging life and suppressing aging. This article provides a comprehensive review of the role of autophagy in health, physiological function, and autophagy-related disease.
    Keywords:  autophagy; autophagy-related gene; cancer; cardiovascular; liver disease; mitophagy; neurodegenerative disease
    DOI:  https://doi.org/10.3390/ijms21238974
  6. Front Immunol. 2020 ;11 603951
    An Interplay Between Autophagy and Immunometabolism for Host Defense Against Mycobacterial Infection.
    Seungwha Paik, Eun-Kyeong Jo.
      Autophagy, an intracellular catabolic pathway featuring lysosomal degradation, is a central component of the host immune defense against various infections including Mycobacterium tuberculosis (Mtb), the pathogen that causes tuberculosis. Mtb can evade the autophagic defense and drive immunometabolic remodeling of host phagocytes. Co-regulation of the autophagic and metabolic pathways may play a pivotal role in shaping the innate immune defense and inflammation during Mtb infection. Two principal metabolic sensors, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) kinase, function together to control the autophagy and immunometabolism that coordinate the anti-mycobacterial immune defense. Here, we discuss our current understanding of the interplay between autophagy and immunometabolism in terms of combating intracellular Mtb, and how AMPK-mTOR signaling regulates antibacterial autophagy in terms of Mtb infection. We describe several autophagy-targeting agents that promote host antimicrobial defenses by regulating the AMPK-mTOR axis. A better understanding of the crosstalk between immunometabolism and autophagy, both of which are involved in host defense, is crucial for the development of innovative targeted therapies for tuberculosis.
    Keywords:  AMP-activated protein kinase; autophagy; host defense; immunometabolism; mammalian target of rapamycin ; mycobacterial infection
    DOI:  https://doi.org/10.3389/fimmu.2020.603951
  7. Pharmaceuticals (Basel). 2020 Nov 28. pii: E432. [Epub ahead of print]13(12):
    The Role of Autophagy in Liver Cancer: Crosstalk in Signaling Pathways and Potential Therapeutic Targets.
    Jianzhou Cui, Han-Ming Shen, Lina Hsiu Kim Lim.
      Autophagy is an evolutionarily conserved lysosomal-dependent pathway for degrading cytoplasmic proteins, macromolecules, and organelles. Autophagy-related genes (Atgs) are the core molecular machinery in the control of autophagy, and several major functional groups of Atgs coordinate the entire autophagic process. Autophagy plays a dual role in liver cancer development via several critical signaling pathways, including the PI3K-AKT-mTOR, AMPK-mTOR, EGF, MAPK, Wnt/β-catenin, p53, and NF-κB pathways. Here, we review the signaling pathways involved in the cross-talk between autophagy and hepatocellular carcinoma (HCC) and analyze the status of the development of novel HCC therapy by targeting the core molecular machinery of autophagy as well as the key signaling pathways. The induction or the inhibition of autophagy by the modulation of signaling pathways can confer therapeutic benefits to patients. Understanding the molecular mechanisms underlying the cross-link of autophagy and HCC may extend to translational studies that may ultimately lead to novel therapy and regimen formation in HCC treatment.
    Keywords:  AMPK; HCC; MAPK; ULK1; autophagy; mTOR; p53
    DOI:  https://doi.org/10.3390/ph13120432
  8. Matrix Biol. 2020 Dec 01. pii: S0945-053X(20)30112-8. [Epub ahead of print]
    Ubiquitin conjugating enzymes in the regulation of the autophagy-dependent degradation pathway.
    Fumiyo Ikeda.
      The ubiquitin-proteasomal system and the autophagy-lysosome system are two major degradation systems in mammalian cells. Ubiquitin not only regulates proteasomal degradation of substrates but also regulates the autophagy pathway. In one type of macroautophagy, called selective autophagy targeting cargos selectively, cargos are recruited to phagophore in a ubiquitin-dependent manner. Ubiquitin can target autophagy regulators for proteasomal degradation, or control protein conformation or interacting partners of these regulators. To understand the regulatory mechanisms of these degradation pathways, it is critical to dissect how the ubiquitin system contributes to them. Since enzymes are key regulators of ubiquitination, in this review, such enzymes in autophagy regulation are discussed, with specific focus on ubiquitin conjugating enzyme E2s, of which roles in autophagy are emerging.
    Keywords:  Autophagy; Degradation; Ubiquitin; Ubiquitin conjugating enzyme (E2)
    DOI:  https://doi.org/10.1016/j.matbio.2020.11.004
  9. J Neurochem. 2020 Dec 01.
    Cellular and physiological functions of C9orf72 and implications for ALS/FTD.
    Weilun Pang, Fenghua Hu.
      The hexanucleotide repeat expansion (HRE) in the C9orf72 gene is the main cause of two tightly linked neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). HRE leads to not only a gain of toxicity from RNA repeats and dipeptide repeats but also reduced levels of C9orf72 protein. However, the cellular and physiological functions of C9orf72 were unknown until recently. Through proteomic analysis, Smith-Magenis Chromosome Regions 8 (SMCR8) and WD repeat-containing protein (WDR41) were identified as binding partners of C9orf72. These three proteins have been shown to form a tight complex, but the exact functions of this complex remain to be characterized. Both C9orf72 and SMCR8 contain a DENN domain, which has been shown to regulate the activities of small GTPases. The C9orf72 complex has been implicated in many cellular processes, including vesicle trafficking, lysosome homeostasis, mTORC1 signaling pathway, and autophagy. C9orf72 deficiency in mice results in exaggerated inflammatory responses and human patients with C9orf72 mutations have neuroinflammation phenotype. Recent studies indicate that C9orf72 regulates trafficking and lysosomal degradation of inflammatory mediators, including toll-like receptors (TLRs) and STING, to affect inflammatory outputs. Further exploration of cellular and physiological functions of C9orf72 will help dissect the pathological mechanism of ALS/FTD caused by C9orf72 mutations.
    Keywords:  ALS; C9orf72; FTLD; SMCR8; WDR41; autophagy; inflammation; lysosome; mTORC1
    DOI:  https://doi.org/10.1111/jnc.15255
  10. Trends Cell Biol. 2020 Nov 30. pii: S0962-8924(20)30226-9. [Epub ahead of print]
    CDK1, the Other 'Master Regulator' of Autophagy.
    Richard I Odle, Oliver Florey, Nicholas T Ktistakis, Simon J Cook.
      Autophagy and cap-dependent mRNA translation are tightly regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signalling complex in response to nutrient availability. However, the regulation of these processes, and mTORC1 itself, is different during mitosis, and this has remained an area of significant controversy; for example, studies have argued that autophagy is either repressed or highly active during mitosis. Recent studies have shown that autophagy initiation is repressed, and cap-dependent mRNA translation is maintained during mitosis despite mTORC1 activity being repressed. This is achieved in large part by a switch from mTORC1- to cyclin-dependent kinase 1 (CDK1)-mediated regulation. Here, we review the history and recent advances and seek to present a unifying model to inform the future study of autophagy and mTORC1 during mitosis.
    Keywords:  CDK1; autophagy; mTORC1; mitosis; translation
    DOI:  https://doi.org/10.1016/j.tcb.2020.11.001
  11. Front Cell Dev Biol. 2020 ;8 594203
    Mitophagy Receptors in Tumor Biology.
    Yangchun Xie, Jiao Liu, Rui Kang, Daolin Tang.
      Mitochondria are multifunctional organelles that regulate cancer biology by synthesizing macromolecules, producing energy, and regulating cell death. The understanding of mitochondrial morphology, function, biogenesis, fission and fusion kinetics, and degradation is important for the development of new anticancer strategies. Mitophagy is a type of selective autophagy that can degrade damaged mitochondria under various environmental stresses, especially oxidative damage and hypoxia. The key regulator of mitophagy is the autophagy receptor, which recognizes damaged mitochondria and allows them to enter autophagosomes by binding to MAP1LC3 or GABARAP, and then undergo lysosomal-dependent degradation. Many components of mitochondria, including mitochondrial membrane proteins (e.g., PINK1, BNIP3L, BNIP3, FUNDC1, NIPSNAP1, NIPSNAP2, BCL2L13, PHB2, and FKBP8) and lipids (e.g., cardiolipin and ceramides), act as mitophagy receptors in a context-dependent manner. Dysfunctional mitophagy not only inhibits, but also promotes, tumorigenesis. Similarly, mitophagy plays a dual role in chemotherapy, radiotherapy, and immunotherapy. In this review, we summarize the latest advances in the mechanisms of mitophagy and highlight the pathological role of mitophagy receptors in tumorigenesis and treatment.
    Keywords:  autophagy; cancer; cell death; mitochondria; mitophagy
    DOI:  https://doi.org/10.3389/fcell.2020.594203
  12. Nat Commun. 2020 11 30. 11(1): 6088
    Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus.
    Xin Zhou, Yanghao Zhong, Olivia Molinar-Inglis, Maya T Kunkel, Mingyuan Chen, Tengqian Sun, Jiao Zhang, John Y-J Shyy, JoAnn Trejo, Alexandra C Newton, Jin Zhang.
      The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.
    DOI:  https://doi.org/10.1038/s41467-020-19937-w
  13. Biochem Biophys Res Commun. 2020 Nov 25. pii: S0006-291X(20)32125-2. [Epub ahead of print]
    AMP-activated protein kinase regulates β-catenin protein synthesis by phosphorylating serine/arginine-rich splicing factor 9.
    Eri Matsumoto, Yu Matsumoto, Jun Inoue, Yuji Yamamoto, Tsukasa Suzuki.
      β-catenin is a multi-functional protein with a central role in regulating embryonic development and tissue homeostasis. The abnormal accumulation of β-catenin, due to disrupted β-catenin degradation or unregulated β-catenin synthesis, causes the development of cancer. A recent study showed that the overexpression of proto-oncogene serine/arginine-rich splicing factor 9 (SRSF9) promotes β-catenin accumulation via binding β-catenin mRNA and enhancing its translation in a manner that is dependent on the mechanistic target of rapamycin (mTOR). However, the regulation of the interaction between SRSF9 and mRNA of β-catenin remains unclear. Here, we show that AMP-activated protein kinase (AMPK) phosphorylates SRSF9 at the RNA-interacting SWQDLKD motif that plays a major role in determining substrate specificity. The phosphorylation by AMPK inhibits the binding of SRSF9 to β-catenin mRNA and suppresses β-catenin protein synthesis caused by SRSF9 overexpression without changing the β-catenin mRNA levels. Our findings suggest that AMPK activators are potential therapeutic targets for SRSF9-derived overproduction of β-catenin in cancer cells.
    Keywords:  AMPK; SRSF9; mTORC1; β-catenin
    DOI:  https://doi.org/10.1016/j.bbrc.2020.11.079
  14. Mol Cell. 2020 Nov 23. pii: S1097-2765(20)30786-3. [Epub ahead of print]
    RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.
    Ayaz Najafov, Hoang Son Luu, Adnan K Mookhtiar, Lauren Mifflin, Hong-Guang Xia, Palak P Amin, Alban Ordureau, Huibing Wang, Junying Yuan.
      The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.
    Keywords:  AMPK; CASP8; MLKL; RIPK1; RIPK3; TSC2; lysosome; mTORC1; neonatal lethality
    DOI:  https://doi.org/10.1016/j.molcel.2020.11.008
  15. FEBS Lett. 2020 Nov 29.
    mTORC1-mediated amino acid signaling is critical for cell fate determination under transplant-induced stress.
    Xiaoyan Cheng, Maolin Ge, Shouhai Zhu, Dan Li, Ruiheng Wang, Qiongyu Xu, Zhihong Chen, Shufeng Xie, Han Liu.
      Transplantation of in vitro-manipulated cells is widely used in hematology. While transplantation is well recognized to impose severe stress on transplanted cells, the nature of transplant-induced stress remains elusive. Here we propose that the lack of amino acids in serum is the major cause of transplant-induced stress. Mechanistically, amino acid deficiency decreases protein synthesis and nutrient consummation. However, in cells with overactive AKT and ERK, mTORC1 is not inhibited and protein synthesis remains relatively high. This impaired signaling causes nutrient depletion, cell cycle block, and eventually autophagy and cell death, which can be inhibited by cycloheximide or mTORC1 inhibitors. Thus, mTORC1-mediated amino acid signaling is critical in cell fate determination under transplant-induced stress, and protein synthesis inhibition can improve transplantation efficiency.
    Keywords:  amino acid; leukemia; mTORC1; metabolism; transplant-induced stress
    DOI:  https://doi.org/10.1002/1873-3468.14008
  16. Int J Mol Sci. 2020 Nov 22. pii: E8840. [Epub ahead of print]21(22):
    The Aging Stress Response and Its Implication for AMD Pathogenesis.
    Janusz Blasiak, Elzbieta Pawlowska, Anna Sobczuk, Joanna Szczepanska, Kai Kaarniranta.
      Aging induces several stress response pathways to counterbalance detrimental changes associated with this process. These pathways include nutrient signaling, proteostasis, mitochondrial quality control and DNA damage response. At the cellular level, these pathways are controlled by evolutionarily conserved signaling molecules, such as 5'AMP-activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR), insulin/insulin-like growth factor 1 (IGF-1) and sirtuins, including SIRT1. Peroxisome proliferation-activated receptor coactivator 1 alpha (PGC-1α), encoded by the PPARGC1A gene, playing an important role in antioxidant defense and mitochondrial biogenesis, may interact with these molecules influencing lifespan and general fitness. Perturbation in the aging stress response may lead to aging-related disorders, including age-related macular degeneration (AMD), the main reason for vision loss in the elderly. This is supported by studies showing an important role of disturbances in mitochondrial metabolism, DDR and autophagy in AMD pathogenesis. In addition, disturbed expression of PGC-1α was shown to associate with AMD. Therefore, the aging stress response may be critical for AMD pathogenesis, and further studies are needed to precisely determine mechanisms underlying its role in AMD. These studies can include research on retinal cells produced from pluripotent stem cells obtained from AMD donors with the mutations, either native or engineered, in the critical genes for the aging stress response, including AMPK, IGF1, MTOR, SIRT1 and PPARGC1A.
    Keywords:  AMD; DNA damage response; PGC-1α; SIRT1; age-related macular degeneration; aging; autophagy; insulin/IGF-1; mitochondrial quality control; the aging stress response
    DOI:  https://doi.org/10.3390/ijms21228840
  17. ACS Sens. 2020 Dec 02.
    Visualization of Autophagy Progression by a Red-Green-Blue Autophagy Sensor.
    Heejung Kim, Hyunbin Kim, Jaesik Choi, Kyung-Soo Inn, Jihye Seong.
      Autophagy is a major degradation process of cytosolic components and misfolded proteins that is crucial for cellular homeostasis and for the pathogenesis of diverse diseases. Autophagy is initiated by the formation of phagophores, which mature to autophagosomes. The autophagosomes then fuse to lysosomes to form autolysosomes. Different stages of autophagy can be deregulated to cause autophagy-related diseases, and thus, an accurate detection of each stage of autophagy progression is critical for efficient therapeutic strategies for these diseases. To identify the different stages of autophagy progression, here, we developed a new autophagy flux sensor, named red-green-blue-LC3 (RGB-LC3). RGB-LC3 is composed of LC3 and red-green-blue (RGB) fluorescent proteins, which were carefully chosen by considering their separate spectral profiles, stability, brightness, and most importantly different pH sensitivities. Utilizing this RGB-LC3 and the predicted pH, we could clearly identify phagophores, autophagosomes, fusion stage, early autolysosomes, and mature autolysosomes in live cells. Furthermore, the RGB-LC3 sensor was successfully applied to distinguish different effects of Aβ monomers and oligomers on autophagy flux. Therefore, we developed a new autophagy flux sensor, RGB-LC3, which may be a valuable tool to further investigate the molecular mechanisms of autophagy and to develop efficient therapeutic strategies for autophagy-related diseases.
    Keywords:  RGB-LC3; autophagic flux; autophagy progression; fluorescent sensor; pH ratiometric sensor
    DOI:  https://doi.org/10.1021/acssensors.0c00809
  18. J Exp Clin Cancer Res. 2020 Nov 30. 39(1): 266
    Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation.
    Lili Quan, Ryuichi Ohgaki, Saori Hara, Suguru Okuda, Ling Wei, Hiroki Okanishi, Shushi Nagamori, Hitoshi Endou, Yoshikatsu Kanai.
      BACKGROUND: Tumor angiogenesis is regarded as a rational anti-cancer target. The efficacy and indications of anti-angiogenic therapies in clinical practice, however, are relatively limited. Therefore, there still exists a demand for revealing the distinct characteristics of tumor endothelium that is crucial for the pathological angiogenesis. L-type amino acid transporter 1 (LAT1) is well known to be highly and broadly upregulated in tumor cells to support their growth and proliferation. In this study, we aimed to establish the upregulation of LAT1 as a novel general characteristic of tumor-associated endothelial cells as well, and to explore the functional relevance in tumor angiogenesis.METHODS: Expression of LAT1 in tumor-associated endothelial cells was immunohistologically investigated in human pancreatic ductal adenocarcinoma (PDA) and xenograft- and syngeneic mouse tumor models. The effects of pharmacological and genetic ablation of endothelial LAT1 were examined in aortic ring assay, Matrigel plug assay, and mouse tumor models. The effects of LAT1 inhibitors and gene knockdown on cell proliferation, regulation of translation, as well as on the VEGF-A-dependent angiogenic processes and intracellular signaling were investigated in in vitro by using human umbilical vein endothelial cells.
    RESULTS: LAT1 was highly expressed in vascular endothelial cells of human PDA but not in normal pancreas. Similarly, high endothelial LAT1 expression was observed in mouse tumor models. The angiogenesis in ex/in vivo assays was suppressed by abrogating the function or expression of LAT1. Tumor growth in mice was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. LAT1-mediated amino acid transport was fundamental to support endothelial cell proliferation and translation initiation in vitro. Furthermore, LAT1 was required for the VEGF-A-dependent migration, invasion, tube formation, and activation of mTORC1, suggesting a novel cross-talk between pro-angiogenic signaling and nutrient-sensing in endothelial cells.
    CONCLUSIONS: These results demonstrate that the endothelial LAT1 is a novel key player in tumor angiogenesis, which regulates proliferation, translation, and pro-angiogenic VEGF-A signaling. This study furthermore indicates a new insight into the dual functioning of LAT1 in tumor progression both in tumor cells and stromal endothelium. Therapeutic inhibition of LAT1 may offer an ideal option to potentiate anti-angiogenic therapies.
    Keywords:  Amino acid transporter; Endothelial cell; Tumor angiogenesis; VEGF-A; mTORC1
    DOI:  https://doi.org/10.1186/s13046-020-01762-0
  19. Int J Mol Sci. 2020 Nov 29. pii: E9094. [Epub ahead of print]21(23):
    Nitrogen Starvation and Stationary Phase Lipophagy Have Distinct Molecular Mechanisms.
    Ravinder Kumar, Muhammad Arifur Rahman, Taras Y Nazarko.
      In yeast, the selective autophagy of intracellular lipid droplets (LDs) or lipophagy can be induced by either nitrogen (N) starvation or carbon limitation (e.g., in the stationary (S) phase). We developed the yeast, Komagataella phaffii (formerly Pichia pastoris), as a new lipophagy model and compared the N-starvation and S-phase lipophagy in over 30 autophagy-related mutants using the Erg6-GFP processing assay. Surprisingly, two lipophagy pathways had hardly overlapping stringent molecular requirements. While the N-starvation lipophagy strictly depended on the core autophagic machinery (Atg1-Atg9, Atg18, and Vps15), vacuole fusion machinery (Vam7 and Ypt7), and vacuolar proteolysis (proteinases A and B), only Atg6 and proteinases A and B were essential for the S-phase lipophagy. The rest of the proteins were only partially required in the S-phase. Moreover, we isolated the prl1 (for the positive regulator of lipophagy 1) mutant affected in the S-phase lipophagy, but not N-starvation lipophagy. The prl1 defect was at a stage of delivery of the LDs from the cytoplasm to the vacuole, further supporting the mechanistically different nature of the two lipophagy pathways. Taken together, our results suggest that N-starvation and S-phase lipophagy have distinct molecular mechanisms.
    Keywords:  Komagataella phaffii; Pichia pastoris; Prl1; autophagic machinery; autophagy; lipid droplets; lipophagy; selective autophagy; vacuole; yeast
    DOI:  https://doi.org/10.3390/ijms21239094
  20. Diabetes. 2020 Dec 04. pii: db200474. [Epub ahead of print]
    Loss of Furin in β cells Induces an mTORC1-ATF4 Anabolic Pathway that Leads to β cell Dysfunction.
    Bas Brouwers, Ilaria Coppola, Katlijn Vints, Bastian Dislich, Nathalie Jouvet, Leentje Van Lommel, Charlotte Segers, Natalia V Gounko, Lieven Thorrez, Frans Schuit, Stefan F Lichtenthaler, Jennifer L Estall, Jeroen Declercq, Bruno Ramos-Molina, John W M Creemers.
      FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, yet its specific role in pancreatic β cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, while PCs that potentially could provide redundancy are expressed at considerably lower levels. β cell-specific Furin knockout (βFurKO) mice are glucose intolerant, due to smaller islets with lower insulin content and abnormal dense core secretory granule morphology. mRNA expression analysis and differential proteomics on βFurKO islets revealed activation of Activating Transcription Factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). βFurKO cells show impaired cleavage or shedding of the V-ATPase subunits Ac45 and prorenin receptor (PRR), respectively, and impaired lysosomal acidification. Blocking the V-ATPase pharmacologically in β cells increases mTORC1 activity, suggesting the involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in β cells lacking Furin, which causes β cell dysfunction.
    DOI:  https://doi.org/10.2337/db20-0474
  21. FEBS Open Bio. 2020 Dec 04.
    Trehalose induces SQSTM1/p62 expression and enhances lysosomal activity and antioxidative capacity in adipocytes.
    Masaki Kobayashi, Hiromine Yasukawa, Tomoya Arikawa, Yusuke Deguchi, Natsumi Mizushima, Misako Sakurai, Shoichi Onishi, Ryoma Tagawa, Yuka Sudo, Naoyuki Okita, Kyohei Higashi, Yoshikazu Higami.
      Adipocytes, which comprise the majority of white adipose tissue (WAT), are involved in obesity-related pathology via various mechanisms, including disturbed lysosomal enzymatic activity and accumulation of oxidative stress. Sequestosome 1 (SQSTM1/p62) is an autophagy marker that participates in antioxidative responses via the activation of nuclear factor erythroid-derived 2-like 2 (NRF2). Trehalose is a non-reducing disaccharide reported to suppress adipocyte hypertrophy in obese mice and improve glucose tolerance in humans. We recently revealed that trehalose increases SQSTM1 levels and enhances antioxidative capacity in hepatocytes. Here, to further evaluate the mechanism behind the beneficial effects of trehalose on metabolism, we examined SQSTM1 levels, autophagy, and oxidative stress in trehalose-treated adipocytes. We initially confirmed that trehalose increases SQSTM1 transcription and protein levels without affecting autophagy in adipocytes. Trehalose also elevated transcription of several lysosomal genes and the activity of cathepsin L, a lysosomal enzyme, independently of the transcription factor EB. In agreement with our data from hepatocytes, trehalose induced the nuclear translocation of NRF2 and the transcription of its downstream antioxidative genes, resulting in reduced cellular reactive oxygen species levels. Moreover, some cellular trehalose was detected in trehalose-treated adipocytes, implying that extracellular trehalose is taken into cells. These observations reveal the mechanism behind the beneficial effects of trehalose on metabolism and suggest its potential for preventing or treating obesity-related pathology.
    Keywords:  Adipocyte; Lysosome; Oxidative Stress; SQSTM1; Trehalose
    DOI:  https://doi.org/10.1002/2211-5463.13055
  22. Cells. 2020 Nov 30. pii: E2562. [Epub ahead of print]9(12):
    Broad and Complex Roles of NBR1-Mediated Selective Autophagy in Plant Stress Responses.
    Yan Zhang, Zhixiang Chen.
      Selective autophagy is a highly regulated degradation pathway for the removal of specific damaged or unwanted cellular components and organelles such as protein aggregates. Cargo selectivity in selective autophagy relies on the action of cargo receptors and adaptors. In mammalian cells, two structurally related proteins p62 and NBR1 act as cargo receptors for selective autophagy of ubiquitinated proteins including aggregation-prone proteins in aggrephagy. Plant NBR1 is the structural and functional homolog of mammalian p62 and NBR1. Since its first reports almost ten years ago, plant NBR1 has been well established to function as a cargo receptor for selective autophagy of stress-induced protein aggregates and play an important role in plant responses to a broad spectrum of stress conditions including heat, salt and drought. Over the past several years, important progress has been made in the discovery of specific cargo proteins of plant NBR1 and their roles in the regulation of plant heat stress memory, plant-viral interaction and special protein secretion. There is also new evidence for a possible role of NBR1 in stress-induced pexophagy, sulfur nutrient responses and abscisic acid signaling. In this review, we summarize these progresses and discuss the potential significance of NBR1-mediated selective autophagy in broad plant responses to both biotic and abiotic stresses.
    Keywords:  NBR1; autophagy; plant heat tolerance; plant stress responses; plant virus interaction; protein aggregates; selective autophagy receptor
    DOI:  https://doi.org/10.3390/cells9122562
  23. Biochem Biophys Res Commun. 2020 Oct 22. pii: S0006-291X(20)31950-1. [Epub ahead of print]
    Cathepsin B is a mediator of organelle-specific initiation of ferroptosis.
    Feimei Kuang, Jiao Liu, Changfeng Li, Rui Kang, Daolin Tang.
      Ferroptosis is a type of non-apoptotic regulated cell death that involves excessive iron accumulation and subsequent lipid peroxidation. Although the antioxidant mechanisms of ferroptosis have been extensively studied recently, little is known about the interactions between the different organelles that control ferroptosis. Here, we show that the translocation of lysosomal cysteine protease cathepsin B (CTSB) into the nucleus is an important molecular event that mediates organelle-specific initiation of ferroptosis in human pancreatic cancer cells. Iron-dependent lysosomal membrane permeability triggers the release of CTSB from the lysosome to nucleus during ferroptosis. Mechanistically, nuclear CTSB accumulation causes DNA damage and subsequent activation of the stimulator of interferon response CGAMP interactor 1 (STING1/STING)-dependent DNA sensor pathway, which ultimately leads to autophagy-dependent ferroptosis. Consequently, the genetic inhibition of CTSB-dependent STING1 activation by RNAi prevents ferroptosis in cell culture and animal models. These new findings not only enhance our understanding of the mechanism by which organelles specifically trigger ferroptosis, but also may provide a potential way to enhance the anticancer activity of ferroptosis therapy.
    Keywords:  Autophagy; Cathepsin B; DNA damage; Ferroptosis; Lysosome; STING1
    DOI:  https://doi.org/10.1016/j.bbrc.2020.10.035
  24. Matrix Biol. 2020 Nov 28. pii: S0945-053X(20)30105-0. [Epub ahead of print]
    Protein quality control by the proteasome and autophagy: A regulatory role of ubiquitin and liquid-liquid phase separation.
    Linlin Lei, Zhixiao Wu, Konstanze F Winklhofer.
      Degradation of dysfunctional, damaged, or misfolded proteins is a crucial component of the protein quality control network to maintain cellular proteostasis. Dysfunction in proteostasis regulation due to imbalances in protein synthesis, folding, and degradation challenges the integrity of the cellular proteome and favors the accumulation of aggregated proteins that can damage cells by a loss of their functions and/or a gain of adverse functions. Ubiquitination is an essential player in proteostasis regulation but also in orchestrating signaling pathways in response to various stress conditions. Both cellular degradation systems, the proteasome and autophagy, employ ubiquitin for selection and targeting of substrates to the degradative machineries. Here we summarize the manifold functions of ubiquitin in protein degradation and discuss its emerging role in the formation of biomolecular condensates through liquid-liquid phase separation, which allows spatiotemporal regulation of protein quality control.
    Keywords:  RAD23B; UBQLN2; biomolecular condensates; p62; phase separation; proteostasis
    DOI:  https://doi.org/10.1016/j.matbio.2020.11.003
  25. Int J Mol Sci. 2020 Nov 26. pii: E8991. [Epub ahead of print]21(23):
    Triangular Relationship between p53, Autophagy, and Chemotherapy Resistance.
    Jingwen Xu, Nipa H Patel, David A Gewirtz.
      Chemotherapy and radiation often induce a number of cellular responses, such as apoptosis, autophagy, and senescence. One of the major regulators of these processes is p53, an essential tumor suppressor that is often mutated or lost in many cancer types and implicated in early tumorigenesis. Gain of function (GOF) p53 mutations have been implicated in increased susceptibility to drug resistance, by compromising wildtype anti-tumor functions of p53 or modulating key p53 processes that confer chemotherapy resistance, such as autophagy. Autophagy, a cellular survival mechanism, is initially induced in response to chemotherapy and radiotherapy, and its cytoprotective nature became the spearhead of a number of clinical trials aimed to sensitize patients to chemotherapy. However, increased pre-clinical studies have exemplified the multifunctional role of autophagy. Additionally, compartmental localization of p53 can modulate induction or inhibition of autophagy and may play a role in autophagic function. The duality in p53 function and its effects on autophagic function are generally not considered in clinical trial design or clinical therapeutics; however, ample pre-clinical studies suggest they play a role in tumor responses to therapy and drug resistance. Further inquiry into the interconnection between autophagy and p53, and its effects on chemotherapeutic responses may provide beneficial insights on multidrug resistance and novel treatment regimens for chemosensitization.
    Keywords:  autophagy; chemoresistance; p53
    DOI:  https://doi.org/10.3390/ijms21238991
  26. J Cell Biol. 2021 Jan 04. pii: e202004179. [Epub ahead of print]220(1):
    A non-canonical Hedgehog pathway initiates ciliogenesis and autophagy.
    Tara Akhshi, William S Trimble.
      Primary cilia function as critical signaling hubs whose absence leads to severe disorders collectively known as ciliopathies; our knowledge of ciliogenesis remains limited. We show that Smo induces ciliogenesis through two distinct yet essential noncanonical Hh pathways in several cell types, including neurons. Surprisingly, ligand activation of Smo induces autophagy via an LKB1-AMPK axis to remove the satellite pool of OFD1. This is required, but not sufficient, for ciliogenesis. Additionally, Smo activates the Gαi-LGN-NuMA-dynein axis, causing accumulation of a portion of OFD1 at centrioles in early ciliogenesis. Both pathways are critical for redistribution of BBS4 from satellites to centrioles, which is also mediated by OFD1 centriolar translocation. Notably, different Smo agonists, which activate Smo distinctly, activate one or the other of these pathways; only in combination they recapitulate the activity of Hh ligand. These studies provide new insight into physiological stimuli (Hh) that activate autophagy and promote ciliogenesis and introduce a novel role for the Gαi-LGN-NuMA-dynein complex in this process.
    DOI:  https://doi.org/10.1083/jcb.202004179
  27. Rejuvenation Res. 2020 Nov 30.
    SUMO wrestles with mitophagy to extend lifespan.
    James Larrick, Jasmine W Larrick, Andrew R Mendelsohn.
      SUMOylation, a conserved protein post-translational modification that performs multiple functions including regulation of nuclear transport and transcription, is implicated in numerous biological processes including aging. RNAi knockdown of the sole SUMO gene, smo-1, in C elegans shortened lifespan, while overexpression in the intestine modestly increased lifespan. Smo-1 is required for mitochondrial fission in a tissue-specific manner. Fission, in turn, is needed for mitophagy to maintain mitochondrial homeostasis during aging. SUMOlyation affects DAF16, which can be directly SUMOylated, and SKN-1, the homolog of mammalian Nrf2. These regulators play key roles in maintaining mitochondrial homeostasis. However, given the modest effect of overexpressing smo-1 on lifespan enhancement and potential interference with other genes that can promote increased lifespan, caution is advised in the translation of this work based on C elegans. Although inhibitors of SUMOlyation have been developed for cancer and activators have been identified, broad-acting biochemical pathway modifiers such as SUMO are often suboptimal drug targets and may not be as promising for anti-aging applications as they may appear.
    DOI:  https://doi.org/10.1089/rej.2020.2406
  28. Front Med. 2020 Dec 01.
    Prohibitin regulates mTOR pathway via interaction with FKBP8.
    Jiahui Zhang, Yanan Yin, Jiahui Wang, Jingjing Zhang, Hua Liu, Weiwei Feng, Wen Yang, Bruce Zetter, Yingjie Xu.
      The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer. The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells. The PI3K-Akt-mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth. Unsurprisingly, it is also one of the most commonly attempted targets for cancer therapy. FK506 binding protein 8 (FKBP8) is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function. We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a "switch" type regulator. We identified through immunoprecipitation-mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1 (PHB1) specifically interacts with FKBP8. Furthermore, the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway, whereas the FKBP8 level in the mitochondria was substantially reduced. Moreover, concomitant with these changes, the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1. Collectively, our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.
    Keywords:  FKBP8; cancer; cell proliferation; mTOR; prohibitin 1
    DOI:  https://doi.org/10.1007/s11684-020-0805-6
  29. Biochem Biophys Res Commun. 2020 Nov 28. pii: S0006-291X(20)32092-1. [Epub ahead of print]
    Autophagy is not involved in lipid accumulation and the development of insulin resistance in skeletal muscle.
    María G Morales-Scholz, Courtney Swinton, Robyn M Murphy, Greg M Kowalski, Clinton R Bruce, Kirsten F Howlett, Christopher S Shaw.
      OBJECTIVE: To investigate the effect of high fat diet-induced insulin resistance on autophagy markers in the liver and skeletal muscle of mice in the fasted state and following an oral glucose bolus.METHODS: Forty C57BL/6J male mice were fed either a high fat, high sucrose (HFSD, n = 20) or standard chow control (CON, n = 20) diet for 16 weeks. Upon trial completion, mice were gavaged with water or glucose and skeletal muscle and liver were collected 15 min post gavage. Protein abundance and gene expression of autophagy markers and activation of related signalling pathways were assessed.
    RESULTS: Compared to CON, the HFSD intervention increased LC3B-II and p62/SQSTM1 protein abundance in the liver which is indicative of elevated autophagosome content via reduced clearance. These changes coincided with inhibitory autophagy signalling through elevated p-mTOR S2448 and p-ULK1S758. HFSD did not alter autophagy markers in skeletal muscle. Administration of an oral glucose bolus had no effect on autophagy markers or upstream signalling responses in either tissue regardless of diet.
    CONCLUSION: HFSD induces tissue-specific autophagy impairments, with autophagosome accumulation indicating reduced lysosomal clearance in the liver. In contrast, autophagy markers were unchanged in skeletal muscle, indicating that autophagy is not involved in the development of skeletal muscle insulin resistance.
    Keywords:  Autophagy; High-fat diet; Insulin resistance; Liver; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.bbrc.2020.11.048
  30. Fundam Clin Pharmacol. 2020 Dec 01.
    Evaluating and modulating TFEB in the control of autophagy: towards new treatments in CNS disorders.
    Anaelle da Costa, Thibaud Metais, Franck Mouthon, Danielle Kerkovich, Mathieu Charvériat.
      TFEB is a mammalian transcription factor that binds directly to the CLEAR consensus sequence (5'-GTCACGTGAC-3') present in the regulatory regions of genes inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase enzyme expression, lysosomal exocytosis and lysosomal housekeeping functions. By modulating these activities, TFEB coordinates on-demand control over each cell's degradation pathway. Thus, a nuclear signaling pathway regulates cellular energy metabolism through TFEB. Our growing understanding of the role of TFEB and CLEAR in the promotion of healthy clearance and subsequent in vitro and in vivo preclinical findings in various animal models of disease supports the conclusion that the pharmacological activation of TFEB could clear toxic proteins in multiple rare and common illnesses including pediatric and adult neurodegenerative disease.
    Keywords:  CNS disorders; TFEB; autophagy; lysosomal functions
    DOI:  https://doi.org/10.1111/fcp.12634
  31. Sci Rep. 2020 Dec 03. 10(1): 21160
    Stability of tuberous sclerosis complex 2 is controlled by methylation at R1457 and R1459.
    Seishu Gen, Yu Matsumoto, Ken-Ichi Kobayashi, Tsukasa Suzuki, Jun Inoue, Yuji Yamamoto.
      Mutations in genes that encode components of tuberous sclerosis complex 2 (TSC2) are associated with tuberous sclerosis complex disease. TSC2 interacts with tuberous sclerosis complex 1 to form a complex that negatively regulates cell growth and proliferation via the inactivation of mechanistic target of rapamycin complex 1. The activity of TSC2 is mainly regulated via posttranslational modifications such as phosphorylation. However, the control of TSC2 activity is not entirely achieved by phosphorylation. In this study, we show that TSC2 is methylated at R1457 and R1459 by protein arginine methyltransferase 1 (PRMT1). Methylation of these two residues can affect the phosphorylation status through protein kinase B (Akt) of TSC2 at T1462 and is essential for TSC2 stability. Taken together, these findings indicate that novel posttranslational modifications are important for the regulation of TSC2 stability through PRMT1-mediated methylation.
    DOI:  https://doi.org/10.1038/s41598-020-78274-6
  32. J Cell Mol Med. 2020 Dec 04.
    Overexpression of c-Fos reverses osteoprotegerin-mediated suppression of osteoclastogenesis by increasing the Beclin1-induced autophagy.
    Xishuai Tong, Miaomiao Chen, Ruilong Song, Hongyan Zhao, Jianchun Bian, Jianhong Gu, Zongping Liu.
      Osteoclastogenesis requires the involvement of transcription factors and degrading enzymes, and is regulated by upstream and downstream signalling. However, c-Fos how regulates osteoclastogenesis through autophagy remain unclear. This study aimed to explore the role of c-Fos during osteoprotegerin (OPG)-mediated suppression of osteoclastogenesis. We found that the number of osteoclasts and the expression of c-Fos, MMP-9, CAⅡ, Src and p62 were decreased after treated with OPG, including attenuation the PI3K/Akt and the TAK1/S6 signalling pathways, but the expression of Beclin1 and LC3Ⅱ were increased. Knockdown of Beclin1 could reverse the expression of c-Fos and MMP-9 by activating the PI3K/Akt signalling pathway, but inhibiting the autophagy and the TAK1/S6 signalling pathway. In addition, inhibition of autophagy using the PI3K inhibitor LY294002 did not rescues OPG-mediated suppression of osteoclastogenesis, but caused reduction of the expression of c-Fos and CAⅡ by attenuating the autophagy, as well as the PI3K/Akt and the TAK1/S6 signalling pathways. Furthermore, continuous activation of c-Fos could reverse OPG-mediated suppression of osteoclastogenesis by activating the autophagy and the PI3K/Akt and the TAK1/S6 signalling pathways. Thus, overexpression of c-Fos could reverse OPG-mediated suppression of osteoclastogenesis via activation of Beclin1-induced autophagy, indicating c-Fos might serve as a new candidate for bone-related basic studies.
    Keywords:  Beclin1; OPG; autophagy; c-Fos; osteoclastogenesis
    DOI:  https://doi.org/10.1111/jcmm.16152
  33. J Cell Sci. 2020 Dec 01. pii: jcs.250241. [Epub ahead of print]
    Mitochondria Control mTORC1 Activity Linked Compartmentalization of eIF4E to Regulate Extracellular Export of microRNAs.
    Susanta Chatterjee, Yogaditya Chakrabarty, Saikat Banerjee, Souvik Ghosh, Suvendra N Bhattacharyya.
      Defective intracellular trafficking and export of microRNAs have been observed in growth retarded mammalian cells having impaired mitochondrial potential and dynamics. Uncoupling Protein 2 mediated depolarization of mitochondrial membrane also results in progressive sequestration of microRNAs with polysomes and lowered their release via extracellular vesicles. Interestingly, impaired miRNA-trafficking process in growth retarded human cells could be reversed in presence of Genipin an inhibitor of Uncoupling Protein 2. Mitochondrial detethering of endoplasmic reticulum, observed in mitochondria depolarized cells, found to be responsible for defective compartmentalization of translation initiation factor eIF4E to endoplasmic reticulum attached polysomes. It causes retarded translation process accompanied by enhanced retention of miRNAs and target mRNAs with endoplasmic reticulum attached polysomes to restrict extracellular export of miRNAs. Reduced compartment specific activity of mTORC1 complex, the master regulator of protein synthesis, in mitochondria defective or ER- detethered cells, causes reduced phosphorylation of eIF4E-BP1 to prevent eIF-4E targeting to ER attached polysome and microRNA export. These data suggest how mitochondrial membrane potential and dynamics, by affecting mTORC1 activity and compartmentalization, determine sub-cellular localization and export of microRNAs.
    Keywords:  EIF4E and mTORC1; Exosomes; Extracellular vesicles; MiRNA; Mitochondria; P-body; Polysome; Processing bodies
    DOI:  https://doi.org/10.1242/jcs.250241
  34. Aging (Albany NY). 2020 Nov 29. 12
    Isoquercitrin induces apoptosis and autophagy in hepatocellular carcinoma cells via AMPK/mTOR/p70S6K signaling pathway.
    Liyan Shui, Weina Wang, Mingjie Xie, Bingjue Ye, Xian Li, Yanning Liu, Min Zheng.
      Hepatocellular carcinoma (HCC) is an aggressive malignancy with high rates of metastasis and relapse. Isoquercitrin (ISO), a natural flavonoid present in the Chinese bayberry and other plant species, reportedly exerts notable inhibitory effects on tumor cell proliferation, though the mechanism is unknown. In the present study, we exposed HepG2 and Huh7 human liver cancer cells to ISO and examined the roles of autophagy and apoptosis in ISO-mediated cell death. We found that ISO exposure inhibited cell viability and colony growth, activated apoptotic pathway, and triggered dysregulated autophagy by activating the AMPK/mTOR/p70S6K pathway. Autophagy inhibition using 3-methyladenine (3-MA) or Atg5-targeted siRNA decreased the Bax/Bcl-2 ratio, caspase-3 activation, and PARP cleavage and protected cells against ISO-induced apoptosis. Moreover, autophagy inhibition reversed the upregulation of AMPK phosphorylation and downregulation of mTOR and p70S6K phosphorylation elicited by ISO. By contrast, application of a broad-spectrum caspase inhibitor failed to inhibit autophagy in ISO-treated cells. These data indicate that ISO simultaneously induced apoptosis and autophagy, and abnormal induction of autophagic flux contributed to ISO-triggered caspase-3-dependent apoptosis.
    Keywords:  AMPK; apoptosis; autophagy; hepatocellular carcinoma; isoquercitrin
    DOI:  https://doi.org/10.18632/aging.202237
  35. J Cell Physiol. 2020 Dec 02.
    mTORC2 negatively controls the maturation process of medullary thymic epithelial cells by inhibiting the LTβR/RANK-NF-κB axis.
    Zhanfeng Liang, Qian Zhang, Xue Dong, Zhaoqi Zhang, Hongxia Wang, Jiayu Zhang, Yong Zhao.
      The differentiation of mature medullary thymic epithelial cells (mTECs) is critical for the induction of central immune tolerance. Although the critical effect of mechanistic target of rapamycin complex 1 (mTORC1) in shaping mTEC differentiation has been studied, the regulatory role of mTORC2 in the differentiation and maturation of mTECs is poorly understood. We herein reported that TEC-specific ablation of a rapamycin-insensitive companion of mTOR (RICTOR), a key component of mTORC2, significantly decreased the thymus size and weight, the total cell number of TECs, and the cell number of mTECs with a smaller degree of reduced cortical thymic epithelial cells. Interestingly, RICTOR deficiency significantly accelerated the mTEC maturation process, as indicated by the increased ratios of mature mTECs (MHCIIhi , CD80+ , and Aire+ ) to immature mTECs (MHCIIlo , CD80- , and Aire- ) in Rictor-deficient mice. The RNA-sequencing assays showed that the upregulated nuclear factor-κB (NF-κB) signaling pathway in Rictor-deficient mTECs was one of the obviously altered pathways compared with wild-type mTECs. Our studies further showed that Rictor-deficient mTECs exhibited upregulated expression of receptor activator of NF-κB (RANK) and lymphotoxin β receptor (LTβR), as well as increased activity of canonical and noncanonical NF-κB signaling pathways as determined by ImageStream and Simple Western. Finally, our results showed that inhibition of NF-κB signaling pathways could partially reverse the accelerated maturation of mTECs in Rictor conditional KO mice. Thus, mTORC2 negatively controls the kinetics of the mTEC maturation process by inhibiting the LTβR/RANK-NF-κB signal axis.
    Keywords:  T cell development; mTORC2; thymic epithelial cells; thymus
    DOI:  https://doi.org/10.1002/jcp.30192
  36. Biol Pharm Bull. 2020 ;43(12): 1983-1986
    mTORC1 Overactivation Leads to Abnormalities in Skeletal Development.
    Sayuki Iwahashi, Kazuya Tokumura, Gyujin Park, Shinsuke Ochiai, Yasuka Okayama, Hiroki Fusawa, Kaname Ohta, Kazuya Fukasawa, Takashi Iezaki, Eiichi Hinoi.
      The mechanistic/mammalian target of rapamycin complex-1 (mTORC1) integrates multiple signaling pathways and regulates various cellular processes. Tuberous sclerosis complex 1 (Tsc1) and complex 2 (Tsc2) are critical negative regulators of mTORC1. Mouse genetic studies, including ours, have revealed that inactivation of mTORC1 in undifferentiated mesenchymal cells and chondrocytes leads to severe skeletal abnormalities, indicating a pivotal role for mTORC1 in skeletogenesis. Here, we show that hyperactivation of mTORC1 influences skeletal development through its expression in undifferentiated mesenchymal cells at the embryonic stage. Inactivation of Tsc1 in undifferentiated mesenchymal cells by paired-related homeobox 1 (Prx1)-Cre-mediated recombination led to skeletal abnormalities in appendicular skeletons. In contrast, Tsc1 deletion in chondrocytes using collagen type II α1 (Col2a1)-Cre or in osteoprogenitors using Osterix (Osx)-Cre did not result in skeletal defects in either appendicular or axial skeletons. These findings indicate that Tsc complex-mediated chronic overactivation of mTORC1 influences skeletal development at the embryonic stage through its expression in undifferentiated mesenchymal cells but not in chondrocytes or osteoprogenitors.
    Keywords:  mechanistic/mammalian target of rapamycin complex-1 (mTORC1); skeletogenesis; tuberous sclerosis complex 1; undifferentiated mesenchymal cell
    DOI:  https://doi.org/10.1248/bpb.b20-00619
  37. Int J Mol Sci. 2020 Nov 25. pii: E8933. [Epub ahead of print]21(23):
    The Protective Effects of the Autophagic and Lysosomal Machinery in Vascular and Valvular Calcification: A Systematic Review.
    Cédric H G Neutel, Jhana O Hendrickx, Wim Martinet, Guido R Y De Meyer, Pieter-Jan Guns.
      BACKGROUND: Autophagy is a highly conserved catabolic homeostatic process, crucial for cell survival. It has been shown that autophagy can modulate different cardiovascular pathologies, including vascular calcification (VCN).OBJECTIVE: To assess how modulation of autophagy, either through induction or inhibition, affects vascular and valvular calcification and to determine the therapeutic applicability of inducing autophagy.
    DATA SOURCES: A systematic review of English language articles using MEDLINE/PubMed, Web of Science (WoS) and the Cochrane library. The search terms included autophagy, autolysosome, mitophagy, endoplasmic reticulum (ER)-phagy, lysosomal, calcification and calcinosis. Study characteristics: Thirty-seven articles were selected based on pre-defined eligibility criteria. Thirty-three studies (89%) studied vascular smooth muscle cell (VSMC) calcification of which 27 (82%) studies investigated autophagy and six (18%) studies lysosomal function in VCN. Four studies (11%) studied aortic valve calcification (AVCN). Thirty-four studies were published in the time period 2015-2020 (92%).
    CONCLUSION: There is compelling evidence that both autophagy and lysosomal function are critical regulators of VCN, which opens new perspectives for treatment strategies. However, there are still challenges to overcome, such as the development of more selective pharmacological agents and standardization of methods to measure autophagic flux.
    Keywords:  aortic valve calcification; autophagy; lysosomes; valvular interstitial cell (VIC); vascular calcification; vascular smooth muscle cell (VSMC)
    DOI:  https://doi.org/10.3390/ijms21238933
  38. Front Cell Dev Biol. 2020 ;8 600950
    Oxidized LDL Causes Endothelial Apoptosis by Inhibiting Mitochondrial Fusion and Mitochondria Autophagy.
    Jia Zheng, Chengzhi Lu.
      Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is an initial step toward atherosclerosis development. Mitochondria damage correlates with ox-LDL-induced endothelial injury through an undefined mechanism. We explored the role of optic atrophy 1 (Opa1)-related mitochondrial fusion and mitophagy in ox-LDL-treated endothelial cells, focusing on mitochondrial damage and cell apoptosis. Oxidized low-density lipoprotein treatment reduced endothelial cell viability by increasing apoptosis. Endothelial cell proliferation and migration were also impaired by ox-LDL. At the molecular level, mitochondrial dysfunction was induced by ox-LDL, as demonstrated by decreased mitochondrial membrane potential, increased mitochondrial reactive oxygen species production, augmented mitochondrial permeability transition pore openings, and elevated caspase-3/9 activity. Mitophagy and mitochondrial fusion were also impaired by ox-LDL. Opa1 overexpression reversed this effect by increasing endothelial cell viability and decreasing apoptosis. Interestingly, inhibition of mitophagy or mitochondrial fusion through transfection of siRNAs against Atg5 or Mfn2, respectively, abolished the protective effects of Opa1. Our results illustrate the role of Opa1-related mitochondrial fusion and mitophagy in sustaining endothelial cell viability and mitochondrial homeostasis under ox-LDL stress.
    Keywords:  DRP1; macrophage; miR-9; mitochondrial fission; ox-LDL
    DOI:  https://doi.org/10.3389/fcell.2020.600950
  39. Autophagy. 2020 Nov 29.
    A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway.
    Sonia R Singh, Moritz Meyer-Jens, Erda Alizoti, W Clark Bacon, Gregory Davis, Hanna Osinska, James Gulick, Silke Reischmann-Düsener, Ellen Orthey, Patrick M McLendon, Jeffery D Molkentin, Saskia Schlossarek, Jeffrey Robbins, Lucie Carrier.
      The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both the UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (UbG76V-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both UbG76V-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with Zfp418 overexpression in HEK293 cells, we evaluated Zfp418 knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from Zfp418 knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activated the promoters of Dapk2 and Fyco1, which are involved in autophagy. RNA-seq from Zfp418 knockdown also revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function.
    Keywords:  ALP; UPS; ZFP418; ZNF418; autophagy; cardiomyocyte proteasome; protein degradation; screen; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2020.1856493
  40. PLoS Genet. 2020 Dec 04. 16(12): e1009255
    Characterization of constitutive ER-phagy of excess membrane proteins.
    Zhanna Lipatova, Valeriya Gyurkovska, Sarah F Zhao, Nava Segev.
      Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.
    DOI:  https://doi.org/10.1371/journal.pgen.1009255
  41. Med Hypotheses. 2020 Nov;pii: S0306-9877(20)31151-8. [Epub ahead of print]144 110243
    Premises among SARS-CoV-2, dysbiosis and diarrhea: Walking through the ACE2/mTOR/autophagy route.
    Ana Patrícia de Oliveira, André Luis Fernandes Lopes, Gabriella Pacheco, Isabela Ribeiro de Sá Guimarães Nolêto, Lucas Antonio Duarte Nicolau, Jand Venes Rolim Medeiros.
      Recently, a new coronavirus (SARS-CoV-2) was discovered in China. Due to its high level of contagion, it has already reached most countries, quickly becoming a pandemic. Although the most common symptoms are related to breathing problems, SARS-CoV-2 infections also affect the gastrointestinal tract culminating in inflammation and diarrhea. However, the mechanisms related to these enteric manifestations are still not well understood. Evidence shows that the SARS-CoV-2 binds to the angiotensin-converting enzyme receptor 2 (ACE2) in host cells as a viral invasion mechanism and can infect the lungs and the gut. Other viruses have already been linked to intestinal symptoms through binding to ACE2. In turn, this medical hypothesis article conjectures that the ACE2 downregulation caused by the SARS-CoV-2 internalization could lead to decreased activation of the mechanistic target of mTOR with increased autophagy and lead to intestinal dysbiosis, resulting in diarrhea. Besides that, dysbiosis can directly affect the respiratory system through the lungs. Although there are clues to other viruses that modulate the ACE2/gut/lungs axis, including the participation of autophagy and dysbiosis in the development of gastrointestinal symptoms, there is still no evidence of the ACE2/mTOR/autophagy pathway in SARS-CoV-2 infections. Thus, we propose that the new coronavirus causes a change in the intestinal microbiota, which culminates in a diarrheal process through the ACE2/mTOR/autophagy pathway into enterocytes. Our assumption is supported by premises that unregulated intestinal microbiota increases the susceptibility to other diseases and extra-intestinal manifestations, which can even cause remote damage in lungs. These putative connections lead us to suggest and encourage future studies aiming at assessing the aforementioned hypothesis and regulating dysbiosis caused by SARS-CoV-2 infection, in order to confirm the decrease in lung injuries and the improvement in the prognosis of the disease.
    Keywords:  Coronavirus; Diarrhea; Dysbiosis; SARS-CoV-2
    DOI:  https://doi.org/10.1016/j.mehy.2020.110243
  42. Autophagy. 2020 Nov 29.
    Analysis of Drosophila Atg8 proteins reveals multiple lipidation-independent roles.
    András Jipa, Viktor Vedelek, Zsolt Merényi, Adél Ürmösi, Szabolcs Takáts, Attila L Kovács, Gábor V Horváth, Rita Sinka, Gábor Juhász.
      Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. In this Brief Report, we use clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. We find that high expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility.
    Keywords:   Drosophila ; Atg8a; Atg8b; autophagy; sperm; testis
    DOI:  https://doi.org/10.1080/15548627.2020.1856494
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